Interaction Relationship Of HSF Family Genes In Amorphophallus Albus

Amorphophallus is the only economic crop which can synthesize a large amount of glucomannan（KGM）,which has extremely high nutrition and economic value.Konjac likes warm and avoid high temperature,sustained high temperature in summer cause a sharp drop in konjac production and a serious decline in quality.At present,there are few reports on the regulation mechanism of konjac heat resistance.Therefore,it is particularly important to study the heat-resistant mechanism of konjac.In order to initially explore the mechanism of konjac heat regulation,this study selected Amorphophallus albus as the test material,which has high heat resistance and excellent quality,cloned the HSFB1 gene and promotes of HSFB1 and HSFA2a,and used the yeast two-hybrid system,pull-down technology and Dual-Glo^?dual luciferase detection system to detected the interactions between HSFB1,HSFA1,HSFA2a,HSP70,preliminary analysis of the function of HSFB1,HSFA1,HSFA2a,HSP70gene,laying the theoretical foundation for the in-depth study of the admissible matrix of the white konjac HSFB1,HSFA1,HSFA2a,HSP70 factor under high temperature stress.The main test results are as follows:1.Cloning and Bioinformatics Analysis of AaHSFB1 GeneWe used Amorphophallus albus as the experimental material,and amplify c DNA of Konjac leaf as a template,and isolated a B1 type HSF gene by homologous cloning and named AaHSFB1.The full-length c DNA is 1365 bp,and the open reading frame（ORF）is 939 bp,and encoded 312 amino acids.Bioinformatics analysis showed that the predicted protein had a molecular weight of 33.94 k Da and a theoretical isoelectric point of 8.48.It had the highest homology with Sorghum bicolor.2.Analysis of the relative expression pattern of AaHSFB1 geneAmorphophallus albus was treated at 41℃for different time,and the expression of AaHSFB1 gene in root,bulb,and leaf was analyzed by q RT-PCR technology.The results revealed that the expression of AaHSFB1 gene in the roots first increased and then decreased,and reached the peak value at 1 h of heat treatment,and the highest expression level in the leaves was reached at 12 h after heat treatment,indicating that AaHSFB1 is sensitive to heat stress.3.Subcellular localization analysis of AaHSFB1 proteinThe p CAMBIA1300-AaHSFB1-GFP fusion expression vector was constructed and transformed into tobacco leaf epidermal cells for transient expression analysis.The results showed that the AaHSFB1 protein was localized in the nucleus.4.Interaction analysis of AaHSFB1,AaHSFA1,AaHSFA2a,and AaHSP70 proteinsThe yeast two-hybrid system detected the interaction of AaHSFB1 with AaHSFA1,AaHSFA2a,and AaHSP70 proteins,and found that none of the yeast fusion strains could grow on DDO/A,QDO/X/A,and QDO media,that is,AaHSFB1 did not interact with AaHSFA1,AaHSFA2a,and AaHSP70 proteins.Used pull-down technology,it was found that none of the HIS-HSFA1,HIS-HSFA2a,and HIS-HSP70 proteins could capture the GST-HSFB1 protein,which was consistent with the yeast two-hybrid results.The yeast two-hybrid system detected the interactions between AaHSFA2a,AaHSFA1,and AaHSP70 proteins,and the interaction between AaHSFA1 and AaHSP70 protein.It was found that the yeast fusion strains both grew white colonies on DDO/A and QDO media and blue colonies on QDO/X/A media,indicating that AaHSFA2a interacts with AaHSFA1 and AaHSP70 proteins,and AaHSFA1 could interact with AaHSP70 protein.The pull-down test found that both HIS-HSFA1 protein could capture GST-HSFA2a protein,and HIS-HSFA2a protein could capture GST-HSP70 protein,and HIS-HSFA1protein could capture GST-HSP70 protein,which is consistent with the results of yeast two-hybrid.5.Cloning and Activity Analysis of AaHSFB1 and AaHSFA2a PromotersBased on the DNA sequences of AaHSFB1 and AaHSFA2a,three specific primers were designed,respectively,and the FPNI-PCR method was used to obtain the promoter of AaHSFB1 and the promoter of AaHSFA2a were 1509bp and 1320bp,respectively,named pr AaHSFB1 and pr AaHSFA2a.It is predicted that in addition to the conservative TATA box and CAAT box,these two promoters also have typical HSE elements,multiple light-responsive elements,multiple hormone-responsive elements,and enhancer-like elements in specific hypoxia responses and other stress-response elements,indicating that they may be regulated by HSFs in the regulation of heat shock.The p BI121-pr AaHSFB1 and p BI121-pr AaHSFA2a vectors were constructed,and positive transgenic Arabidopsis plants were obtained by Agrobacterium-mediated method,and promoter activity analysis was performed by GUS staining method,the results showed that both pr AaHSFB1 and pr AaHSFA2a were high temperature inducible promoters.6.Analysis of pr AaHSFB1,pr AaHSFA2a interaction with AaHSFA1,AaHSP70 proteinsUsed double luciferase technology,p Green II0800-LUC-Aapr HSFB1Agrobacterium liquid was mixed with p Green II62-SK-AaHSFA1,p Green II62-SK-AaHSFA2a,p Green II62-SK-AaHSP70,respectively,p Green II0800-LUC-Aapr HSFA2a Agrobacterium liquid was mixed with p Green II62-SK-AaHSFA1,p Green II62-SK-AaHSFB1,p Green II62-SK-AaHSP70,respectively,the test results showed that only the ratio of firefly luciferase to Renilla luciferase of the mixed strains of p Green II0800-LUC-Aapr HSFA2a and p Green II62-SK-AaHSFA1 was significantly different from the negative control.The ratio of firefly luciferase to Renilla luciferase of other fusion strains was not significantly different from that of the negative control,indicating that pr AaHSFA2a only interacts with the AaHSFA1 protein,while pr AaHSFB1 did not interaction with AaHSFA1,AaHSFA2a and AaHSP70 proteins.