Function And Regulatory Mechanism Analysis Of Stress Adaptability Of 14-3-3 Protein Genes HaFT-1 And HaFT-9 In Haloxylon Ammodendron

Haloxylon ammodendron has strong stress tolerance and can adapt to harsh ecological environment such as drought,saline alkali and extreme high temperature.It is the largest afforestation species in desert area in China.Therefore,it is of great practical significance to study the stress adaptation mechanism of H.ammodendron for ecological management and economic construction in desert area.14-3-3 protein is a kind of molecular chaperone widely existing in eukaryotes.Previous studies have shown that the expression of 14-3-3 protein genes Ha FT-1 and Ha FT-9 is induced by various stresses in H.ammodendron.In this study,we conducted a comprehensive analysis from three aspects:cloning of Ha FT-1 and Ha FT-9promoters and their stress response analysis;function and regulate mechanism of Ha FT-1 gene in stress adaptability;and the function of Ha FT-9 gene in stress adaptability.The results are as follows:1.The upstream regulatory sequence of 5’end of Ha FT-1 and Ha FT-9 genes was amplified by chromosome walking technique,and 961 bp and 879 bp promoter sequences were obtained respectively.Sequence analysis showed that both promoters contained not only basic core elements of promoter,but also a variety of stress response elements.GUS recombinant vectors of Ha FT-1 and Ha FT-9 gene promoters were constructed and transiently transformed into tobacco,cotton and H.ammodendron seedlings.GUS staining results showed that the promoters of Ha FT-1 and Ha FT-9 genes were significantly responsive to heat,dehydration,Na Cl stress and ABA treatment;under heat stress,GUS expression regulated by the promoters of Ha FT-1 and Ha FT-9 genes was tissue-specific.QRT-PCR results showed that the expression trend of Ha FT-1 and Ha FT-9 genes in H.ammodendron seedlings under heat,dehydration,Na Cl stress and ABA treatment was basically consistent with the above GUS staining results.The recombinant vectors prp-haft-1::Gus and prp-haft-9::Gus containing promoter were transferred into wild-type（WT）Arabidopsis,and the T₁generation transgenic Arabidopsis lines were screened.2.In H.ammodendron seedlings,expression of Ha FT-1 and Ha FT-9 during secondary heat stress and subsequent normal temperature recovery was promoted by heat stress trainig,while methylation rate of Ha FT-1 promoter during secondary heat stress was reduced,indicating that Ha FT-1 and Ha FT-9 had the characteristics of heat stress memory gene expression.Subcellular localization showed that Ha FT-1 and Ha FT-9 proteins were located in the cytoplasm.3.The screened T₃generation of Arabidopsis homozygous lines overexpressing Ha FT-1 gene were used for phenotype analysis.Ha FT-1 lines seeds had higher heat tolerance and green seedling rate than WT Arabidopsis seeds,but the germination and green seedling rate of Ha FT-1 lines seeds did not show heat stress memory characteristics.After heat stress training,survival rate of Ha FT-1 seedlings was higher than that of WT seedlings,and the cell death rate was lower than that of WT seedlings.Transcriptome and q RT-PCR analysis showed that the expression patterns of genes related to protein quality control,autophagy,apoptosis inhibition,antioxidation and proline synthesis in Ha FT-1 seedlings were different from WT.After heat stress,proline,SOD activity and chlorophyll content of Ha FT-1 were higher than those of WT.These results showed that Ha FT-1 gene overexpression inhibited cell death under secondary heat stress,promoted the expression of related genes,increased the accumulation of proline and other substances,suggesting its important role in regulation of adaptation to heat stress.4.Seed germination and phenotype of Ha FT-1 lines were analyzed under salt stress.The results showed that the germination rate of Ha FT-1 lines was higher than that of WT.The proline content of Ha FT-1 lines was higher than that of WT.The Expression of BI-1,ATG18a,JMJ16 and P5C2 in Ha FT-1lines showed stress memory.These results indicated that Ha FT-1 played a positive role in regulation of salt stress adaptability.Under ABA treatment,the root length and rosette diameter of Ha FT-1 lines were significantly longer than those of WT seedlings,and the stomatal opening of detached leaves was larger than that of WT seedlings.These results suggest that Ha FT-1 overexpression makes Arabidopsis less sensitive to ABA.5.The plant expression vector of Ha FT-9 was constructed and transformed into WT Arabidopsis.The homozygous lines were obtained for phenotype analysis.The Germination rate of Ha FT-9 lines was lower than that of WT under heat stress,salt stress and drought stress.Survival rate of Ha FT-9 lines seedlings was higher than that of WT seedlings under heat stress.Root length and rosette diameter of Ha FT-9 lines seedlings were significantly longer than those of WT seedlings under salt stress.These results suggest that Ha FT-9 played a positive role in regulation of heat stress adaptability.