蒺藜苜蓿快中子突变体esn1共生固氮特性的遗传定位与转录组研究

The understanding on the genetic mechanisms of symbiotic nitrogen fixation in legumes is an important cornerstone to realize the environment-friendly and sustainable agriculture in the future. Medicago truncatula and Sinorhizobium meliloti could form a symbiotic association resulting in the formation on the host roots of new organs, nodules, in which biological nitrogen fixation occurs. Any abnormality of the complicatedly dynamic molecular recognition during the establishment of the symbiosis between hosts and microsymbionts would lead to failure in symbiotic nitrogen fixation. In this project, we will use a fast neutron-derived M. truncatula material which was identified as an early senescent nodule1 (esn1) mutant, adopt genomic microarray technology to position the mutation locus on the chromosome; use PCR techniques to determine whether the chromosomal mutation is linked with the phenotype or not; employ RNA-Seq approach to investigate the transcriptome of 9dpi (days post inoculation) nodules and analyze the possible premature reasons for esn1 at transcript level; take all experiment results to draw out the candidates for MtESN1. This project aims to reveal the molecular mechanism of early senescence in esn1 nodules and lay the solid foundation for MtESN1 cloning. The study could expand our understanding on the biological mechanism of symbiotic nitrogen fixation in M.truncatula and provide new molecular target to manipulate to improve the efficiency of symbiotic nitrogen fixation in legumes.

对豆科植物生物固氮遗传机理的认识是实现未来农业环境友好和可持续发展的重要基石。蒺藜苜蓿和中华根瘤菌之间的共生结合可导致共生固氮发生场所即新器官根瘤的形成。共生关系建立过程中，宿主和微共生体之间存在复杂的分子动态识别，其中任何环节的异常都会导致共生固氮的失败。本项目以早期筛选鉴定到的蒺藜苜蓿根瘤早衰快中子突变体esn1为实验材料，拟采用基因组芯片杂交技术对该突变体的突变位点进行染色体定位；利用PCR技术确认定位到的突变位点与突变表型是否具有遗传连锁效应；采用RNA-Seq技术对接种9天的根瘤进行转录组分析，以在转录水平解析esn1早衰的原因；同时，结合基因组芯片杂交数据和转录组数据，限定MtESN1的候选基因。该研究旨在揭示esn1早衰的分子机制并为该基因的克隆奠定基础，可进一步深化我们对蒺藜苜蓿共生固氮生物发生机制的认识，并为改良豆科植物共生固氮效率提供新的分子靶点。

豆科植物和根瘤菌之间共生固氮遗传基础的阐释是通过基因工程技术实现非豆科植物共生固氮的必要先决条件之一。而其遗传基础的阐释有赖于相关突变体对应功能基因的遗传定位和克隆。本项目以蒺藜苜蓿快中子共生固氮缺陷突变esn1为研究材料，通过比较基因组芯片杂交技术研究其突变遗传位点的染色体定位，结果显示在蒺藜苜蓿Chr4中存在大片段的DNA删除，进一步的遗传连锁分析发现，该DNA删除区域与固氮缺陷表型具有紧密连锁效应，表明MtESN1位于相应的DNA删除区域内。通过RNA-Seq技术的转录组研究发现，esn1突变体根瘤的代谢与转录与对照根瘤差异巨大，发生了较强的胁迫响应。通过分析比较基因组杂交芯片结果和转录组结果，将MtESN1的候选基因限定在7个基因内。该研究不但增加了为MtESN1基因的克隆奠定了基础，还为同类研究的遗传定位提供了新的技术途径和策略。项目执行过程中，授权发明专利和实用新型专利各1项。