Cloning And Functional Analysis Of Stress Related Genes TaDjA1 And TaSAP7-A From Wheat(Triticum Aestivum L.)

Wheat(Triticum aestivum L.)is one of the most important food crops in our country.However,the global climate change and adverse stresses,such as drought,salinity and extreme temperature,seriously restrict the wheat production.Excavation and utilization of excellent stress-resistant gene resources is the basis of improvement stress tolerance for wheat.The Dna J proteins are ubiquitous molecular chaperones in the plant cell.Dna J proteins function alone or in association with Hsp70 in protein folding,refolding and protein complex depolymerization.It is very important that Dna J proteins maintain stabilization of protein and protein complex under stress conditions.SAPs are the A20/AN1 zinc-finger proteins which involve in abiotic stress tolerance and development in plants.In this study,the cDNA library responsed to drought and heat stresses from wheat was used to screen TaDjA1 gene.Based on sequence polymorphism of TaDjA1 gene,functional markers were developed.The elite alleles in TaDjA1 gene were identified through association analysis.Meanwhile,the functions of TaDjA1-B were also studied by using gene expression pattern analysis,subcellular localization,transgenic Arabidopsis and interacting protein analysis.In addition,the phenotype of transgenic Arabidopsis was identified to clarify the function of TaSAP7-A in stress condition.The main results are summarized as follows:1.The reference sequences of TaDjA1 from A,B and D subgenomes were screened out by sequence alignment using cDNA sequence in wheat genome database.Three alleles,TaDjA1-A,TaDjA1-B and TaDjA1-D were isolated by specific primers designed from wheat A,B and D subgenomes.All three alleles have seven exons and six introns.The length of cDNA sequences is 1266 bp and the similarity is 99.5%.The length of amino acids sequences is 421 and the similarity is 100%.Using Chinese Spring nullisomic-tetrasomic lines,TaDjA1-A,TaDjA1-B and TaDjA1-D were located on chromosome homologous group 6.2.TaDjA1 includes J domain,G/F domain,zinc finger domain and uncharacterized C terminal domain.TaDjA1 belongs to A type Dna J protein and appears to be conserved in the J domain.The typical HPD module was detected in the J domain.3.One SNP(C/T)was detected at the third intron in the coding region of TaDjA1-A gene and it was a rare SNP variation site.There were 4 SNPs in the upstream sequence of TaDjA1-A gene.One marker named as SNP-1149 was developed.Significant association was noted between the marker and 1,000-grain weight.The SNP(G)was considered as the superior allelic variation to increase 1,000-grain weight.4.No evidence for nucleotide variation was found in the coding region of TaDjA1-B gene,but 5 SNPs and 2In Dels were detected in the upstream sequence of TaDjA1-B gene.On the basis of these SNPs,two markers were developed,named as SNP-1431 and SNP-1254.Three haplotypes of TaDjA1-B were identified in a natural population based on the two markers.Association analysis revealed that the haplotypes Hap-6B-2and Hap-6B-3 were considered as the superior haplotypes to decrease plant height and increase chlorophyll contents in the flag leaves at jointing and grain filling stages.5.Three SNPs were detected at the fifth intron,the sixth exon and the seventh exon in the coding region of TaDjA1-D gene,respectively.Among them,the SNP(A/T)at the sixth exon led to change of amino acids(Phe/Tyr).There were 2 SNPs in the upstream sequence of TaDjA1-D gene.All of them were rare SNP variation sites.6.TaDjA1-B was a chloroplast target protein and involved in response to H2O2 and heat stress.TaDjA1-B transgenic Arabidopsis enhanced heat stress tolerance.Overexpression of TaDjA1-B reduced ROS accumulation and increased the antioxidant enzyme activities in the transgenic Arabidopsis.In addition,TaDjA1-B can interact with Hsp70 in plants.7.TaSAP7-A transgenic Arabidopsis enhanced susceptibility to salt and osmotic stresses and accelerated detached leaves chlorophyll degradation.Three haplotypes were identified in a natural population based on the sequence polymorphism of TaSAP7-A.The haplotype Hap-5A-2 was considered as the superior haplotype to decrease plant height and increase 1,000-grain weight and chlorophyll contents.In conclusion,the present developed functional markers of TaDjA1,which associated with plant height,1,000-grain weight and chlorophyll contents in the flag leaves at jointing and grain filling stages,could be helpful for marker-assisted selection in wheat.TaDjA1-B transgenic Arabidopsis significantly enhanced heat stress tolerance.Therefore,it could be used as candidate gene to improve the tolerance to heat stress in crops.TaSAP7-A transgenic Arabidopsis enhanced susceptibility to salt and osmotic stresses.The result expanded the understanding of the SAP gene function in wheat.