Cloning And Functional Analysis Of An ABA Receptor Gene TaPYL3 From Bread Wheat (Triticum Aestivum L.)

Hexaploid bread wheat?Triticum aestivum L.?is one of the most crucial crops cultivated and consumed by humans in the world.Compared to other countries China is the largest producer and consumer of wheat.The wheat production is variable and also below the potential yield.There is major decline in wheat production due to biotic stresses and environmental conditions,especially drought stress.Hence finding candidate genes tolerant to drought stress in germplasm resources and understanding the gene regulation mechanism will provide a new approach to enhance drought tolerance in wheat.PYL?pyrabactin resistance?PYR?like?/PYR?pyrabactin resistance?/RCAR?regulatory component of ABA receptor?consists of 14 family members in Arabidopsis that acts as ABA receptor,is involved in activation of ABA signaling pathway under abiotic stress.In the present study we cloned TaPYL3 from the A genome of wheat based on AtPYL3 sequence from Arabidopsis thaliana.By analyzing gene expression patterns under multiple abiotic stresses in wheat seedlings and observing the phenotype under abiotic stresses in TaPYL3 overexpressed transgenic Arabidopsis T₃ lines,it was inferred that TaPYL3 may responds to salt stresses.Association of two TaPYL3 polymorphic sites with agronomic traits was studied based on the derived cleaved amplified polymorphic sequence?dCAPS?markers developed from the gene sequence.The main research findings are as follows:1.TaPYL3 was isolated from wheat genome A,with the total fragment size 3115 bp,the cDNA coding region is 606 bp.Using diploid species and Chinese Spring nullisomic-tetrasomic lines TaPYL3gene showed location on chromosome 3A.Using a DH?doubled haploid?population,TaPYL3 gene was mapped in chromosome 3A region flanked by markers Ax-111529842?34.5 cM?and Xwmc21?65.4cM?.2.The PYL/PYR family proteins in Arabidopsis are very closely related in sequence and structure.Amino acid sequence analysis showed that TaPYL3 is a conserved gene and when compared to AtPYL family members in phylogenetic analysis,TaPYL3 is a homologous protein with AtPYL members.3.Quantitative RT-PCR analysis indicated that TaPYL3 gene was down-regulated in all treatments except in ABA and heat treatment in wheat.ABA treatment upregulation pattern observed in roots at 6h,whereas down-regulation pattern observed in leaves,and under heat stress down-regulation in root while up-regulation in leaves was observed.4.TaPYL3 gene was transferred into Arabidopsis,different stress treatments were given to T₃ lines to observe change in phenotype.Compared with control plants,the transgenic lines showed tolerance to NaCl stress and ABA treatment,with better root growth.5.Association analysis results showed dCAPS functional marker P3AM1 significantly associated with spikelet per spike,chlorophyll content,and P3AM2 functional marker significantly associated with grains per spike,yield per plant and thousand kernel weight.These results revealed that TaPYL3 gene is associated significantly with agronomical traits under abiotic stresses.Allele wise geographical distribution in agro ecological zones of China depicts that these novel traits associated alleles were positively selected during wheat breeding process.Taken together these results suggested that TaPYL3 gene involved in the response to abiotic stress.The favourable alleles significantly contribute to agronomic traits.The molecular markers will facilitate the marker assisted breeding,and will help breeders to develop drought tolerant breeding lines and speed-up the wheat breeding program.