多油辣木高效抗虫转基因研究

Crop pests are the severely hindrance in terms of the crop production in a sustainable, stable and healthy way. Moringa oleifera (Moringa) is a remarkable species with high nutritional value as food and biofuel due to its good biomass production, which can be grown in hillside and drought tolerant. Moringa is an economically important multi-purpose tree for the food safety and biofuel development in the view of China’s tactic. However, insects such as Noorda blitealis, have been severely hindered the Moringa production sustainable and environment friendly worldwide. Breeding Moringa carrying insect-resistant genes is the new method to control this pest. In our earlier research, 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. On the basis of the earlier work, this project will, on one hand, focus on the optimization for the Agrobacterium-mediated transformation system in order to modify the Moringa genetically using either LBA4404 or EHA105 as the engineered bateria. The Moringa transformants integrated genes of interest either sck or Bt will be obtained. On the other hand, the genetic stability of the binary insect-resistant transgenic moringa at DNA, RNA and Protein levels, the resistance of Moringa to Noorda blitealis, the transgenic Moringa’s performance in the progenies will be also addressed. Therefore, the outcome of this project will ultimately provide valuable materials for breeding genetically modified Moringa with high insect resistance in the future.

新资源植物辣木耐贫瘠耐干旱，既可作为粮食也是荒山旱地速生树，从国家粮食安全和生物燃料考虑，发展辣木具有国家战略意义。但是，辣木瑙螟Noorda blitealis危害非常严重，培育抗虫辣木新品种十分迫切。我们前期研究通过细胞内定位技术，目标蛋白定位到内质网提高外源蛋白稳定性，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等研究取得较好进展；辣木种质资源与育种得到国家现代农业产业技术体系稳定支持。本次申请利用修饰的CpTI和Bt基因构建高效抗虫载体，三种类型多油辣木的下胚轴、花药愈伤组织或胚性愈伤组织为受体，LBA4404或EHA105介导，优化转化参数，建立多油辣木高效遗传转化体系。并在DNA、RNA和蛋白活性水平进行分子检测，开展离体和活体抗虫性鉴定及综合农艺性状评价，筛选获得综合农艺性状优良的转基因抗虫辣木株系。最终，为培育出高效抗虫转基因辣新品种奠定基础。

辣木全球年产值近400亿美元，不仅可作为粮食而且还是荒山旱地速生树，从国家粮食安全和生物燃料两个层面看，发展辣木产业具有国家战略意义。商业化生产中，辣木瑙螟Noorda blitealis危害十分严重，急需培育抗虫品种。项目31860414围绕建立辣木高效遗传转化体系和获得转基因抗虫优良辣木株系，四年来开展了十个方面的工作。基因修饰及高效植物表达载体构建；辣木茎段、叶片、下胚轴和幼胚诱导胚性愈伤组织；辣木花药培养；辣木愈伤组织诱导与分化阶段内源激素的动态变化；建立辣木叶片组织培养再生体系；PKM1愈伤组织诱导和分化阶段对卡那霉素耐受性；建立农杆菌介导高效转化辣木技术体系；辣木种子EMS诱变获得新种质；获得辣木雄性不育新材料；境内外辣木种质资源收集与评价。.项目获得修饰的cpti基因sck (Signal-cpti-KDEL)和融合蛋白基因sbck(Signal-Bt-cpti-KDEL)，nptII为筛选标记，35s和actin驱动sck和sbck，构建高效植物表达载体，获得工程菌EHA105。成功诱导辣木茎段、叶片、下胚轴和幼胚等4种外植体获得胚性愈伤组织。MS和B5为基本培养基，添加不同激素（NAA、2,4-D、6-BA和IBA）及其激素配比等诱导辣木花药，获得双单倍体愈伤组织。明确辣木叶片、茎段和花药愈伤诱导和分化过程中吲哚乙酸（IAA）、脱落酸（ABA）、玉米素（ZT）和赤霉素（GA3）含量及比值的变化；高含量的ABA、低含量的ZT和高IAA/GA3共同抑制了辣木愈伤组织分化。辣木叶盘诱导芽的成功率在0-2.38%，最终获得完整植株的叶盘近1%。辣木愈伤组织诱导和分化阶段合适的卡那霉素筛选浓度分别是70mg/L和50mg/L，辣木愈伤组织分化阶段对卡那霉素更敏感。初步建立辣木叶片组织培养再生体系和农杆菌介导高效转化辣木技术体系。辣木种子EMS诱变获得新种质2000份。获得辣木雄性不育新材料2份，组织切片结果表明其雄蕊退化，雌蕊正常。收集境内外辣木资源326份，涵盖辣木13个种；为辣木育种奠定基础。依托本项目培养毕业硕士研究生8名，本科生25名。参加国内学术交流2次，报告2次；参加国际学术会议交流1次，报告1次，发表论文1篇。获授权发明专利1项，发表学术论文8篇。