棉花NBS-LRR类基因RNA干扰载体的构建及烟草转化

【目的】初步获知棉花NBS-LRR类基因的生物学意义,构建干扰载体。【方法】以抗枯萎病棉花中棉12号为材料,用霍格兰营养液培养至三叶期,进行枯萎病菌接菌诱导处理,以不接菌为对照。利用cDNA-AFLP技术筛选差异基因片段,在GenBank中进行序列同源性比对,找到抗枯萎病基因片段,该基因片段的序列与GenBank中报道的序列同源性达到72%。将此基因片段以正向和反向插入到pPZP35S,经限制性酶切和质粒PCR鉴定。【结果】已成功构建了干扰载体。采用农杆菌侵染法将干扰载体转入烟草中。【结论】为研究棉花NBS-LRR类基因的功能打下基础,为通过转基因技术深入研究该基因在棉花抗病机理创造条件。

Construction of the RNAi Vector of Cotton NBS - LRR Genes and Tobacco Transformation

【Objective】The purpose of this program was to get an initial knowledge of the biological significance of the gene and construct interfering carriers.【Method】In this study,resistance to Fusarium wilt of cotton in 12 was taken as the experimental material which was cultured with Hoagland nutrient solution to the three-leaf stage,and then the induction of Fusarium inoculation treatment was done with non-inoculation as control.cDNA-AFLP was used to screen the differential gene fragments in order to have a sequence homology comparison in the GeneBank and find the gene that is resistant to Fusarium wilt.The sequence of the gene fragment that we found reached 72%homology with the one reported in the GeneBank. This found gene fragment was inserted into pPZP35S in forward and reverse direction and identified by restriction digestion and plasmid PCR.【Result】The interference carrier has been successfully built.Using Agrobacterium infection into tobacco law will interfere with the carrier,which laid the foundation for studying cotton NBS-LRR class gene.【Conclusion】This study created condition for the adoption of transgenic technology in-depth study of the mechanism of disease resistance genes in cotton.