Cloning And Application Of Root-Specific Promoters For Cotton Verticillium Resistance Breeding

Cotton is an important economic crop in China.Verticillium wilt caused by Verticillium dahliae has been seriously affecting the yield and quality of Cotton.Due to the lack of germplasm resources of cotton against Verticillium wilt,the improvement of traditional breeding and cultivation techniques not effectively prevent and control cotton Verticillium wilt.Therefore,the development of new disease-resistant varieties using genetic engineering technology has become the most effective way to improve cotton resistance to Verticillium wilt at this stage.The Root-specific promoter allows the foreign gene expressed specifically in the root of the Recipient plant,thereby reducing the waste caused by non-specific expression of the foreign gene in the recipient plant and preventing the interference of the multi-site expression of the foreign gene on the normal growth of the recipient plant,but for the Cotton Root-specific promoters studies also relatively rare.In this study,we designed primers by the reported Root-specific genes,four promoters(MIC-3,WRKY54,TIP-2 and PRP-2)were cloned from Cotton by PCR amplification.The results of promoter sequence analysis indicated that all four promoters contained Root-specific expression elements such as ROOTMOTIFTAPOX1,OSE1ROOTNODULE and OSE2ROOTNODULE.It is hypothesized that the four promoters are root-specific promoters,all of which are capable of driving foreign genes to be specifically expressed in plant roots.This study uses the binary expression vector pCOMBIA1301 as a template,the constitutive promoter CaMV35S in the pCOMBIA1301 vector was replaced by four cotton root-specific promoters by restriction enzyme-ligation,obtain a recombinant expression vector for driving the expression of the GUS gene by a cotton root-specific promoter,named MIC-3::GUS,WRKY54::GUS,TIP-2::GUS and PRP-2::GUS.And transformed into Arabidopsis thaliana by Agrobacterium tumefaciens-mediated method.Histochemical staining of transgenic plants,the results indicate that the GUS reporter gene is mainly expressed in the roots of transgenic plants,indicating that the four promoters(MIC-3,WRKY54,TIP-2 and PRP-2)have strong Root-specific expression.The BTD-S is a synthetic antibacterial peptide gene designed by my laboratory,which has significant disease resistance to cotton Verticillium wilt.This study uses the binary expression vector MIC-3::GUS as a template,the GUS reporter gene in the MIC-3::GUS expression vector was replaced with the synthetic antimicrobial peptide gene BTD-S by restriction enzyme-ligation.Thus,a recombinant expression vector for driving the artificial antimicrobial peptide gene BTD-S by the MIC-3 promoter was obtained,named MIC-3::BTD-S.And transformed intoArabidopsis thaliana by Agrobacterium tumefaciens-mediated method.Quantitative RT-PCR analysis of BTD-S antimicrobial peptide gene expression levels in transgenic plants showed that BTD-S antimicrobial peptide gene was mainly expressed in the roots of transgenic plants.The root expression leaves was significantly higher than other parts,indicating that MIC-3 promoter can specific expression of the BTD-S antimicrobial peptide gene in plant roots.Identification of transgenic plants against in vitro bacteriostasis showed that the crude protein extract of the transgenic plants could effectively inhibit the growth of Verticillium dahliae and spores,indicating the MIC-3 promoter can drive the BTD-S antimicrobial peptide gene to produce Cotton Verticillium resistance in the Root of the Recipient plant...