Polymorphism And Functional Analysis Of Stress Associated Protein Gene TaSAP7 From Wheat(Triticum Aestivum L.)

Wheat(Triticum aestivum L.)is one of the most important food crops in the world.The lack of water resources and soil salinization seriously restrict the wheat production.Excavation and utilization of excellent stress-resistant genes are a very important approach for wheat improvement and production.Stress associated proteins(SAPs)are the A20/AN1 zinc-finger proteins which involve in abiotic stresse tolerance and development in plants.There are abundant allelic variations in wheat germplasm.Excavating favorable stress-tolerant alleles and developing functional markers will provide the basis for marker-assisted selection breeding.Association analysis possesses the advantages of shorter research time and higher mapping resolution,therefore it is considered as a powerful approach for identifying functional alleles.In this study,elite alleles in TaSAP7 gene were identified through developing functional markers and association analysis.In addition,the functions of TaSAP7-A were also preliminarily studied by using gene expression pattern analysis,subcellular localization,transcriptional activity assay and yeast two-hybrid library screening.The main results are summarized as follows:1.The reference sequences of TaSAP7 were screened out by sequence alignment using conserved domain in wheat genome database.Three alleles,TaSAP7-A,TaSAP7-B and TaSAP7-D were isolated by specific primers designed from wheat genomes A,B and D.All three alleles have two AN1 domains.The similarity of amino acids sequences among three alleles was 95.03%.Using Chinese Spring nullisomic-tetrasomic lines,TaSAP7-A,TaSAP7-B and TaSAP7-D were located on chromosome homologous group 5.TaSAP7-B was mapped on chromosome 5B flanked by AX-99395950(2.86 cM)and Xwmc380(5.26 cM)using a DH population including 150 lines derived from Hanxuan 10 × Lumai 14.2.The full-length of TaSAP7-A gene was 3.5 kb including the upstream sequence(2990 bp)and coding region(549 bp).There was no intron in the coding region.Using a highly diverse population consisted of 32 wheat accessions,35 SNPs and 4 InDels were detected.Among them,2 SNPs(A/G and T/C)were in the coding region which led to changes of amino acids(Asn/Asp and Val/Ala).Based on these 2 SNP sites,two markers were developed,named as SNP-464 and SNP-2044.Association analysis revealed that two markers were associated with grain number per spike and spikelet number per spike.Three haplotypes of TaSAP7-A were identified in a natural population based on the two markers.The haplotype Hap3A made a positive contribution to the grain number per spike and spikelet number per spike.3.The full-length of TaSAP7-B gene was 1.9 kb including the upstream sequence(1308 bp)and coding region(543 bp).No evidence for intron and nucleotide variation was found in the coding region,but one SNP(C/T)and one InDel(A/-)were detected in the upstream sequence.On the basis of the SNP(C/T),one marker was developed,named as SNP-260.Significant association was noted between the marker SNP-260 and agronomic traits of the natural population,including plant height,peduncle length,length of penultimate internode,number of spike per plant and 1,000-grain weight.The SNP(C)was considered as the superior allelic variation to decrease plant height and increase 1,000-grain weight.The distribution proportion of the superior allelic variation in Chinese 10 major wheat zones was significantly enlarged from landraces to modern cultivars.The frequencies ditribution during the breeding process revealed that the superior allelic variation was positivly selected by breeders.4.The full-length of TaSAP7-D gene was 2.9 kb including the upstream sequence(2313 bp)and coding region(549 bp).No evidence for intron and nucleotide variation were found in the coding region,but one SNP(C/T)was detected in the upstream sequence.On the basis of the SNP in the upstream sequence,one marker was developed,named as SNP-340.All of accessions in the natural population were C allele in the variation site except Chinese Spring cultivar possessing T allele.It was a rare SNP variation site.5.TaSAP7-A was involved in response to exogenous ABA and several abiotic stress,such as cold,PEG,and high salinity stresses.Subcellular localization revealed that TaSAP7-A was located in nucleus and cytoplasm in tobacco leaves.The full-length and two AN1 truncations of TaSAP7-A had no transcriptional autoactivation activity by using yeast system.TaS10B,interaction with TaSAP7-A,was obtained by yeast two-hybrid library screening.Protein structure prediction showed TaS10B was a 26S protease regulatory subunit.The present developed functional markers of TaSAP7s,which associated with grain number per spike,spikelet number per spike,plant height and 1,000-grain weight,could be helpful for marker-assisted selection in wheat.The results including expression pattern analysis,subcellular localization,transcriptional autoactivation analysis and interaction protein screening,provided the basis for further biological studies of TaSAP7-A.