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Maize is one of the most vital staple crops worldwide. G proteins modulate plentiful signaling pathways, and G protein-coupled receptor-type G proteins (GPCRs) are highly conserved membrane proteins in plants. However, researches on maize G proteins and GPCRs are scarce. In this study, we identified three novel GPCR-Type G Protein (GTG) genes from chromosome 10 (Chr 10) in maize, designated as ZmCOLD1-10A, ZmCOLD1-10B and ZmCOLD1-10C. Their amino acid sequences had high similarity to TaCOLD1 from wheat and OsCOLD1 from rice. They contained the basic characteristics of GTG/COLD1 proteins, including GPCR-like topology, the conserved hydrophilic loop (HL) domain, DUF3735 (domain of unknown function 3735) domain, GTPase-activating domain, and ATP/GTP-binding domain. Subcellular localization analyses of ZmCOLD1 proteins suggested that ZmCOLD1 proteins localized on plasma membrane (PM) and endoplasmic reticulum (ER). Furthermore, amino acid sequence alignment verified the conservation of the key 187th amino acid T in maize and other wild maize-relative species. Evolutionary relationship among plants GTG/COLD1 proteins family displayed strong group-specificity. Expression analysis indicated that ZmCOLD1-10A was cold-induced and inhibited by light. Together, these results suggested that ZmCOLD1 genes had potential value to improve cold tolerance and to contribute crops growth and molecular breeding.

Maize is one of the most vital staple crops worldwide. G proteins modulate plentiful signaling pathways, and G protein-coupled receptor-type G proteins (GPCRs) are highly conserved membrane proteins in plants. However, researches on maize G proteins and GPCRs are scarce. In this study, we identified three novel GPCR-Type G Protein (GTG) genes from chromosome 10 (Chr 10) in maize, designated as ZmCOLD1-10A, ZmCOLD1-10B and ZmCOLD1-10C. Their amino acid sequences had high similarity to TaCOLD1 from wheat and OsCOLD1 from rice. They contained the basic characteristics of GTG/COLD1 proteins, including GPCR-like topology, the conserved hydrophilic loop (HL) domain, DUF3735 (domain of unknown function 3735) domain, GTPase-activating domain, and ATP/GTP-binding domain. Subcellular localization analyses of ZmCOLD1 proteins suggested that ZmCOLD1 proteins localized on plasma membrane (PM) and endoplasmic reticulum (ER). Furthermore, amino acid sequence alignment verified the conservation of the key 187th amino acid T in maize and other wild maize-relative species. Evolutionary relationship among plants GTG/COLD1 proteins family displayed strong group-specificity. Expression analysis indicated that ZmCOLD1-10A was cold-induced and inhibited by light. Together, these results suggested that ZmCOLD1 genes had potential value to improve cold tolerance and to contribute crops growth and molecular breeding.

Background More than a dozen of fungal immunomodulatory proteins (FIPs) have been identified to date, most of which are from Ganoderma species. However, little is known about the similarities and differences between different Ganoderma FIPs’ bioactivities. In the current study, two FIP genes termed FIP-gap1 and FIP-gap2 from G. applanatum, along with LZ-8 and FIP-gsi, another two representative Ganoderma FIP genes from G. lucidum and G. sinense were functionally expressed in Pichia. Subsequently, bioactivities of four recombinant Ganoderma FIPs were demonstrated and compared. Results All the four Ganoderma FIP genes could be effectively expressed in P. pastoris GS115 at expression levels ranging from 197.5 to 264.3 mg L− 1 and simply purified by one step chromatography using HisTrap™ FF prepack columns. Amino acid sequence analysis showed that they all possessed the FIP conserved fragments. The homologies of different Ganoderma FIPs were from 72.6 to 86.4%. In vitro haemagglutination exhibited that FIP-gap1, FIP-gsi and LZ-8 could agglutinate human, sheep and mouse red blood cells but FIP-gap2 agglutinated none. Besides, the immunomodulation activities of these Ganoderma FIPs were as: rFIP-gap2 > rFIP-gap1 > rLZ-8 and rFIP-gsi in terms of proliferation stimulation and cytokine induction on murine splenocytes. Additionally, the cytotoxic activity of different FIPs was: rFIP-gap1 > rLZ-8 > rFIP-gsi > rFIP-gap2, examined by their inhibition of three human carcinomas A549, Hela and MCF-7. Conclusions Taken together, four typical Ganoderma FIP genes could be functionally expressed in P. pastoris, which might supply as feasible efficient resources for further study and application. Both similarities and differences were indeed observed between Ganoderma FIPs in their amino acid sequences and bioactivities. Comprehensively, rFIP-gaps from G. applanatum proved to be more effective in immunomodulation and cytotoxic assays in vitro than rLZ-8 (G. lucidum) and rFIP-gsi (G. sinense). Electronic supplementary material The online version of this article (10.1186/s12896-018-0488-0) contains supplementary material, which is available to authorized users.

Fungal immunomodulatory proteins (FIPs) have been identified from a series of fungi, especially in Ganoderma species. However, little is known about the FIPs from G. applanatum. In this study, two novel FIP genes, termed as FIP-gap1 and FIP-gap2, were cloned from G. applanatum, characterized and functionally expressed after codon optimization in Pichia pastoris GS115. Results showed that FIP-gap1 and FIP-gap2 comprised 342-bp encoding peptides of 113 amino acids, which shared a high homology with other Ganoderma FIPs. The yield of recombinant FIP-gap1 and FIP-gap2 increased significantly after codon optimization and reached 247.4 and 197.5 mg/L, respectively. Bioactivity assay in vitro revealed that both rFIP-gap1 and rFIP-gap2 could agglutinate mouse, sheep, and human red blood cells. Besides, rFIP-gap1 and rFIP-gap2 obviously stimulated the proliferation of mouse splenocytes and enhanced IL-2 and IFN-γ release. Cytotoxicity detection indicated that IC50 of rFIP-gap1 towards A549 and HeLa cancer cells were 29.89 and 8.34 μg/mL, respectively, whereas IC50 of rFIP-gap2 to the same cancer cells were 60.92 and 41.05 μg/mL, respectively. Taken together, novel FIP gaps were cloned and functionally expressed in P. pastoris, which can serve as feasible and stable resources of rFIP gaps for further studies and potential applications.

Haemagglutination examination of four recombinant Ganoderma FIPs including rFIP-gap1, rFIP-gap2, rLZ-8 and rFIP-gsi (all final concentration at 5 μg mL− 1) towards human (hRBCs), sheep (sRBCs) and mouse (mRBCs) red blood cells, respectively. PBS and PHA (5 μg mL− 1) served as negative and positive controls. All results were from biological duplicate tests. (DOCX 1042 kb)

Homology between different Ganoderma FIPs based on their peptide sequence alignment using a DNAman software (Version 8.0). FIP-gap1 and FIP-gap2, FIP-gat, FIP-gbo, FIP-gja, FIP-gmi, FIP-gsi, FIP-gts, LZ-8 and LZ-9 represented FIPs from G. applanatum, G. atrum, G. boninense, G. japonicum, G. microsporum, G. sinense, G. tsugae and G. lucidum, respectively. (DOCX 411 kb)

Schematic map of synthetic Ganoderma FIP genes. Four codon-optimized FIP genes were synthesized (in green) by Sango (Shanghai, China), in which His-tag sequences (in yellow) were also inserted before stop codons. (DOCX 74 kb)

Codon adaptation indexes (CAI) for Ganoderma FIP genes in P. pastoris. FIP-gap1, FIP-gap2, LZ-8, and FIP-gsi represent FIP genes from G. applanatum, G. lucidum and G. sinense. (DOCX 13 kb)

Based on the galactinol synthase (AnGolS1) fragment sequence from a cold-induced Suppression Subtractive Hybridization (SSH) library derived from Ammopiptanthus nanus (A. nanus) seedlings, AnGolS1 mRNA (including the 5′ UTR and 3′ UTR) (GenBank accession number: GU942748) was isolated and characterized by rapid amplification of cDNA ends polymerase chain reaction (RACE–PCR). A substrate reaction test revealed that AnGolS1 possessed galactinol synthase activity in vitro and could potentially be an early-responsive gene. Furthermore, quantitative real-time PCR (qRT-PCR) indicated that AnGolS1 was responded to cold, salts and drought stresses, however, significantly up-regulated in all origans by low temperatures, especially in plant stems. In addition, the hybridization signals in the fascicular cambium were strongest in all cells under low temperature. Thus, we propose that AnGolS1 plays critical roles in A. nanus low-temperature stress resistance and that fascicular cambium cells could be involved in AnGolS1 mRNA transcription, galactinol transportation and coordination under low-temperature stress.