Identification Of Intronic Regulator In Stripe Rust Resistance Gene WKS1of Wheat

Wheat is one of the most widely planted crops worldwide, provides about20%of totalcalories consumed by humankind. Wheat stripe rust, caused by Puccinia striiformis West. f.sp. tritici (Pst), is a worldwide airborne fungal disease. Spores of Pst easily travel longdistances in the air, cause rust epidemics in wheat field under mild and humid conditions,and result in significant yield reduction. Currently, stripe rust is among the most importantwheat diseases in China, posing great threat to food security.WKS1is the first stripe rust resistance gene isolated from wheat, conferring high levelresistance under high-temperature conditions. The first intron of WKS1carries two putativeregulatory elements, including a hairpin structure and a microRNA target site. Thesedomains might regulate WKS1expression. To further understand molecular regulation ofthe WKS1gene, we developed four plasmid constructs that included the wild copy and threemodified alleles of the WKS1gene. Corresponding transgenic plants were produced incommon wheat ‘Bobwhite’, and they will be used to study the function of those putativeintrogenic elements in WKS1. Major results are summarized as follows:1. Construction of GUS common vectorIn order to prepare GUS fusion expression, a Gateway-based common vector wasconstructed. It carried both BAR and HPTII as selection markers, and a Gateway:GUSfusion matrix where target gene and its native promoter can be placed in the Gatewayregion. Transgenic plants of this construct can be confirmed using BASTA resistance andX-Gluc staining.2. Construction of WKS1:GUS fusion vectorsTo identify putative regulatory elements in the1stintron of WKS1, the hairpin structure,the microRNA target site, or the1stintron were deleted in a6152bp fragment includingpromoter, exon1, intron1and exon2of the WKS1gene, and were cloned in frame into theGateway:GUS fusion matrix. Four diffient constructs were developed including vector A(wild copy control), vector B (microRNA site removed), vector C (hairpin structureremoved), and vector D (intron1removed).3Generation of transgenic wheat A total of27putative transgenic lines were obtained, in which vector A, B, C and Deach corresponded to15,7,1and4independent plants, respectively. X-Gluc staining andPCR amplification confirmed there were3,1and2positive transgenic plants from vector A,B and D, respectively. Transgenic plants will be used to investigate regulatory roles ofintrogenic elements of the WKS1gene.