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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。
Sugarcane is an important energy crop and sugar crop, facing growing problems of pests and diseases. Because sugarcane has a very complex genetic background and lacks the necessary germplasm, it is difficult to breed insect-resistant and herbicide-polymerized varieties by crossing. Genetic engineering provides a new way. In this study, the conserved sequences of ScMV-CP and ScYLV-CP genes of sugarcane mosaic virus (Mosaic Virus) were cloned by BLAST. As the interference sequence, we designed a restriction endonuclease site, biosynthesis and ligation to obtain two Conserved sequence fusion fragment (MYCP). And insert it into the intermediate carrier pHANNIBAL forward and reverse to form a bivalent virus interference structure. The bivalent virus interfering structural expression cassette was then joined with the Cry1AC gene expression cassette by adding the SmaⅠ endonuclease site to the pHANNIBAL; the divalent viral interference structure and the Cry1AC gene expression cassette fragment were recovered by NotⅠ digestion, ligated to the bar (PART27-MYCP-Cry1AC) was constructed on the pART27-Z vector expressing the gene expression vector. The constructed tetraploid plant expression vector was transferred into embryogenic callus of sugarcane variety FN15 by particle bombardment and resistant regenerated sugarcane plants were obtained through PPT (glufosinate) screening. After PCR, quantitative analysis and Bt protein test paper , And obtained the tetravalent transgenic sugarcane plant which integrates the four target genes at the same time and provides a material for cultivating new multi-resistant sugarcane varieties.