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Additional file 6. The vector maps used in this paper.

Additional file 5 The primers of the promoters of AtHsps used in yeast one hybrid.

Additional file 3. Original, unprocessed versions of the blots in Figs. 6 and 7.

Additional file 4 The primers for Arabidopsis Hsp genes related to thermotolerance in qRT-PCR.

Heat shock transcription factors (Hsfs) play an important role in regulating heat stress response in plants. Our previous study found that there were 82 non-redundant Hsfs in wheat, 18 of which belonged to subclass A2. In this study, we cloned an A2 member, TaHsfA2-1, which encoded a protein of 346 amino acid residues in wheat. The fusion protein TaHsfA2-1-GFP was localized in the nucleus under normal growth conditions. TaHsfA2-1 was expressed in nearly all the measured tissues, most highly in mature leaves. The expression level of TaHsfA2-1 can be enhanced by heat stress, PEG stress, and signal molecules such as H2O2 and SA. Yeast cells transformed with TaHsfA2-1 improved thermotolerance compared to those with the empty vector. TaHsfA2-1-overexpressing Arabidopsis displayed a better growth state with more green leaves than wild-type seedlings after heat stress. Accordingly, the chlorophyll content and survival rate in the transgenic lines were higher than in the wild type, and relative conductivity in the transgenic lines was lower than in the wild type. Further research found that TaHsfA2-1-overexpressing Arabidopsis up-regulated the expression of some heat shock protein genes (Hsps) compared to wild type after heat stress. These results suggested that TaHsfA2-1 is a new gene that improves thermotolerance in plants by mediating the expression of Hsps. A functional gene was provided for molecular breeding in the subsequent research.

High temperature directly affects the yield and quality of crops. Plant Hsfs play vital roles in plant response to heat shock. In the present study, ZmHsf05 was isolated from maize (Zea mays L.) using homologous cloning methods. The sequencing analysis demonstrated that CDS of ZmHsf05 was 1080 bp length and encoded a protein containing 359 amino acids. The putative amino acid sequence of ZmHsf05 contained typical Hsf domains, such as DBD, OD, NLS and AHA motif. Subcellular localization assays displayed that the ZmHsf05 is localized to the nucleus. ZmHsf05 was expressed in many maize tissues and its expression level was increased by heat stress treatment. ZmHsf05 rescued the reduced thermotolerance of the athsfa2 mutant in Arabidopsis seedlings. Arabidopsis seedlings of ZmHsf05-overexpressing increased both the basal and acquired thermotolerances. After heat stress, the ZmHsf05-overexpressing lines showed enhanced survival rate and chlorophyll content compared with WT seedlings. The expression of Hsps was up-regulated in the ZmHsf05-overexpressing Arabidopsis lines after heat stress treatment. These results suggested that ZmHsf05 plays an important role in both basal and acquired thermotolerance in plants.

Antagonists and sonication treatment relieved the structural barriers of Agrobacterium entering into cells; hindered signal perception and transmission; alleviated defense responses and increased cell susceptibility to Agrobacterium infection. Soybean gene expression analysis was performed to elucidate the general response of soybean plant to Agrobacterium at an early stage of infection. Agrobacterium infection stimulated the PAMPs-triggered immunity (BRI1, BAK1, BZR1, FLS2 and EFR) and effector-triggered immunity (RPM1, RPS2, RPS5, RIN4, and PBS1); up-regulated the transcript factors (WRKY25, WRKY29, MEKK1P, MKK4/5P and MYC2) in MAPK pathway; strengthened the biosynthesis of flavonoid and isoflavonoid in the second metabolism; finally led to a fierce defense response of soybean to Agrobacterium infection and thereby lower transformation efficiency. To overcome it, antagonist α-aminooxyacetic acid (AOA) and sonication treatment along with Agrobacterium infection were applied. This novel method dramatically decreased the expression of genes coding for F3'H, HCT, β-glucosidase and IF7GT, etc., which are important for isoflavone biosynthesis or the interconversion of aglycones and glycon; genes coding for peroxidase, FLS2, PBS1 and transcription factor MYC2, etc., which are important components in plant-pathogen interaction; and genes coding for GPAT and α-L-fucosidase, which are important in polyesters formation in cell membrane and the degradation of fucose-containing glycoproteins and glycolipids on the external surface of cell membrane, respectively. This analysis implied that AOA and sonication treatment not only relieved the structural membrane barriers of Agrobacterium entering into cells, but also hindered the perception of 'invasion' signal on cell membrane and intercellular signal transmission, thus effectively alleviated the defense responses and increased the cell susceptibility to Agrobacterium infection. All these factors benefit the transformation process; other measures should also be further explored to improve soybean transformation.

Based on the information of 25 heat shock transcription factor (Hsf) homologues in maize according to a genome-wide analysis, ZmHsf06 was cloned from maize leaves and transformed into Arabidopsis thaliana (L. Heynh.) (ecotype, Col-0). Three transgenic positive lines were selected to assess the basic and acquired thermotolerance and drought-stress tolerance under stresses and for some physiological assays. The sequence analysis indicates that ZmHsf06 contained the characteristic domains of class A type plant Hsfs. The results of qRT–PCR showed that the expression levels of ZmHsf06 were elevated by heat shock and drought stress to different extents in three transgenic lines. Phenotypic observation shows that compared with the Wt (wild-type) controls, the overexpressing ZmHsf06 of Arabidopsis plants have enhanced basal and acquired thermotolerance, stronger drought-stress tolerance and growth advantages under mild heat stress conditions. These results are further confirmed by physiological and biochemical evidence that transgenic Arabidopsis plants exhibit higher seed germination rate, longer axial-root length, higher activities of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT), higher leaf chlorophyll content, but lower relative electrical conductivity (REC), malondialdehyde (MDA) and osmotic potential (OP) than the Wt controls after heat shock and drought treatments. ZmHsf06 may be a central representative of maize Hsfs and could be useful in molecular breeding of maize or other crops for enhanced tolerances, particularly during terminal heat and drought stresses.

Human cytomegalovirus (HCMV) infection is an ubiquitous herpesvirus disease in human populations. It is rarely pathogenic to healthy adults, yet it may cause severe outcome to organ transplant recipients, the immunocompromised individuals and pregnant women. Using DNA from HCMV AD169 strain as template, the UL32 gene encoding pp150 protein fragment and the UL57 gene encoding MDBP protein fragment were amplified by PCR technique. After the construction of cloning vector pMD18-T-UL32, pMD18-T-UL57, pMD18-T-UL32/UL57 and expression vector pET-11a-UL32/UL57, the recombinant fusion proteins pp150/MDBP were induced with IPTG in BL21 host strain. The results showed that the relative molecular weight of recombinant fusion proteins pp150/MDBP is about 27 kD, the product of fusion proteins takes 17.45% in the total proteins in host bacteria, the analytical result was positive to the fusion proteins pp150/MDBP via Western blot technique, while the purified recombinant fusion proteins have strong antigenicity detected by ELISA and protein chip compared with whole virus antigens from HCMV. It was demonstrated that when used for the detection of serum from the clinical patients it has the same detection rate compared with the whole virus antigen. It needs further research for application.

To identify tumor associated genes of rectal cancer and to probe the application possibility of gene expression profiles for the classification of tumors.Rectal cancer tissues and their paired normal mucosa were obtained from patients undergoing surgical resection of rectal cancer. Total RNA was extracted using Trizol reagents. First strand cDNA synthesis was indirectly labeled with aminoallyl-dUTP and coupled with Cy3 or Cy5 dye NHS mono-functional ester. After normalization to total spots, the genes which background subtracted intensity did not exceed 2 SD above the mean blank were excluded. The data were then sorted to obtain genes differentially expressed byor= 2 fold up or down in at least 5 of the 21 patients.In the 21 rectal cancer patients, 23 genes were up-regulated in at least 5 samples and 15 genes were down-regulated in at least 5 patients. Hierachical cluster analysis classified the patients into two groups according to the clinicopathological stage, with one group being all above stage II and one group all below stage II.The up-regulated genes and down-regulated genes may be molecular markers of rectal cancer. The expression profiles can be used for classification of rectal cancer.