Functional Study Of Heat Shock Transcription Factor And Heat Shock Protein In Response To High Temperature Stress On CMS-Based F₁ In Soybean（Glycine Max（L.） Merr.）

Compared with conventional soybean,pollen development of CMS-based F₁ in soybean is more sensitive to high-temperature（HT）.Heat shock transcription factor（HSF）and heat shock protein（HSP）are very important factors for plants to respond to high temperature,which are important regulatory factors to resist HT stress.Previous studies in our laboratory found that heat shock transcription factors GmHSFA2,GmHSFB2 and heat shock proteins GmHSP90a,GmHSP20a,Gms HSPa were induced by HT in the flower buds of HT-tolerant CMS-based F₁（NF₁）in soybean,while inhibited by HT in the flower buds of HT-sensitive CMS-based F₁（YF₁）in soybean,but the molecular mechanism of their response to HT stress was not clear.Therefore,in this study,heat shock transcription factors GmHSFA2,GmHSFB2 and heat shock proteins GmHSP90a,GmHSP20a,Gms HSPa were selected as the research objects,and their roles in the regulatory network of plant response to HT stress were studied by means of bioinformatics analysis,gene cloning,yeast one hybrid,genetic transformation of overexpressed Arabidopsis and soybean.The main results are as follows:1.Bioinformatics analysis showed that GmHSFB2 had the typical functional domain of plant HSF family and had a close relationship with soybean HSFB-2b protein.Functional studies showed that the survival rate of transgenic Arabidopsis plants of overexpressing GmHSFB2 gene was significantly higher than that of wild type（WT）Arabidopsis seedlings after HT stress at seedling stage.After HT stress at flowering stage,anther dehiscence of transgenic Arabidopsis of overexpressing GmHSFB2 gene was normal,while WT had anther indehiscence;The fertile pollen rate of transgenic Arabidopsis of overexpressing GmHSFB2 gene was significantly higher than that of WT.Reactive oxygen staining showed that the DAB and NBT staining of transgenic Arabidopsis leaves and inflorescences of overexpressing GmHSFB2 gene were lighter than that of WT after HT treatment for 2 hours and the recovery under Normal-temperature（NT）for 2 hours.In conclusion,GmHSFB2 gene may play a positive role in the regulatory network of Arabidopsis thaliana in response to HT stress.2.Bioinformatics analysis showed that GmHSP90a and Gms HSPa had high homology with Gs HSP83 and Gss HSP protein sequences of Glycine soja,respectively.There were typical homeopathic elements such as CAAT-box in the promoter region of GmHSP90a.The functional study of transgenic Arabidopsis showed that the survival rate of transgenic Arabidopsis seedlings of overexpressing GmHSFB2 and Gms HSPa genes were significantly higher than that of WT after HT stress at seedling stage.After HT stress at flowering stage,anther dehiscence of transgenic Arabidopsis of overexpressing GmHSP90a and Gms HSPa gene was normal,while WT had anther indehiscence;The fertile pollen rate of transgenic Arabidopsis of overexpressing GmHSP90a and Gms HSPa gene was significantly higher than that of WT.Reactive oxygen staining showed that the DAB and NBT staining of transgenic Arabidopsis leaves and inflorescences of overexpressing GmHSP90a and Gms HSPa genes were lighter than that of WT after HT treatment for 2 hours and the recovery under NT for 2hours.The functional study of transgenic soybean showed that before HT treatment,fertile pollen of transgenic soybeans of overexpressing GmHSP90a and Gms HSPa genes and Williams82 soybean were basically fertile;after HT treatment,the fertile pollen rate of transgenic soybeans of overexpressing GmHSP90a and Gms HSPa genes and Williams82 soybean gradually decreased with the increase of HT treatment time,but the fertile pollen rate of transgenic soybeans was higher than that of Williams82soybean,and the fertile pollen rate of transgenic soybean of overexpressing GmHSP90a gene was higher than that of transgenic soybean of overexpressing Gms HSPa gene.These results suggest that GmHSP90a and Gms HSPa genes may respond to HT stress in Arabidopsis and soybean,thereby improving their heat tolerance.3.The heat tolerance experiment of transgenic Arabidopsis of overexpressing GmHSFA2 and GmHSP20a genes was carried out at flowering stage,the results showed that anther dehiscence of transgenic Arabidopsis of overexpressing GmHSFA2 and GmHSP20a gene was normal,while WT had anther indehiscence;The fertile pollen rate of transgenic Arabidopsis of overexpressing GmHSFA2 and GmHSP20a gene was significantly higher than that of WT after HT stress,the transgenic Arabidopsis of overexpressing GmHSFA2 and GmHSP20a genes had anther dehiscence and most pollen was fertile,while WT had anther indehiscence and only a few pollen was fertile,which was consistent with the results of heat tolerance test of transgenic Arabidopsis of overexpressing GmHSFB2,GmHSP90a and Gms HSPa genes.The results of yeast one hybrid showed that there was interaction between GmHSFA2 gene and promoter of GmHSP20a and GmHSP90a.These results suggest that GmHSFA2 can respond to HT stress and regulate the expression of GmHSP20a and GmHSP90a,thus improving the heat tolerance of soybean during the reproductive development.