甘蔗黄叶病毒与花叶病毒CP基因RNAi载体构建与转化甘蔗研究

Construction of ihpRNA expression vector of CP Genes of SCYLV and SCMV and Genetic Transformation in Sugarcane

甘蔗黄叶病和甘蔗花叶病是我国甘蔗最主要的病毒病,感病品种产量下降和品质变劣,传统方法难以防治。RNA干扰技术使培育抗病毒甘蔗品种成为可能。本研究根据甘蔗黄叶病毒和侵染甘蔗的高粱花叶病毒结构与功能特性,广泛收集不同来源病毒分离物CP基因序列,经过比对选取保守序列作为干扰序列。通过在序列两端添加酶切位点,合成并连接成双价甘蔗黄叶病和花叶病毒干扰序列,然后构建到中间载体p HANNIBAL上,形成双价RNAi干扰结构,最后插入到p ART27上形成干扰表达载体。利用基因枪轰击甘蔗品种福农15号愈伤组织进行转化,经G418筛选获得抗性再生植株。通过提取DNA和RNA分析,证实双价RNAi干扰结构不仅以不同拷贝整合到甘蔗基因组中,而且得到了转录表达。

Diseases caused by Sugarcane mosaic virus （SCMV） and Sugarcane yellow leaf virus （SCYLV） are widespread virus diseases in the growth of sugarcane in China. Sugarcane varieties infected the viruses produce less tonnage and sugar potential. Conventional methods don＇t work to control the diseases. The RNA interference technology makes it possible to create antiviral sugarcane varieties by genetic engineering. Based on the viral structure and functional characteristics of SrMV and SCYLV, sequences of the coat protein CP genes were collected and conserved sequences were selected as the RNA interference sequences after alignment. The two RNAi fragments were combined by designing PCR primers with restriction enzyme sites and constructed into vector pHANNIBAL to form the binary ihpRNA antiviral structure. The structure was then inserted the expression vector pART27. The expression vector was transformed into the calli of sugarcane cuhivar FN15 via gene gun bombardment. Sugarcane plants were regenerated from G418 screening media. The regenerated plants were detected in DNA and RNA levels, which verifying the binary ihpRNA antiviral structure was not only integrated into the host genome in copies, but also transcribed successfully.