四价抗病虫除草剂RNAi植物表达载体构建与转化甘蔗研究

甘蔗是重要的能源作物和糖料作物,面临病虫害日益严重问题。由于甘蔗遗传背景非常复杂,且缺乏必要种质,难以通过杂交培育抗病虫、除草剂聚合品种。基因工程技术提供了新方法。本研究利用BLAST比对获得甘蔗花叶病毒ScMV-CP基因和黄叶病毒ScYLV-CP基因的保守序列作为干扰序列,通过设计内切酶位点、生物合成和连接,获得全长687 bp的两个保守序列融合片段（MYCP）。并将其正向和反向插入中间载体pHANNIBAL上,形成双价病毒干扰结构。再通过在pHANNIBAL上添加SmaⅠ内切酶位点,将双价病毒干扰结构表达框和Cry1AC基因表达框结合;利用NotⅠ酶切,回收双价病毒干扰结构和Cry1AC基因表达框片段,连接到添加Bar基因表达框的表达载体pART27-Z上,构建了抗病虫及除草剂四价植物表达载体（pART27-MYCP-Cry1AC）。采用基因枪法将构建好的四价植物表达载体转入甘蔗品种FN15的胚性愈伤组织中,通过PPT（草丁膦）筛选获得了抗性再生甘蔗植株,经过PCR、定量以及Bt蛋白试纸检测,获得同时整合四种目的基因并表达的四价转基因甘蔗植株,为培育多抗甘蔗新品种提供了材料。

Construction of Tetravalent Expression Vector Carrying Cry1Ac,Bar and RNAi Fragments Resistant to Virus Diseases and Genetic Transformation in Sugarcane

Sugarcane is an important crop for sugar and biofuel. Sugarcane production is badly affected by plant diseases and insects. Sugarcane is a highly heterozygous allopolyploid plant with a complex genetic background and short of germplasm resources resistant to diseases, insects and herbicide. Therefore, it is very difficult to get novel varieties with integrated traits through crossbreeding. Genetic engineering technology offers a new way for integrated breeding. Consensus sequences of CP gene of ScMV and CP gene of ScYLV were analysised by BLAST and selected out as RNAi fragments to target genes respectively. The RNAi fragments against to ScMV and ScYLV were linked together to form a fusion fragment of 687 bp（MYCP）. According to the restriction enzyme sites, the fusion fragment was inserted into pHANNIBAL forwardly and reversely to form a bivalent RNAi structure, and then the gene Cry1AC was introduced into the vector with addition of Sma Ⅰ. Finally, after digestion with Not Ⅰ and gel recovery the fragment of Cry1AC and the bivalent RNAi structure were introduced into pART27-Z in which Npt Ⅱ had been repl aced by Bar to form tetravalent expression vector pART27-MYCP-Cry1AC with genes resistant to insects, herbicide, ScMV and ScYLV. The tetravalent vector was transformed into sugarcane calli of FN15 via gene gun. The calli were subjected to PPT stress and regenerated. Tetravalent transgenic sugarcane plants were identified and expression of the target genes was also detected by PCR, quantitative PCR and protein strips, which provide materials for breeding new varieties with multiple resistance.