Characteristics,Function Under High Temperature Stress Of AaHSFC2a Gene And The Study Of Interaction Protein Of Amorphophallus Albus

Amorphophallus is the only cash crop so far that can provide large quantities of konjac glucomannan.As a soluble dietary fiber,glucomannan has been widely used in food,cosmetics,medicine,chemical industry and other fields.However,Amorphophallus likes to be warm and humid,avoiding high temperature environment.The continuous high temperature in summer will directly affect the growth and yield of Amorphophallus,resulting in a decline in Amorphophallus yield and poor quality.Therefore,the thermal mechanism of Amorphophallus is studied,clarify its regulation network,will lay a theoretical basis for the breeding of Amorphophallus heat-resistant varieties.In this study,Amorphophallus albus with stronger heat resistance and the best quality was used as the test material.A C-type HSF was amplified from the Amorphophallus albus c DNA using homologous cloning and 3’RACE technology,and it was named AaHSFC2 a after NCBI-BLAST comparison analysis.By constructing transient expression vector p CAMBIA1300-AaHSFC2a-GFP and transferring into tobacco leaf epidermal cells,the localization of AaHSFC2 a protein was verified.Meanwhile,the promoters of AaHSFC2 a and AaHSP70 were obtained by FPNI-PCR,and the transcriptional activities of the promoters were analyzed by GUS staining.The interaction between HSFC2 a,HSP90 and HSFA1,HSFA2 c,HSFB1,and HSP70 were detected by the yeast two-hybrid system,yeast one-hybrid system and dual luciferase system.These laid a theoretical foundation for in-depth study of the heat-resistant regulatory network of Amorphophallus albus.The main research results are as follows:1.Cloning and bioinformatics analysis of AaHSFC2 a geneThe open reading frame(ORF)of AaHSFC2 a is 1056 bp,encoding a total of 351 amino acids,and has a typical DBD domain,OD domain and NLS domain.Using g DNA as a template,a 1160 bp sequence was obtained.Comparative analysis revealed that the AaHSFC2 a gene contains one intron and two exons,and the intron region ranges from 281 bp to 384 bp,with a total length of 104 bp.2.Analysis of the relative expression pattern of AaHSFC2 a gene in different tissues of Amorphophallus albusThe results indicated that the AaHSFC2 a gene was expressed in all three tissues of the Amorphophallus albus,and the lowest expression level was in the bulbs.From the expression point of view,The expression of the AaHSFC2 a gene peaked in leaves and roots at 24 h and 0.5 h after heat treatment,respectively,indicating that AaHSFC2 a gene had a response under high temperature stress.3.Localization analysis of AaHSFC2 a protein in tobacco epidermal cellsthe empty p CAMBIA1300-GFP was used as a control,and it was found that the AaHSFC2 a protein was localized in the cell nucleus and the cell cytoplasm.4.Effect of overexpression of AaHSFC2 a gene on heat tolerance of ArabidopsisThe results of heat shock treatment found that both wild and transgenic Arabidopsis seedlings could grow normally without high temperature treatment and46 ℃ treatment for 1 h,but died after treated at 46 ℃ for 5 h.When the temperature was adjusted to 41 ℃ for 12 h,wild-type Arabidopsis did not survive,while transgenic Arabidopsis seedlings had a certain survival rate,indicating that the AaHSFC2 a gene can enhance the heat tolerance of Arabidopsis.5.Interaction analysis between AaHSFC2 a,AaHSP90 and AaHSFA1,AaHSFA2 c,AaHSFB1,AaHSP70 proteinsThe yeast two-hybrid system detected the interaction bewteen AaHSFC2 a and AaHSFA1,AaHSFA2 c,AaHSFB1 and AaHSP70 proteins,and found that none of the yeast fusion strains could grow on DDO/A,QDO/X/A,and QDO media,indicating that,AaHSFC2 a did not interact with AaHSFA1,AaHSFA2 c,AaHSFB1 and AaHSP70 proteins.However,The yeast fusion strain AaHSFC2 a and AaHSP90 grows white colonies on DDO/A and QDO medium,and blue colonies on QDO/X/A medium,It shows that AaHSFC2 a and AaHSP90 protein interact in yeast cells.Similarly,AaHSP90 interacts with AaHSFA1,AaHSFA2 c,and AaHSFB1 proteins in yeast cells,while AaHSP90 and AaHSP70 proteins do not interact in yeast cells.6.Cloning and activity analysis of AaHSFC2 a and AaHSP70 promotersA total of 1073 bp of AaHSFC2 a promoter and 1062 bp of AaHSP70 promoter were obtained by FPNI-PCR method,named pr AaHSFC2 a and pr AaHSP70 respectively.Plant CARE analysis found that there are multiple transcription factor binding sites on the two promoters,but the HSE element only exists on the AaHSP70 promoter,not found on AaHSFC2 a.At the same time,GUS staining test showed that the AaHSP70 promoter was heat inducible.7.Interaction analysis of pr AaHSP70 and AaHSFA1,AaHSFA2 c,AaHSFB1,AaHSFC2 a,AaHSP90Using yeast one-hybrid technique,The co-transformed strains of pr AaHSP70 and AaHSFA1,AaHSFA2 c are blue on the chromogenic medium,while the co-transformed strains of pr AaHSP70 and AaHSFB1,AaHSFC2 a,and AaHSP90 are not blue on the chromogenic medium.The results showed that the HSE element of the pr AaHSP70 promoter interacted with AaHSFA1,AaHSFA2 c,but did not interact with AaHSFB1,AaHSFC2 a,AaHSP90.Further verify the result with the dual luciferase system.