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SUMMARY To enhance the breeding of new scab‐resistant apple cultivars, a comprehensive understanding of the mechanisms governing major scab resistance genes is essential. Rvi12_Cd5 was previously identified as the best candidate gene for the Rvi12 scab resistance of the crab apple “Hansen's baccata #2” by gene prediction and in silico analysis. In the present study, Rvi12_Cd5 was used to transform the scab‐susceptible apple cultivar “Gala Galaxy.” Two constructs were prepared: the first carrying Rvi12_Cd5 under the control of a 35S promoter and E9 terminator, and the second carrying Rvi12_Cd5 under the control of its native promoter and terminator. All the transgenic lines were analyzed for T‐DNA integration, copy number, and expression of Rvi12_Cd5 and phenotypically evaluated for scab resistance. The “Gala Galaxy” lines carrying the 35S promoter expressed Rvi12_Cd5 at a high level, showing partial to high resistance against a mixed inoculum of Venturia inaequalis, with symptoms ranging from class 0 to 3b on the Chevalier scale. The transgenic lines carrying the native promoter showed a lower expression of Rvi12_Cd5 compared with the 35S lines. Nevertheless, the low expression was sufficient to induce a resistance level comparable to that of the transgenic lines carrying the 35S promoter. These results indicate that Rvi12_Cd5 confers scab resistance to a susceptible apple cultivar and that even a low level of gene transcript can trigger a plant response to V. inaequalis infection. After HcrVf2 and Vr2‐C, Rvi12_Cd5 is the third major apple scab resistance gene being functionally proven. [ABSTRACT FROM AUTHOR]

Background: The 12-oxo-phytodienoic acid reductase (OPR) enzyme is crucial for the synthesis of jasmonates (JAs), and is involved in the plant stress response. However, the OPR gene family in sweetpotato, an important horticultural crop, remains unidentified. Results: In this study, we employed bioinformatics techniques to identify nine IbOPR genes. Phylogenetic analysis revealed that these genes could be divided into Group I and Group II. Synteny analysis indicated that IbOPR evolution was driven by tandem duplication, whole-genome duplication (WGD), and segmental duplication events. The promoter sequences of IbOPRs were found to be associated with stress and hormonal responses. Additionally, we successfully cloned four IbOPRs from "Haida HD7791" and "Haida HD7798" using homologous cloning technology. These sequences were 1203 bp, 1200 bp, 1134 bp, and 1137 bp in length and encoded 400, 399, 377, and 378 amino acids, respectively. The protein sequence similarity between the salt-tolerant variety "Haida HD7791" and the salt-sensitive variety "Haida HD7798" was determined to be 96.75% for IbOPR2, 99.75% for IbOPR3, 92.06% for IbOPR6, and 98.68% for IbOPR7. Phylogenetic analysis categorized IbOPR2 and IbOPR3 proteins into Group II, while IbOPR6 and IbOPR7 proteins belonged to Group I. Subcellular localization experiments showed IbOPR2 protein present in the peroxisome, while IbOPR3, IbOPR6, and IbOPR7 proteins were found in the cytoplasm and nucleus. Salt stress induction experiments demonstrated that IbOPR2, IbOPR3, and IbOPR7 were significantly upregulated only in 'Haida HD7791' after 6 h. In contrast, IbOPR6 was induced in 'Haida HD7798' at 6 h but inhibited in 'Haida HD7791' at later time points (12, 24, 48, and 72 h), highlighting functional differences in salt stress responses. Conclusions: Our findings suggest that IbOPR2 may play a crucial role in sweetpotato's response to salt stress by participating in JAs synthesis. These results provide a foundation for future functional analyses of OPR genes in sweetpotato. [ABSTRACT FROM AUTHOR]

The full-length cDNA sequence of Penaeus monodon glucose transporter-2 (PmGLUT2) was cloned in this study using the RACE method. Quantitative real-time PCR was used to analyze the differential expression of PmGLUT2 during the development of P. monodon larvae in different tissues and under low salinity stress. The PmGLUT2 cDNA exhibited a total length of 2018 base pairs, with 94 base pairs located in the 5' untranslated region (UTR) and 352 base pairs in the 3' UTR. Additionally, the sequence contained 29 base poly (A) tails and 1572 base pairs within the open reading frame (ORF), capable of encoding 523 amino acids. Through a comparative analysis of the amino acid sequences of PmGLUT2 and LvGLUT2, it was determined that PmGLUT2 had 94.77% homology with Tret1 gene of Litopenaeus vannamei, and 60.54% homology with GLUT2 gene of L. vannamei. The results indicated that there was a fluctuation in PmGLUT2 expression levels from zygote to postlarva development, with initial reduction followed by an increase, although the difference was not statistically significant. According to the molting analysis, the highest expression level of PmGLUT2 was observed in the hepatopancreas during the premolt period, while the gill and gut exhibited peak expression levels during the intermolt period. PmGLUT2 was found to be most abundant in lymph tissue, followed by gill tissue, and least abundant in ootheca, according to the tissue expression analyses. Following 96 hours of acute salt stress, there was a notable inhibition in the expression of PmGLUT2 in the hepatopancreas and gills. Additionally, the expression level in the gills at 96 hours was significantly lower compared to the baseline level at 0 hours. [ABSTRACT FROM AUTHOR]

[Objective] To clone the full-length sequence of the squalene synthase gene and perform bioinformatics and expression pattern analysis from Lonicera macranthoides. [Method] Total RNA was extracted from Lonicera macranthoides, and the full-length cDNA sequence of LmSS gene was cloned using RT-PCR and RACE techniques, bioinformatics analysis was conducted on the gene sequence using relevant software, the relative expression of the gene in stem, leaf, and different flower period was determined by using Real-time PCR. [Result] The open reading frame (ORF) of LmSS gene was 1 245 bp, encoding 414 amino acids. It belongs to a hydrophilic protein and is located in the cytoplasm. LmSS gene has a typical polyisoprene synthase active domain, which has high homology with other plant SS genes. The expression of LmSS gene is tissue-specific in different flowering stages and organs of Lonicera macranthoides. [Conclusion] This study successfully cloned the SS gene in Lonicera macranthoides, laying a foundation for further research on its function and providing a research basis for exploring the biosynthesis and regulatory mechanism of saponins in Lonicera macranthoides. [ABSTRACT FROM AUTHOR]

Leaf senescence is the final stage of leaf development, where the nutrients and energy of senescent leaves are redistributed to developing tissues or organs for plant growth, reproduction, and defense. Outer leaves are photosynthetic organs that usually senesce at the late heading stage in Chinese cabbage, and premature leaf senescence often reduces leafy head yield and quality. In this study, 11 premature leaf senescence mutants were screened from an ethyl methanesulfonate-mutagenized population of the double haploid line 'FT' in Chinese cabbage. At the early heading stage, the mutants exhibited edge yellowing within its outer leaves, and at the mature stage, its leafy head weight decreased significantly. Genetic analysis revealed that the mutated trait of all 11 mutants corresponds to single gene recessive inheritance. Semi-diallel cross tests showed that 5 of the 11 were allelic mutants. MutMap and Kompetitive Allele Specific PCR genotyping revealed that BraA01g001400.3C was the candidate gene, which is orthologous of Arabidopsis SUPPRESSOR OF rps4-RLD 1, encoding an immune regulator, so we named it as BrSRFR1. All the BrSRFR1 in the five allelic mutants exhibited single nucleotide polymorphisms at different positions on their exons and led to premature translation termination, which confirmed that defect in BrSRFR1 led to premature leaf senescence. These results verify the role of BrSRFR1 on leaf senescence and provide a new insight into the mechanisms of leaf senescence in Chinese cabbage, which reveals a novel function of SRFR1 in plant development. [ABSTRACT FROM AUTHOR]

Copyright of Bulletin of Botanical Research is the property of Bulletin of Botanical Research Editorial Department and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Environmental pollution is growing every day in terms of the increase in population, industrialization, and urbanization. Shewanella azerbaijanica is introduced as a highly potent bacterium in metal bioremediation. The mtrC gene was selected as a cloning target to improve electron flux chains in the EET (extracellular electron transfer) pathway. Using the SDM (site-directed mutagenesis) technique, the unique gene assembly featured the mtrC gene sandwiched between two napD/B genes to disrupt the nitrate reduction pathway, which serves as the primary metal reduction competitor. Shew-mtrC gene construction was transferred to expression plasmid pET28a (+) in the expression host bacteria (E. coli BL21 and S. azerbaijanica), in pUC57, cloning plasmid, which was transferred to the cloning host bacteria E. coli Top10 and S. azerbaijanica. All cloning procedures (i.e., synthesis, insertion, transformation, cloning, and protein expression) were verified and confirmed by precise tests. ATR-FTIR analysis, CD, western blotting, affinity chromatography, SDS-PAGE, and other techniques were used to confirm the expression and structure of the MtrC protein. The genome sequence and primers were designed according to the submitted Shewanella oneidensis MR-1 genome, the most similar bacteria to this native species. The performance of recombinant S. azerbaijanica bacterium in metal bioremediation, as sustainable strategy, has to be verified by more research. [ABSTRACT FROM AUTHOR]

Background: Cabbage (Brassica oleracea L. var. capitata) is an important crop within the Brassica oleracea species and is extensively cultivated worldwide. In recent years, outbreaks of downy mildew caused by Hyaloperonospora parasitica have resulted in substantial losses in cabbage production. Despite this, there have been limited studies on genes associated with resistance to downy mildew in cabbage. Results: This study identified sister lines exhibiting significant differences in disease resistance and susceptibility. Using bulked segregant analysis followed by sequencing (BSA-seq) and linkage analysis, the cabbage resistance locus BoDMR2 was accurately mapped to an approximately 300 kb interval on chromosome 7. Among the candidate genes identified, several single nucleotide polymorphisms (SNPs) and a 3-bp insertion were found within the conserved domain of the Bo7g117810 gene, encoding a leucine-rich repeat domain protein, in susceptible genotypes. Additionally, real-time quantitative polymerase chain reaction (RT‒qPCR) analysis revealed that the expression level of Bo7g117810 in resistant specimens was 2.5-fold higher than that in susceptible specimens. An insertion‒deletion (InDel) marker was designed based on the identified insertion in susceptible materials, facilitating the identification and selection of downy mildew-resistant cabbage cultivars. Conclusions: This study identifies Bo7g117810 as a potential candidate gene associated with adult-stage resistance to downy mildew in cabbage, supported by observed differences in gene sequence and expression levels. Furthermore, the development of an InDel marker I1-3, based on its mutation, provides valuable resources for breeding resistant cabbage cultivars. [ABSTRACT FROM AUTHOR]

Copyright of Science & Technology of Food Industry is the property of Science & Technology of Food Industry Editorial Office and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Background and purpose: SARS-CoV-2 causes a severe respiratory disease known as COVID-19 and is responsible for a global viral pandemic. The SARS-CoV-2 receptor binding domain (RBD) is located on the spike protein, which identifies and binds to the angiotensin-converting enzyme 2 (ACE2) receptor. The RBD is an important target for developing virus-neutralizing antibodies, vaccines, and inhibitors. Experimental approach: In this study, recombinant SARS-CoV-2 RBD was expressed in E. coli BL21 (DE3) and purified and its binding activity was determined. Purification was conducted using the Ni-NTA column. ELISA. flow cytometry assays were set to evaluate the binding ability of recombinant RBD to different anti-RBD antibodies and native ACE2 receptors on HEK293A cells, respectively. Findings/Results: The SDS-PAGE analysis revealed the corresponding band at 27 kDa in the culture after induction with 0.7 mM IPTG, while the corresponding band was not observed in the culture without IPTG induction. ELISA results showed that antibodies produced in the human sera could bind to the recombinant RBD protein and the commercial anti-RBD antibody. Also, flow cytometry analysis revealed that the recombinant RBD could bind to human ACE2 on the surface of HEK293A cells. Conclusion and implication: Our outcomes displayed that the recombinant RBD expressed in the E. coli strain has biological activity and can be used as an antigen for the development of diagnosis kits and vaccines as well as a tool for screening drugs against SASR-CoV-2. [ABSTRACT FROM AUTHOR]

MORF4L2 is a transcription factor involved in heterochromatin assembly and histone modification by forming the NuA4 histone acetyltransferase complex, which plays an important role in cell growth, proliferation, and apoptosis. In order to explore the function of MORF4L2 in red deer （Cervus elaphus） velvet antler, qPRC was used to detect the mRNA expression level of MORF4L2 gene in different tissues of deer antler. The CDS sequence of the MORF4L2 gene was cloned by PCR. Similarity analysis of mRNA sequences of the MORF4L2 gene was performed by multiple species comparison and phylogenetic tree was constructed. The structure and physicochemical properties of MORF4L2 encoding protein were predicted and analyzed by bioinformatics method. The results showed that the mRNA relative expression of MORF4L2 gene was the highest in precartilaginous layer of deer antler. The mRNA sequence of the MORF4L2 gene in red deer is highly conserved, with the highest similarity to that in C. canadensis. The total length of the CDS region of MORF4L2 gene in red deer is 864 bp, encoding 287 amino acids, and the theoretical isoelectric point is 9. 73, which is a basic protein. It was predicted that there was no signal peptide present in the MORF4L2 protein of red deer, and there were 30 potential phosphate sites. The protein structure is mainly composed of random coils and alpha helixes, where proteins are mainly located in the nucleus. This study provided basic data for the important role of MORF4L2 in the growth and development of the red deer velvet antler. [ABSTRACT FROM AUTHOR]

espl1 (extra spindle pole bodies like 1), a cysteine endopeptidase, is a mitotic key player in chromosomal segregation and centriole duplication during mitosis and meiosis. Considering the espl1 gene has not been reported in aquatic organisms, we reported the isolation and expression of the espl1 gene from loach. In this study, the full-length cDNA of espl1 was cloned for the first time in loach. In loach, the full-length cDNA of espl1 consisted of 6948 bp, the open reading frame (ORF) is 6240 bp, and the espl1 gene encodes 2139 amino acids. Moreover, the deduced amino acid sequences of espl1 in loach shared the highest identity with those of Cyprinus carpio (78.39%) and Sinocyclocheilus anshuiensis (78.27%), and the sequence homology among the various separases is confined to the C-terminal region. Furthermore, tissue-specific checking results indicated that the espl1 gene of the loach gene was highly expressed in the ovary and testis, especially in stage IV oocytes and stage IV testis by Real-time quantitative PCR (qPCR). Then, whole-mount in situ hybridization analyses revealed the expression of espl1 in the early development of loach. We found that the positive signal of espl1 was observed in the notochord during the early embryo development of loach. Last but not least, when treated with luteinizing hormone-releasing hormone analog (LHRH-A2), the mRNA expression of espl1 was significantly increased in the testes and ovaries. These observations suggest that the espl1 gene had a distinct and important role in the gonads of Loach. This study will be of value for further studies into the function of the espl1 gene in fish. [ABSTRACT FROM AUTHOR]

Gene cloning is an important step in investigating gene structure and function. To verify gene sequence, Sanger sequencing is used, which may produce several overlapping sequencing files that need to be merged before alignment to the target gene sequence is performed. Previously, we reported the Python program to Merge Sanger sequences (), which ran in command line and relied heavily on EMBOSS suite. In this updated version of the program, we have made several remarkable improvements. It provides a graphical user interface (GUI) written with tkinter, which is convenient and stable. It does not require users to rename the input sequences before performing merging. With regard to the implementation, the updated version utilizes Python function (Align.PairwiseAligner) to align adjacent sequences, which is more flexible (can adjust program parameter i.e., the number of first-time consecutive matching bases). The new version of the program makes merging Sanger sequences much more convenient and facilitates gene study. [ABSTRACT FROM AUTHOR]

The Cytochrome P450 (CYP450) family is the largest enzyme protein family in plants, distributed across various organs and involved in significant catalytic activities in primary and secondary metabolic processes. In this study, we cloned the PtCYP721A57 gene, characterized its open reading frame (ORF), and conducted comprehensive analyses including physicochemical properties, evolutionary relationships, subcellular localization, prokaryotic expression, and correlation between the relative expression of different parts and the content of tenuifolin, hormones, and abiotic stress response associated with the encoded protein. The ORF of PtCYP721A57 was 1,521 bp, with a secondary structure predominantly composed of α-helices and random coils. Subcellular localization experiments confirmed the presence of PtCYP721A57 in the endoplasmic reticulum. For prokaryotic expression, we constructed the recombinant plasmid pET28a-PtCYP721A57 using pET28a as the vector, which was then transformed into BL21(DE3). Induction with Isopropyl β-D-1-thiogalactopyranoside (IPTG) at temperatures of 16 and 25 °C and varying concentrations (0.1, 0.2, 0.5, 1, 2 mM) resulted in the formation of inclusion bodies, with higher expression observed at 25 °C. Our qPCR analyses revealed that PtCYP721A57 exhibited the highest expression in the cortex of Polygala tenuifolia, followed by roots and xylem, correlating with the observed tenuifolin content distribution. Induction with abscisic acid (ABA) and chitosan (CHT) initially decreased PtCYP721A57 expression followed by a subsequent increase, peaking at 48 h. Similarly, drought stress induced a gradual increase in PtCYP721A57 expression, also peaking at 48 h. NaCl treatment for 6 h significantly upregulated PtCYP721A57 expression. In conclusion, our study provides foundational insights into the PtCYP721A57 gene in Polygala tenuifolia, laying the groundwork for further exploration of its role in the biosynthesis pathway of triterpenoid saponins. [ABSTRACT FROM AUTHOR]

Copyright of Acta Prataculturae Sinica is the property of Acta Prataculturae Sinica Editorial Office and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Copyright of Acta Edulis Fungi is the property of Acta Edulis Fungi and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Copyright of Acta Agriculturae Zhejiangensis is the property of Acta Agriculturae Zhejiangensis Editorial Office and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Averrhoa carambola, a key tropical and subtropical economic tree in the Oxalidaceae family, is distinguished by its unique pentagram-shaped fruit. This study investigates the developmental processes shaping the polarity of A. carambola fruit and their underlying hormonal and genetic mechanisms. By analyzing the Y1, Y2, and Y3 developmental stages—defined by the fruit diameters of 3–4 mm, 4–6 mm, and 6–12 mm, respectively—we observed that both cell number and cell size contribute to fruit development. Our findings suggest that the characteristic pentagram shape is established before flowering and is maintained throughout development. A hormonal analysis revealed that indole-3-acetic acid (IAA) and abscisic acid (ABA) show differential distribution between the convex and concave regions of the fruit across the developmental stages, with IAA playing a crucial role in polar auxin transport and shaping fruit morphology. A transcriptomic analysis identified several key genes, including AcaGH3.8, AcaIAA20, AcaYAB2, AcaXTH6, AcaYAB3, and AcaEXP13, which potentially regulate fruit polarity and growth. This study advances our comprehension of the molecular mechanisms governing fruit shape, offering insights for improving fruit quality through targeted breeding strategies. [ABSTRACT FROM AUTHOR]

Abstract Background The 12-oxo-phytodienoic acid reductase (OPR) enzyme is crucial for the synthesis of jasmonates (JAs), and is involved in the plant stress response. However, the OPR gene family in sweetpotato, an important horticultural crop, remains unidentified. Results In this study, we employed bioinformatics techniques to identify nine IbOPR genes. Phylogenetic analysis revealed that these genes could be divided into Group I and Group II. Synteny analysis indicated that IbOPR evolution was driven by tandem duplication, whole-genome duplication (WGD), and segmental duplication events. The promoter sequences of IbOPRs were found to be associated with stress and hormonal responses. Additionally, we successfully cloned four IbOPRs from "Haida HD7791" and "Haida HD7798" using homologous cloning technology. These sequences were 1203 bp, 1200 bp, 1134 bp, and 1137 bp in length and encoded 400, 399, 377, and 378 amino acids, respectively. The protein sequence similarity between the salt-tolerant variety "Haida HD7791" and the salt-sensitive variety "Haida HD7798" was determined to be 96.75% for IbOPR2, 99.75% for IbOPR3, 92.06% for IbOPR6, and 98.68% for IbOPR7. Phylogenetic analysis categorized IbOPR2 and IbOPR3 proteins into Group II, while IbOPR6 and IbOPR7 proteins belonged to Group I. Subcellular localization experiments showed IbOPR2 protein present in the peroxisome, while IbOPR3, IbOPR6, and IbOPR7 proteins were found in the cytoplasm and nucleus. Salt stress induction experiments demonstrated that IbOPR2, IbOPR3, and IbOPR7 were significantly upregulated only in 'Haida HD7791' after 6 h. In contrast, IbOPR6 was induced in 'Haida HD7798' at 6 h but inhibited in 'Haida HD7791' at later time points (12, 24, 48, and 72 h), highlighting functional differences in salt stress responses. Conclusions Our findings suggest that IbOPR2 may play a crucial role in sweetpotato's response to salt stress by participating in JAs synthesis. These results provide a foundation for future functional analyses of OPR genes in sweetpotato.

Background and purpose: SARS-CoV-2 causes a severe respiratory disease known as COVID-19 and is responsible for a global viral pandemic. The SARS-CoV-2 receptor binding domain (RBD) is located on the spike protein, which identifies and binds to the angiotensin-converting enzyme 2 (ACE2) receptor. The RBD is an important target for developing virus-neutralizing antibodies, vaccines, and inhibitors. Experimental approach: In this study, recombinant SARS-CoV-2 RBD was expressed in E. coli BL21 (DE3) and purified and its binding activity was determined. Purification was conducted using the Ni-NTA column. ELISA. flow cytometry assays were set to evaluate the binding ability of recombinant RBD to different anti-RBD antibodies and native ACE2 receptors on HEK293A cells, respectively. Findings/Results: The SDS-PAGE analysis revealed the corresponding band at 27 kDa in the culture after induction with 0.7 mM IPTG, while the corresponding band was not observed in the culture without IPTG induction. ELISA results showed that antibodies produced in the human sera could bind to the recombinant RBD protein and the commercial anti-RBD antibody. Also, flow cytometry analysis revealed that the recombinant RBD could bind to human ACE2 on the surface of HEK293A cells. Conclusion and implication: Our outcomes displayed that the recombinant RBD expressed in the E. coli strain has biological activity and can be used as an antigen for the development of diagnosis kits and vaccines as well as a tool for screening drugs against SASR-CoV-2.

Abstract Background Cabbage (Brassica oleracea L. var. capitata) is an important crop within the Brassica oleracea species and is extensively cultivated worldwide. In recent years, outbreaks of downy mildew caused by Hyaloperonospora parasitica have resulted in substantial losses in cabbage production. Despite this, there have been limited studies on genes associated with resistance to downy mildew in cabbage. Results This study identified sister lines exhibiting significant differences in disease resistance and susceptibility. Using bulked segregant analysis followed by sequencing (BSA-seq) and linkage analysis, the cabbage resistance locus BoDMR2 was accurately mapped to an approximately 300 kb interval on chromosome 7. Among the candidate genes identified, several single nucleotide polymorphisms (SNPs) and a 3-bp insertion were found within the conserved domain of the Bo7g117810 gene, encoding a leucine-rich repeat domain protein, in susceptible genotypes. Additionally, real-time quantitative polymerase chain reaction (RT‒qPCR) analysis revealed that the expression level of Bo7g117810 in resistant specimens was 2.5-fold higher than that in susceptible specimens. An insertion‒deletion (InDel) marker was designed based on the identified insertion in susceptible materials, facilitating the identification and selection of downy mildew-resistant cabbage cultivars. Conclusions This study identifies Bo7g117810 as a potential candidate gene associated with adult-stage resistance to downy mildew in cabbage, supported by observed differences in gene sequence and expression levels. Furthermore, the development of an InDel marker I1-3, based on its mutation, provides valuable resources for breeding resistant cabbage cultivars.

High-yield strains of cold-active lipase are screened to provide production information for the industrial development of lipase. A cold-active lipase-producing strain was isolated from Xing'an larch forest soil in Mohe County using the Rhodamine B plate method. Its morphological, physiological, and biochemical characteristics were identified, and combined with the 16S rDNA gene sequence, the strain was determined to be Bacillus thuringiensis. The lipase gene Lip240 was cloned using the Bacillus thuringiensis genome as a template, and heterologous expression and enzymatic properties of Lip240 were analyzed. The results showed that the recombination lipase Lip240 had an optimal reaction temperature of 30 ℃, and could maintain more than 60% activity when treated at 4~30 ℃ for 6 hours, thus a cold-active lipase. Besides, its optimal pH was 8.0. Additionally, Ca2+, Mn2+, Fe2+, Fe3+, and Cr3+ had a certain enhancing effect on the activity of Lip240. Notably, Lip240 had a good organic solvent tolerance, while SDS could significantly (P

Abstract [Objective] The study aims to clone the fructokinase (FRK ) gene of red pitaya (Hylocereus polyrhizus), detect its expression and enzyme activity, and analyze its physiological function in soluble sugar accumulation during fruit development. [Methods] HpFRK1 gene was cloned from ‘Zihonglong’ fruits, and bioinformatics and subcellular localization analysis were carried out. Gene expression was detected by qRT-PCR. Recombinant protein was obtained by E.coli induction and enzyme activity was detected. [Results] The open reading frame (ORF) of HpFRK1 was 993 bp in length, encoding 330 amino acids with a phosphofructokinase type B (pfkB) domain. HpFRK1 had the closest genetic relationship with sugar beet BvFRK, both of which belonged to cytoplasm localized FRKs. HpFRK1 was expressed in different developmental stages of mature stems and fruits. The expression level of HpFRK1 was the highest at 20 days after flowering and the lowest at 30 days after flowering (fruit ripening), and was gradually decreased with fruit development. Subcellular localization assay showed that HpFRK1 was mainly localized in the nucleus and cytoplasm. HpFRK1 recombinant protein specifically catalyzed fructose phosphorylation (K m=11.01 mmol/L). [Conclusion] HpFRK1 specifically catalyzes fructose phosphorylation in the cytoplasm and negatively regulates fruit soluble sugar accumulation during the development of red-fleshed pitaya fruits

Abstract [Objective] As a peony breeding material, Paeonia delavayi has abundant flower color resources. Studying the regulatory effects of its MYB transcription factors on flower color will benefit molecular breeding of peony flower color. [Methods] Using the flowers of yellow and red P . delavayi as materials for hand sectioning. Based on the transcriptomic data of P . delavayi, a MYB transcription factor coding gene PdMYB57 was obtained through sequence alignment. Function of this gene was verified through gene cloning, phylogenetic tree construction, qRT-PCR, transient expression, and HPLC. [Results] PdMYB57 gene has an ORF of 798 bp, encoding an unstable hydrophilic protein of 265 amino acids. PdMYB57 was clustered with the Arabidopsis SG6 subfamily, as well as MYB transcription factors in P . qiui PqMYB113, P . suffruticosa PsMYB57/PsMYB58, and Vitis vinifera VvMYBA1/VvMYBA2. PdMYB57 had a R2R3 conserved domain [R/K] Px [P/A/R] xx [F/Y] motif. PdMYB57 gene was highly expressed in sepals of red P . delavayi and leaves of yellow P . delavayi. HPLC analysis showed that tobacco leaves transiently expressed PdMYB57 gene contained cyanidin-3-O-rutinoside (Cy3R). [Conclusion] PdMYB57 gene encodes an R2R3-MYB transcription factor. PdMYB57 gene expression is tissue-specific and promotes anthocyanin synthesis in plants.

Abstract [Objective] This study aims to explore the function of VuARI2 gene in response to low temperature stress, by cloning and expressing VuARI2 gene in Arabidopsis. [Methods] VuARI2 gene was cloned from the stem of bog bilberry by RACE and RT-PCR and conducted a comprehensive bioinformatics analysis. Expression of VuARI2 in different tissues after cold acclimation was analyzed by RT-qPCR. Furthermore, the transgenic Arabidopsis was analyzed for the expression of VuARI2, physiological indexes, and freezing resistance. [Results] VuARI2 was successfully cloned,with an open reading frame of 1 770 bp, encoding 589 amino acids. The multi-sequence alignment and phylogenetic tree indicated that ubiquitin ligase VuARI2 was closely related to the amino acid sequence of ARI2 proteins in wild camellia, coffee tree, grape, and riverside grape. RT-qPCR showed that the expression of VuARI2 in leaves and flower buds of bog bilberry was higher than that in roots and stems, and the expression of VuARI2 in stems and flower buds was induced under low temperature. After cold acclimation, expression levels of VuARI2 gene in transgenic plants were higher in wild type, and chlorophyll content was lower than the wild type. Proline content and soluble sugar content were higher than the wild type. Superoxide dismutase activity, peroxidase activity, and catalase activity were higher than the wild type, while relative conductivity and malondialdehyde content were lower than the wild type. After cold acclimation, the survival rate of transgenic plants was higher than the wild type,and the relative conductivity was lower than the wild type at freezing temperature,enhancing freezing resistance of plants. [Conclusions] VuARI2 gene is cloned from bog bilberry and expressed in Arabidopsis. Overexpress of VuARI2 gene enhances the cold resistance of plants.

To explore substitutes for natural tropomyosin as basic materials for the diagnosis and treatment of shrimp allergy, total RNA was extracted from Macrobrachium nipponense, and the full-length sequence of the tropomyosin gene was cloned by rapid amplification of cDNA ends (RACE). Specific primers were designed based on the cDNA sequence of tropomyosin and the expression plasmid pET-30a-TM was constructed. Finally, recombinant tropomyosin was expressed under the induction of isopropyl-β-D-thiogalactopyranoside (IPTG). The results showed that the full-length cDNA sequence of tropomyosin was 1 658 bp in length (GenBank accession number: OP974621). Its open reading frame (ORF) was 855 bp in length, encoding 284 amino acids, with a predicted protein isoelectric point of 4.70 and a predicted molecular mass of 32.8 kDa. The highest expression was obtained after 4 h of induction with a final IPTG concentration of 1 mmol/L at 37 ℃. The recombinant tropomyosin mainly existed as a soluble form in the supernatant of cell lysate with a molecular mass of approximately 38 kDa.

Abstract [Objective] WRKY is one of the plant specific transcription factor families, involved in various plant life activities. However, currently there are few reports related to the WRKY70 gene in strawberry. Analyzing the role of WRKY70 homologous genes in strawberry in response to stress will help accelerate the application of molecular breeding technology and cultivate new strawberry germplasm resources. [Methods] The FaWRKY70 gene was cloned from ‘Benihoppe’ strawberry fruits using homologous cloning method. Its conserved domain, physicochemical properties, protein structure, and evolutionary relationship were analyzed using bioinformatics. Expression pattern analysis was performed using qRT-PCR. [Results] The FaWRKY70 gene had a length of 1 020 bp and encodes 339 amino acids. Homologous gene alignment revealed that FaWRKY70 had a high degree of amino acid sequence similarity with species in the same family such as apple and peony. The homologous genes were mostly related to plant responses to biotic and abiotic stress, suggesting that FaWRKY70 may be involved in resistance to stress. FaWRKY70 was expressed in different organs of strawberry, with significant differences. The expression level was highest in flowers and lowest in fruits. Under salicylic acid treatment, the FaWRKY70 gene was quickly responded, reaching its highest expression level after 3 hours, and gradually decreased thereafter. Under the treatment with methyl jasmonate, the FaWRKY70 gene showed a slight degree of induction and an overall downward trend. [Conclusion] FaWRKY70 is involved in growth and hormone signal transduction through different response modes in strawberry.

【Objective】 The DHNS (1,4-dihydroxy-2-naphthoyl-CoA synthase) gene in potatoes is a potentially important in the biosynthesis of glycoalkaloid metabolism in Solanaceae plants. Researching and verifying of the function of the potato DHNS gene can provide a source of genes and materials for the selection of low-glycoalkaloid potato varieties (lines) . 【Method】 The full-length sequence of ScDHNS cDNA was cloned from the wild potato species Solanum chacoense using the RACE technique. This sequence was analyzed for bioinformatics and subcellular localization, and its function was verified by constructing an overexpression vector pBWA (V) HS-DHNS to transform cultivated potato. 【Result】 The read open frame of ScDHNS cDNA sequence is 1 023 bp, encodes 340 amino acids, has a molecular weight of 37.34 kD and an isoelectric point of 8.592, and contains a typical ECH domain. It belongs to the enoyl hydratase/acetyl-CoA superfamily and is present in the genomes of plants such as Brachypodium distachyon and Medicago truncatula with homologous genes, showing gene expansion and contraction events. After the overexpression of the ScDHNS gene, it was found that the expressions of the transformed plants ScDHNS and SGT1 were significantly upregulated, and the expressions were notably higher than those in wild-type (WT) potato plants. Additionally, the total glycoalkaloid content in the corresponding transformed plants was significantly higher than that in WT potato plants, reaching a maximum of 364.3 mg/kg, which is 2.4 times that of the control. Subcellular localization results indicates that ScDHNS is localized in the peroxisome. 【Conclusion】 The ScDHNS gene in potato may regulate the expression of the key gene SGT1, which is involved in glycoalkaloid synthesis. It influences glycoalkaloid synthesis through the β-oxidation and mevalonate pathways. This gene is significantly associated with the subcellular compartmentalization of glycoalkaloids and has considerable application value in the cultivation of potato varieties with reduced glycoalkaloid content. [ABSTRACT FROM AUTHOR]

This study proposed a two-step process optimization for enhanced cumulative isoprene production. This involves establishing an isoprene biosynthetic pathway in Escherichia coli BL21 DE-3 via up-regulation of native deoxy xylulose 5-phosphate (DXP) synthase (Ec DXS), isopentenyl pyrophosphate isomerase (Ec IPPI /Ec IDI) and introducing isoprene synthase (Pm IspS) from kudzu (Pueraria montana), followed by the process conditions (incubation temperature, time, and inducer concentration) optimization using the Box-Behnken design (BBD) approach. BBD showed the maximum cumulative isoprene production (160.15 mg L-1), productivity (11.18 mg L-1 h-1), and yield (7.69 mg gdcw-1) at the optimized process conditions (incubation temperature 27.32 ºC, incubation time 14.25 h, and inducer concentration 0.453 mM). The transgenic E. coli isoprene production, -productivity, and -yield were 1.52-, 1.46-, and 1.24-fold higher than the unoptimized condition (incubation temperature 30 ºC, incubation time 16 h, and inducer concentration 0.10 mM). This work demonstrates that fine tuning of MEP pathway in addition with process conditions optimization is an efficient strategy for improving isoprene production from engineered E. coli. [Display omitted] • Isoprene, a high-value natural product, entirely being produced by petroleum route. • Recombinant E. coli has potential for sustainable production of isoprene. • DXS, IPPI, and IspS were overexpressed under IPTG inducible promoter. • RSM based statistical model was used for optimizing culture growth conditions. • Engineered E. coli produced 160.15 mg L-1 of isoprene at optimal values of variables. [ABSTRACT FROM AUTHOR]

Drought is a significant environmental limiting factor that restricts the growth of Quercus wutaishanica Mayr. The MYB transcription factor plays a wide role in controlling the growth of plants. In this study, the QwMYB108 gene was cloned and the bioinformatics was analyzed, and we examined how QwMYB108 responded to various gradient drought stresses. The results demonstrated that QwMYB108 encoded 275 amino acids using an 828 bp open reading frame. Subcellular localization indicated that the gene was located in the nucleus. Phylogenetic analysis showed that QwMYB108 was close to Q. robur, and that the highest level of expression was found in leaves, which was significantly different from other tissues. The expression of QwMYB108 increased as the stress degree rose when drought stress was present, and there was a significant difference between severe drought stress and other gradient stress. In this study, the function of QwMYB108 in drought stress response was investigated, and the drought response function gene of Q. wutaishanica was further explored to provide a theoretical basis. [ABSTRACT FROM AUTHOR]

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[Objectives] This study was conducted to understand the structure and function of MsrA protein. [Methods] With Vibrio alginolyticus HY9901 as the object of study, primers were designed to amplify the full-length gene of msrA, and its bioinformatics analysis was carried out. [Results] The full length of msrA gene was 639 bp, encoding 212 amino acids, and its theoretical molecular weight was about 23 729.60 Da. The protein had a stable structure, and it was hydropho-bic overall. The structure of signal peptides at the N terminal of the amino acid sequence was predicted, and it was found that there was no signal peptide cleavage site and no transmembrane region. The amino acid sequence of MsrA contained multiple signal binding sites. Protein subcellular localization showed that MsrA protein was most likely located in the cytoplasm. Homology analysis showed that MsrA of V. alginolyticus had high homology with other Vibrio species, and the highest homology with V. alginolyticus. In the prediction of functional domains, MsrA had the function of methionine sulfoxide reduction. In secondary structure prediction, MsrA contained random coils at a proportion of 46.70%, which was the highest. The similarity between the tertiary structure model of MsrA and template Q87SW6. 1. A was 89. 15%. PTM analysis showed that MsrA protein had many PTM modification sites such as phosphorylation and glycosylation sites. [Conclusions] This study provides some reference value for further study on the role of MsrA in bacterial antioxidant stress. [ABSTRACT FROM AUTHOR]

【Objective】The purpose of this study was to clone the FLS2 gene from wild eggplant species (Solanum sisymbriifolium Lam.) in Yunnan province, and to analyzed the physicochemical properties, subcellular localization and phylogenetic evolution of its encoded protein, as well as the expression, in order to preliminarily explore the biological functions of the wild eggplant FLS2 gene under the stress of verticillium wilt.【Method】In this study, the FLS2 gene, named SsFLS2, was cloned based on the transcriptome data (S. sisymbriifolium inoculation with verticillium pathogens) . The physical and chemical properties of SsFLS2 gene were analyzed by using bioinformatic analysis software, and real-time fluorescence quantitative PCR (RT-qPCR) was used to detect the expression of S. sisymbriifolium roots, stems and leaves, as well as the expression at different times after inoculation of verticillium pathogens.【Result】The results showed that SsFLS2 gene was 3655 bp in length, has a complete open reading frame (ORF), and encoded 1126 amino acids. The molecular formula of the encoded protein was C5596H8814N1494O1627S4, the theoretical molecular weight was 124.34 kD, the theoretical isoelectric point (pI) was 7.33, and the total mean hydrophilicity coefficient was 0.081. The secondary structure of SsFLS2 was mainly composed of α-helix (40.23%), random coil (42.81%), β-folding (3.73%), and extended chain (13.23%), and there was a transmembrane structure, localized on the cell membrane, in which the sites that can be phosphorylated and exceed the threshold line, a total of 151. The results of homologous sequence alignment and phylogenetic evolutionary analysis showed that the amino acid sequence of SsFLS2 protein was most closely related to the potato (Solanum tuberosum) homologous protein (XP 006358149.2) . RT-qPCR assay indicated that SsFLS2 gene was expressed in roots, stems, and leaves of S. sisymbriifolium, and that the relative expression level of SsFLS2 gene was highly significant higher in roots and leaves than in stems. The relative expression level of SsFLS2 gene was substantially and highly significantly higher at 24 h in both treatment and control groups than at other time points within 72 h after inoculation with verticillium pathogens.【Conclusion】In this study, SsFLS2 gene was successfully cloned from S. sisymbriifolium, and and its expression as well as the physicochemical properties of its encoded protein were analyzed. The results showed that SsFLS2 gene was closely correlated to the response of S. sisymbriifolium to verticillium wilt stress, which lays a theoretical foundation for further study on the role of SsFLS2 gene in verticillium wilt resistance in S. sisymbriifolium. [ABSTRACT FROM AUTHOR]

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Copyright of Acta Agriculturae Zhejiangensis is the property of Acta Agriculturae Zhejiangensis Editorial Office and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Copyright of Acta Agriculturae Zhejiangensis is the property of Acta Agriculturae Zhejiangensis Editorial Office and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Copyright of Acta Agriculturae Zhejiangensis is the property of Acta Agriculturae Zhejiangensis Editorial Office and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

[Objective] To explore the biosynthetic pathway of ergothioneine in Naematelia aurantialba and Stereum hirsutum. [Method] The ergothioneine synthase gene Egt1 and Egt2 of N. aurantialba and S. hirsutum were cloned by PCR amplification technology, respectively, and their functions were analyzed by bioinformatics software. High performance liquid chromatography（HPLC）was used to identify the intermediate products of hercynine, ergothioneine and their contents in two species. [Result] The complete DNA sequences of Egt1 and Egt2 genes of two species were successfully cloned. The analysis using bioinformatics software revealed that Egt1 of the two species contained functional binding domains such as EgtD and SAM-dependent methyltransferase. Egt2 contained the binding sites for pyridoxal phosphate （LPL）and cysteine desulfurase. Further analysis indicated that Egt1 and Egt2 shared similar functional domains and substrate binding sites with model fungi（Schizosaccharomyces pombe and Neurospora crassa）. This result showed that Egt1 and Egt2 may have similar gene functions as these model fungi. HPLC analysis revealed the presence of hercynine and ergothioneine in N. aurantialba blastospore（JEYB）, S. hirsutum fermentum broth（ShFJY）, S. hirsutum mycelium（ShJST）and N. aurantialba fruiting bodies（JEZST）. Additionally, the ergothioneine content in the JEZST was found to be the highest at 113.19 μg/g, which was 7.45 times, 26.14 times, and 27.74 times higher than that of the JEYB, ShFJY, and ShJST, respectively. [Conclusion] The Egt1 and Egt2 genes of N. aurantialba and S. hirsutum were identified for the first time. It is hypothesized that the biosynthetic pathway of N. aurantialba and S. hirsutum are involved the catalysis of histidine by the Egt1 enzyme, resulting in the formation of hercynine. Subsequently, the Egt1 enzyme catalyzes the conversion of hercynine into hercynylcysteine sulfoxide. Finally, the Egt2 enzyme catalyzes the transformation of hercynylcysteine sulfoxide into ergothioneine. [ABSTRACT FROM AUTHOR]

[Objective] Cytochrome P450 monooxygenase 98（CYP98）is a key rate-limiting enzyme in the phenylpropanoid pathway, and it is proposed to investigate whether it is involved in the biosynthesis of the phenylpropanoid pathway and in the anti-disease function in Hornworts, so as to provide a reference for the future study of the evolution of the early terrestrial plants and physiological mechanisms of adaptation to adversity stress. [Method] The full-length sequence of FfCYP98 cDNA was cloned from Folioceros fuciformis using RACE, analyzed for bioinformatics and subcellular localization, and functionally verified by constructing an overexpression vector to transform the Arabidopsis thaliana mutant FfCYP98-OE1. [Result] The FfCYP98 cDNA sequence, with an open reading frame of 1305 bp, is evolutionarily closest to the Bryophytes Anthoceros angustus and Physcomitrium patens CYP98, and subcellular localization results show that FfCYP98 is localized to both the nucleus and the cytoplasm. The overexpression of the FfCYP98 gene revealed that the expressions of total phenolics, total flavonoids, and C4H,4CL and C3H in the phenylpropanoid pathway was significantly higher in FfCYP98-OE1 plants than in A. thaliana WT plants. In addition, infestation of F. fuciformis and A. thaliana FfCYP98-OE1 plants with Botrytis cinerea revealed that the expression of FfCYP98 was significantly up-regulated, that FfCYP98-OE1 plants died significantly slower than WT plants, and that the expressions of total phenolics, total flavonoids, and four genes, C4H, HCT, C3H, and CAD in the phenylpropanoid pathway were significantly higher in A. thaliana FfCYP98-OE1 plants than in WT plants. [Conclusion] FfCYP98 may be involved in the biosynthesis of phenolics and flavonoids in the phenylpropanoid pathway by regulating the expressions of genes related to the synthesis of phenylpropanoid pathway, and may be related to disease resistance of plants. [ABSTRACT FROM AUTHOR]

[Objective] The heading date of rice（Oryza sativa）is an important agronomic trait and crucial for the regional adaptability and yields. Identification and functional analysis of heading date-associated genes may provide important gene resources for the breeding in rice. [Method] BSA-seq was performed to identify target gene of a yellowish and early heading mutant huangzao 1（hz1）. RT-qPCR was used to analyze the expression profile of target genes. The subcellular localization of HZ1 was determined by transient transformation between rice protoplasts and tobacco cells. Meanwhile, the heading dates, chlorophyll content and hydrogen peroxide content were measured to analyze the phenotype of hz1 mutant. [Result] Field phenotypic observations found that hz1 showed early heading phenotype. The heading date of hz1 mutant was the same under long-day（LD）and short-day（SD）conditions, which were 43 d and 26 d earlier than those of wild type（WT）respectively, indicating that hz1 was a photoperiod-insensitive mutant. Moreover, hz1 demonstrated a yellowish phenotype, with a decrease in chlorophyll content compared with WT. Genetic analysis revealed that hz1 was controlled by a recessive gene. BSA-seq（Bulk segregation analysis with whole-genome sequencing）of F2 pools indicated that the causal gene was linked with molecular markers on the chromosome 6 with 17.8 Mb region. Further analysis demonstrated that LOC_Os06g40080 site with a T-DNA insertion was completely linkage with hz1 mutant. LOC_Os06g40080 site was an identified SE5 gene encoding heme oxygenase 1（HO1）. Expression pattern analysis revealed that HZ1/SE5 was highly expressed in the leaves and dhad diurnal rhythm expression. Subcellular localization assay showed that HZ1/SE5 protein was localized in chloroplasts. Gene expression analysis showed that HZ1/SE5 regulated the expression of florigen genes Hd3a and RFT1 to modulate the heading date in rice. In addition, HZ1/SE5 can also regulate the expression of genes associated with chlorophyll synthesis pathway to modulate the change of chlorophyll levels. [Conclusion] hz1 mutant is a photoperiodic insensitive mutant due to the mutation of heme oxygenase gene SE5. HZ1/SE5 may regulate the expression of the florigen genes and chlorophyll synthesis-associated genes to influence the heading date and leaf yellowish in rice. [ABSTRACT FROM AUTHOR]

Various environmental stresses induce the production of reactive oxygen species (ROS), which have deleterious effects on plant cells. Glutathione (GSH) is an antioxidant used to counteract reactive oxygen species. Glutathione is produced by glutamylcysteine synthetase (GCS) and glutathione synthetase (GS). However, evidence for the GCS gene in sweetpotato remains scarce. In this study, the full-length cDNA sequence of IbGCS isolated from sweetpotato cultivar Xu18 was 1566 bp in length, which encodes 521 amino acids. The qRT-PCR analysis revealed a significantly higher expression of the IbGCS in sweetpotato flowers, and the gene was induced by salinity, abscisic acid (ABA), drought, extreme temperature and heavy metal stresses. The seed germination rate, root elongation and fresh weight were promoted in T3 Arabidopsis IbGCS-overexpressing lines (OEs) in contrast to wild type (WT) plants under mannitol and salt stresses. In addition, the soil drought and salt stress experiment results indicated that IbGCS overexpression in Arabidopsis reduced the malondialdehyde (MDA) content, enhanced the levels of GCS activity, GSH and AsA content, and antioxidant enzyme activity. In summary, overexpressing IbGCS in Arabidopsis showed improved salt and drought tolerance. Headings: •IbGCS gene was cloned from the sweetpotato cultivar Xu18. • The multiple abiotic stresses significantly increased the expression of IbGCS gene. • Overexpression of IbGCS significantly improved the salt and drought stress tolerance [ABSTRACT FROM AUTHOR]

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Copyright of Journal of Northwest A & F University - Natural Science Edition is the property of Editorial Department of Journal of Northwest A&F University (Natural Science Edition) and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Gene cloning is an important step in investigating gene structure and function. To verify gene sequence, Sanger sequencing is used, which may produce several overlapping sequencing files that need to be merged before alignment to the target gene sequence is performed. Previously, we reported the Python program to Merge Sanger sequences (https://peerj.com/articles/11354/), which ran in command line and relied heavily on EMBOSS suite. In this updated version of the program, we have made several remarkable improvements. It provides a graphical user interface (GUI) written with tkinter, which is convenient and stable. It does not require users to rename the input sequences before performing merging. With regard to the implementation, the updated version utilizes Python function (Align.PairwiseAligner) to align adjacent sequences, which is more flexible (can adjust program parameter i.e., the number of first-time consecutive matching bases). The new version of the program makes merging Sanger sequences much more convenient and facilitates gene study.

The Cytochrome P450 (CYP450) family is the largest enzyme protein family in plants, distributed across various organs and involved in significant catalytic activities in primary and secondary metabolic processes. In this study, we cloned the PtCYP721A57 gene, characterized its open reading frame (ORF), and conducted comprehensive analyses including physicochemical properties, evolutionary relationships, subcellular localization, prokaryotic expression, and correlation between the relative expression of different parts and the content of tenuifolin, hormones, and abiotic stress response associated with the encoded protein. The ORF of PtCYP721A57 was 1,521 bp, with a secondary structure predominantly composed of α-helices and random coils. Subcellular localization experiments confirmed the presence of PtCYP721A57 in the endoplasmic reticulum. For prokaryotic expression, we constructed the recombinant plasmid pET28a-PtCYP721A57 using pET28a as the vector, which was then transformed into BL21(DE3). Induction with Isopropyl β-D-1-thiogalactopyranoside (IPTG) at temperatures of 16 and 25 °C and varying concentrations (0.1, 0.2, 0.5, 1, 2 mM) resulted in the formation of inclusion bodies, with higher expression observed at 25 °C. Our qPCR analyses revealed that PtCYP721A57 exhibited the highest expression in the cortex of Polygala tenuifolia, followed by roots and xylem, correlating with the observed tenuifolin content distribution. Induction with abscisic acid (ABA) and chitosan (CHT) initially decreased PtCYP721A57 expression followed by a subsequent increase, peaking at 48 h. Similarly, drought stress induced a gradual increase in PtCYP721A57 expression, also peaking at 48 h. NaCl treatment for 6 h significantly upregulated PtCYP721A57 expression. In conclusion, our study provides foundational insights into the PtCYP721A57 gene in Polygala tenuifolia, laying the groundwork for further exploration of its role in the biosynthesis pathway of triterpenoid saponins.

【Objective】Trehalase (Tre) is a key enzyme in trehalose metabolism of Conopomorpha sinensis Bradley, which plays an important role in energy metabolism and growth development by specifically hydrolyzing trehalose into glucose. The study aims to clone two trehalase genes (CsTre1 and CsTre2) from Conopomorpha sinensis Bradley, to clarify its expression patterns in different developmental stages and tissues, and to analyze the molecular characteristics of the two genes and their enzyme proteins.【Method】Based on the transcriptome data of C. sinensis, the full-length cDNA sequences of CsTre1 and CsTre2 were cloned with the rapid amplification of cDNA ends (RACE)-PCR. Bioinformatics analysis was performed with software such as ORF Finder, ProtParam, SignalP 4.1, ProtScale, NetPhos2.0 Server and IQ TREE. The mRNA expression patterns of CsTre1 and CsTre2 in different developmental stages and tissues of C. sinensis were detected by using real-time quantitative PCR (RT-qPCR).【Result】The open reading frame of CsTre1 was 1 701 bp, encoding 566 amino acids, and the protein molecular weight was 64.53 kD. The open reading frame length of CsTre2 was 1 821 bp, encoding 606 amino acids, and the protein molecular weight was 69.08 kD. Signal peptide prediction analysis showed that both front-ends of CsTre1 and CsTre2 had a signal peptide, with positions of 1-16 and 1-17, respectively. The analysis on the secondary structure of the sequence showed that both CsTre1 and CsTre2 were mainly composed of α-helix and random coil, CsTre1 had 24 Sers, 15 Tyrs, and 10 Thrs that may serve as binding sites for protein kinases, while CsTre2 had 27 Sers, 10 Tyrs, and 13 Thrs that may serve as binding sites for protein kinases. The RT-qPCR results revealed that CsTre was expressed throughout all developmental stages of C. sinensis. In the expression pattern of adult stage, the expression level of CsTre1 was much higher than that of CsTre2, and the expression level of male adults of CsTre1 dropped sharply after the fourth day.【Conclusion】The study successfully cloned two trehalose genes of Conopomorpha sinensis Bradley. According to the results of analysis on their molecular characteristics and expression patterns, CsTre1 may be the main gene regulating trehalose metabolism in Conopomorpha sinensis Bradley. The research results provide important clues for elucidating the function of trehalase genes, which lay a solid foundation for the development pest control strategies.

Abstract [Objective] Tartaric acid not only affects the taste of grape berries but also determines the color, texture, microbial stability, and aging potential of the wine. It is of great value to explore the transcription factors and mechanisms that regulate tartaric acid biosynthesis, regulate the accumulation of tartaric acid in grapes through biotechnology or to cultivate wine grape varieties with high tartaric acid accumulation. [Methods] In this study, using ‘Traminer’ and ‘Marselan’ wine grapes as experimental materials, we cloned the tartaric acid related transcription factor VvbHLH79 by PCR. We performed bioinformatic and subcellular localization analyses of VvbHLH79. Furthermore, we measured the tartaric acid content and VvbHLH79 expression levels in grape berries at different developmental stages using HPLC and qRTPCR techniques. We also constructed a plant expression vector and the expression and tartaric acid content of VvbHLH79 were detected after transient transformation of young grape berries using Agrobacteriummediated fruiting pedicle infection, in order to clarify the role of the VvbHLH79 gene in regulating the content of tartaric acid in grape berries. [Results] (1) A grapevine VvbHLH79 gene was obtained by RNA-seq, with a CDS of 831 bp, amino acids of 277, molecular weight of 66.22 kD, and isoelectric point of 5.15. It belonged to the class of unstable hydrophilic proteins without signal peptides and transmembrane structures. Positions 147-232 of amino acid sequence contained the bHLH-SF conserved domain, which belonged to the bHLH transcription factor family. The protein was localized in the cell nucleus. (2) The grapevine VvbHLH79-encoded protein had the highest sequence similarity with the bHLH89-encoded protein in riverbank grape (accession number: XP_034699340.1), with a sequence identity of 99.64%. (3) There was a gradual decrease of VvbHLH79 expression during the development of ‘Traminer’ grape fruits, decreasing 67.25% from veraison to harvest. The accumulation of tartaric acids occurred mainly before color change of the grapes, with a rapid decrease after color change. (4) Transient expression of VvbHLH79 in low tartaric acid grapes resulted in a increase in tartaric acids, whereas silencing of VvbHLH79 in high tartaric acid grapes resulted in a decrease in tartaric acids, confirming that the VvbHLH79 gene positively regulated tartaric acid content. [Conclusion] The VvbHLH79 transcription factor may play a role in the tartaric acid biosynthesis in grape berries.