转双价抗虫基因甘蔗遗传稳定性及去抗性选择标记研究

Sugarcane is an important source for sugar and recently renewable bioenergy. Insects such as stem borer Sesamia inferens, have been severely hindered the sugarcane production sustainable and environment friendly in China. Breeding sugarcane carrying insect-resistant genes is the new method to control sugarcane pests. However, the genetic stability of the insect-resistant transgenic sugarcane and the safety of the selection marker are the new challenge. In an earlier project NSFC 30671330, for the sake of enhancing the protein accumulation and the stability of the transgene, sequences inclusion of signal pepetide and KDEL were added to the ends of cowpea trypsin inhibitor gene cpti or the merged gene Bt-cpti which yielded the modified gene sck (Signal-cpti-KDEL) and sbck (Signal-Bt-cpti-KDEL) exploiting triple-primer PCR techniques. The expression cassettes of the modified genes were framed by the matrix attachment region (MAR) of maize at both ends, aims to prevent positional effects of the transgene. Marker gene and genes of interest were integrated into two T-DNA independently facilitating the removal of the selection marker gene via self-seed setting as the T-DNA separation with the chromosomes segregation or crossover in the progenies, which was helpful for the GMO safety. The efficient expression binary insect-resistant vectors integrated either sbck or Bt+sck for monocotylendon including sugarcane was constructed, driven by promoters Ubi1 (actin) and selected with neomycin phosphotransferase gene II (nptII) or hygromycin gene (hpt). Transformants were obtained via an optimized Agrobacterium-mediated transformation system, selected with geneticin 418/paromomycin or hygromycin B using embryogenic callus as targets. Transgenic sugarcane plants were confirmed by PCR, Southern blot, Northern blot and protein activity assay, followed by both in vivo and in vitro bioassay, and finally the high insect-resistant independent transgenic lines were generated. On the basis of the earlier work, this project will, on one hand, focus on the genetic stability of the binary insect-resistant transgenic sugarcane at DNA, RNA and Protein levels, the tolerance of stem borer to the toxic proteins, the transgenic sugarcane performance in the progenies. On the other hand, remove the selection marker gene of the insect-resistant transgenic sugarcane via self-seed setting as well as molecular screening such as PCR in the segregation progenies. Therefore, the outcome of this project will ultimately provide valuable materials for breeding selection marker free genetically modified sugarcane with high insect resistance in the future.

甘蔗是重要糖料和能源作物,生产中虫害问题比较突出。培育抗虫转基因甘蔗是应对甘蔗害虫的重要新举措，但又面临转基因遗传稳定性和标记基因安全性问题。前期研究，通过细胞内定位技术修饰抗虫基因，目标蛋白定位到内质网提高外源蛋白稳定性；目标抗虫基因两侧添加MAR序列，克服转基因位置效应提高转基因稳定性；抗性筛选标记基因和抗虫基因表达结构分别置于两个独立的T-DNA区，为通过染色体分离或交换等后续研究获得无抗性筛选标记抗虫转基因甘蔗提供了基础材料。本次申请在前期双T-DNA系统、农杆菌介导，抗性筛选、分子检测、抗虫性鉴定，筛选获得转双价抗虫基因甘蔗基础上，研究转双价抗虫基因甘蔗后代，DNA、RNA和蛋白水平以及抗虫性遗传稳定性、主要工农艺性状。光周期诱导转基因甘蔗开花、自交分离、分子检测和FISH分析等，筛选获得去除抗性选择标记的抗虫转基因甘蔗，为培育无抗性选择标记的高效抗虫转基因甘蔗新品种奠定基础。

甘蔗是重要糖料和能源作物,生产中虫害问题比较突出。培育抗虫转基因甘蔗是应对甘蔗害虫的重要新举措，但又面临转基因遗传稳定性和标记基因安全性问题。项目围绕转抗虫基因甘蔗遗传稳定性和去除抗性选择标记，在目标抗虫基因两侧添加MAR序列，克服转基因位置效应提高转基因稳定性；抗性筛选标记基因和抗虫基因表达结构分别位于两个独立的T-DNA区，为探索通过染色体分离或交换等后续研究获得无抗性筛选标记抗虫转基因甘蔗提供基础材料。项目主要开展二方面研究。一是从DNA、RNA和蛋白水平，研究cry1Ac和sck转双价抗虫基因甘蔗无性系后代中的整合与表达，以及抗虫性和主要工农艺性状表现，探明转双价抗虫基因甘蔗的遗传稳定性。二是通过光周期诱导高效抗虫转基因甘蔗株系开花，自交结实，获得分离后代，在苗期进行PCR扩增及ImmunoStrip检测，筛选整合cry1Ac和sck双价抗虫基因，但不含有抗性选择标记基因的转基因甘蔗植株，对获得的已经去除了抗性选择标记基因的转基因甘蔗株系进行抗虫性鉴定等，最终获得无抗性选择标记的转双价抗虫基因甘蔗。.研究结果表明转双价抗虫基因甘蔗后代遗传相对稳定，抗虫性和主要工农艺性状表现基本正常。自交结实后代实生苗，苗期PCR检测结果表明实生苗存在T-DNA分离，可以选择到无抗性选择标记的转双价抗虫基因甘蔗株系，进一步的抗虫性鉴定和工农艺性状分析还有待后续研究继续完成。虽然基因编辑技术也可以获得无选择标记基因的植株，但是现有技术还不能编辑出赋予抗虫性状的植株。通过项目实施，建立的农杆菌介导双T-DNA转化甘蔗系统，对于开展其它自交结实作物的去除抗性筛选标记基因研究有一定借鉴价值。甘蔗光周期诱导开花积累的成功经验可为甘蔗杂交育种提供参考。CRISPR/Cas9系统定点编辑水稻Wx基因的成功经验，可为甘蔗基因编辑系统的建立提供参考。依托本项目培养毕业硕士研究生4名，本科生7人。参加国内学术交流3次，大会报告2次；参加国外学术交流1次，墙报交流1次；到省外高校学术讲座1次。发表学术论文6篇。