Resistance of hrfl Transgenic Wheat to Fusarium Graminearum and Construction of Rice RNA Silencing Vector
|School||Nanjing Agricultural College|
|Keywords||Hrf1 transgenic wheat Scab resistance RNA silencing Construction of vector WRKY30|
Wheat Wheat scab (Fusarium head blight, FHB), caused by Fusarium graminearum (Fusarium graminearum Schw.), is a worldwide wheat fungal disease. hrp gene cluster encoding a particular gene product, that is an unique nature harpin protein elicitor. Exogenous application of harpins can stimulate many useful phenotypes. In this study, we used two infection methods of cutting hull and directing injection spike to transfer hrfl gene into wheat. In Agrobacterium EHA105-mediated, we transferred hrfl gene into Yangmai 158, an important wheat variety in Jiangsu Province.599 transgenic lines were positive detected by PCR analysis and Kanamycin Sulfate resistance selection. The transformation efficiency of two infection methods was 0.8% and 1.5%,respectively.This method didn’t require induce callus,compared with traditional pollen tube pathway method.The analysis results showed that the control efficacy of T1 generation to F.graminearum was 28.3% and 78 strains were 0-class disease resistance. The control efficacy of T2 generation was 59.7% and 34 strains were 0-class disease resistance. These results suggested that hrfl gene might offer new opportunities for disease resistance to F. graminearumRNA silencing is widespread in the organism and is an ancient mechanism for gene regulation. Because of efficient, specific characteristics of regulating gene expression, RNA silencing has become a powerful tool to study gene function. Usually, an inverted repeat sequence containing intron is taken into plants, transcripted in plants, formated a hairpin structure and produced double-stranded RNA, then caused gene silencing. In this study, based on binary vector pCAMBIA1301,we used a rice efficient constitutive promoter Ubi, rice 5-enol-pyruvylshikimate-3-phosphate synthase(EPSP synthase) first intron and nos terminator to construct a common RNA silencing vector pCAMBIA1301::Ubi::Intron::Nos, and introduced restriction sites Xhol, AatⅡ, SpeⅠto expand its scope of application. When we used this vector to build a silent carrier, we only needed to insert into the inverted repeat sequence of positive fragments between Ubi and Intron, and insert into reverse fragments between intron and nos. It is easy to get the transgenic vector or overexpression vector by cutting the intron between promoter and terminator and replace the corresponding gene sequences. We also constructed WRKY30 gene silencing vector pCAMBIA1301:: Ubi::WRKY30A::Intron::WRKY30B::Nos, overexpression vector pCAMBIA1301:: WRKY30::Nos and position vector pCAMBIA1301::WRKY30::eGFP::Nos.Because of transitive RNA silencing, we constructed silent vector selected the WRKY30 gene 5’-UTR region sequences 386bp as target to construct a hairpin structure, which expected a better silencing effect.