转基因甘蔗外源cry1Ac和cry2A基因在线粒体基因组中的整合机制

The stem borer is the most harmful pest in sugarcane cultivation and causes serious loss. Due to the lack of insect-resistant gene in sugarcane gene pool, modern sugarcane has poor resistance to pest. The improvement of pest resistance is one of the two successful traits in transgenic plants with large-scale cultivation. Transgenic cry sugarcane is efficient in the improvement of pest-resistance, while the integration of foreign gene in genome affects its expression level and stability. It appeared to be a tendency that the exogenous cry1Ac in two transgenic sugarcane lines from particle bombardment integrated into the mitochondrial genome in our previous study. Is there an universal tendecy of integration bias? And what the integration mechanism is? Thus, transgenic lines of two genotypes FN15 and ROC22 possessing cry1Ac/cry2A (both gene two receptor), which exhibited different resistant phenotype, were selected for study. Firstly, the flanking sequences of foreign cry1Ac/cry2A transgenic lines will be investigated by molecular cloning i.e. genome walking and hi-tail PCR in this study. Secondly, the mitochondrial genome of both the cry1Ac/cry2A lines and their non-transgenic lines will be sequenced and assembled by the PacBio RSII. Then, the comparative analysis of the flanking sequences and the mitochondrial genome will be performed to analyze the insertion site, the integration patterns, integrative trend of the foreign genes, which will result in the revelation of the integration mechanism in transgenic sugarcane. Thus, the project has important scientific significance.

螟虫是甘蔗生产上最严重的害虫。由于甘蔗基因池中缺乏优异抗虫基因，导致甘蔗抗虫性差，而抗虫性是转基因作物改良并规模化种植最成功的两个性状之一，甘蔗导入cry基因能有效提高其抗虫性，外源基因在基因组中的整合影响其表达水平和稳定性。前期研究发现：2个基因枪法获得的cry1Ac转基因甘蔗株系中，外源基因趋向整合到线粒体基因组。该整合偏向性是否具有普遍性？其整合机制如何？为此，项目拟对不同基因型受体（FN15和ROC22）的cry1Ac和cry2A群体，借助分子克隆，鉴定其外源基因旁侧序列；通过高通量测序组装，得到不同转基因株系和受体对照系的线粒体基因组，比对分析明确外源基因在线粒体基因组中的插入位点、整合模式及整合的偏向性，揭示外源基因的整合机制。项目具有重要的科学意义。

甘蔗是最重要的糖料作物，螟虫是甘蔗生产上最严重的害虫。由于甘蔗基因池中缺乏优异抗虫基因，导致甘蔗抗虫性差，而抗虫性是转基因作物改良并规模化种植最成功的两个性状之一，甘蔗导入cry基因能有效提高其抗虫性，外源基因在基因组中的整合影响其表达水平和稳定性。前期研究发现外源cry1Ac基因趋向于整合到甘蔗的线粒体基因组的偏向性。该整合偏向性是否具有普遍性？为了回答这个科学问题，项目把高通量测序、分子克隆等技术与旨在解决甘蔗产业螟害严重的抗螟虫育种材料相结合，旨在明确外源基因的遗传稳定性及整合偏向性，揭示其整合机制，项目具有重要的科学意义。主要结果和结论如下：（1）改良了hi-tail PCR技术体系，其扩增cry基因旁侧序列更高效，克隆了部分转基因株系的外源cry1Ac和cry2A基因的旁侧序列。（2）利用高通量测序获得受体蔗和转基因系的线粒体基因组序列信息：采用Illumina Hiseq XTen 和 PacBio Sequel测序平台，组装获得了2个受体蔗和3个转cry基因甘蔗的线粒体基因组序列信息、基因组的完整完成图，发现甘蔗线粒体基因组均由两个独立的环状DNA组成，共线性分析显示线粒体基因组高度保守；甘蔗线粒体基因组的序列丰富甘蔗基因资源库，同时为线粒体基因组序列演化相关研究奠定了基础。（3）明确了受体蔗和部分转基因蔗线粒体基因组的组成及差异，通过生物信息学分析获得了部分外源基因及其旁侧序列在线粒体基因组中的位点。（4）基于受体和转基因株系温室螟虫胁迫下的转录组学分析、表达谱分析、螟害率调查等表型数据联合分析，发现外源cry基因导入和部分内源抗性相关基因（CDPKs, OPDAs, MAPKs, JAR1, JAZs ,MYC2和DIMBOA/DIBOA）在提高转cry1Ac基因甘蔗抗虫性上具有协同性。本项目研究发现基因枪法轰击得到的转基因甘蔗外源cry1Ac/cry2A基因部分株系确有整合到线粒体基因组中的偏向性，但是否具有普遍性仍不能下结论，还需要进一步扩大群体进行探究。项目实施培养硕士研究生2人、发表SCI论文4篇。