低氮条件下玉米穗位叶持绿QTL qlnSG1b的克隆和功能分析

Maize ear leave was the most important to the supply of carbohydrates to kernel. Nitrogen deficiency significantly reduced ear leaf chlorophyll content, thus influence the photosynthetic rate, eventually lead to a drop in grain yield. It has important theoretical and practical significance to enhance the stay-green phenotype of maize ear leaves in nitrogen-deficient condition. In the previous research one single segment substitution line (SSSL) population derived from a cross between an N-efficient inbred line Xu178 and an N-inefficient inbred line Zong3 was used to identify the QTL associated with nitrogen efficiency. One SSSL named 1314 contained major QTL qlnSG1b for ear leave stay-green in nitrogen-deficient condition was selected by field trial. Moreover, QTLs for shoot biomass, shoot nitrogen content and root traits in nitrogen-deficient condition were also detected in this SSSL. In the present project, based on a methodology termed bulked segregant analysis (BSA), whole-genome sequencing data are derived from pooled F2 segregants after crossing 1314 line and recurrent parent Xu178 and are analyzed for single nucleotide polymorphisms (SNPs). SNPs with statistically significant deviations from SNP index in the substitute region of 1314 line were identified. The candidate gene will be defined combined with the results of RNA-Seq. And its function verification will be conducted by constructing overexpression vector and CRISPR-Cas9 vector to reveal the relationship between ear leaf stay-green and nitrogen use efficiency. And then the candidate-gene based association analyses were carried out to discover the excellent alleles for breeding ear leaf stay-green cultivar and increase nitrogen use efficiency of maize.

玉米穗位叶对籽粒碳水化合物的供应最重要，施氮不足显著降低穗位叶的叶绿素含量，最终导致产量的降低。因此，提高低氮条件下玉米穗位叶的持绿性对提高玉米氮肥利用效率具有重要意义。前期通过两年大田实验筛选出一个包含低氮下玉米穗位叶持绿性QTL qlnSG1b的单片段代换系1314，并且1314同时检测到低氮下生物量、氮含量、氮累积量，以及根系性状的QTL。本研究从1314代换系与轮回亲本构建的BC5F2分离群体中挑选极端表型单株，构建DNA混池，利用NGS-BSA方法找到目的区间内的差异突变位点，结合前期RNA-Seq结果，确定控制目标性状的候选基因。通过构建超量表达载体和CRISPR-Cas9敲除载体对其功能进行转基因验证，揭示低氮条件下穗位叶持绿性与氮效率之间的关系。利用候选基因的关联分析发掘优异等位基因位点，并开发分子标记，为培育低氮下穗位叶持绿的玉米品种、提高玉米氮利用率提供优异的基因资源。

玉米穗位叶对籽粒碳水化合物的供应极为重要，施氮不足显著降低穗位叶的叶绿素含量，最终导致产量的降低。因此，提高低氮条件下玉米穗位叶的持绿性对提高玉米氮肥利用效率具有重要意义。本研究以携带低氮穗位叶持绿主效QTL qlnSG1b单片段代换系1314和轮回亲本许178为研究材料，分别在水培条件下对两个材料的转录组进行分析，结果表明相对于许178而言，在低氮胁迫下1314叶片中有更多的参与抗氧化、植物激素合成和物质转运等过程的基因表达量上调，且根系中有更多的参与呼吸作用、抗氧化过程和氨基酸代谢的基因表达量上调。在大田条件下对亲本及F1的穗位叶持绿性、光合作用以及碳氮代谢相关酶活性进行分析，低氮条件下代换系1314的叶绿素含量和光合速率显著低于许178和F1，并且低氮条件下其穗位叶中氮代谢关键酶GOGAT和GDH酶活性也显著低于轮回亲本许178和F1。但是PEPC酶活性差异不显著。表明1314所含的持绿QTL与氮代谢关系密切。构建1314和许178的F2分离群体，对4890个F2单株在低氮条件下玉米穗位叶持绿表型进行鉴定，筛选极端表型单株，构建DNA混池，进行BSA分析，将低氮条件下玉米穗位叶持绿qlnSG1b的定位到第6染色体2.39 Mb区间内，其中包含322个基因。同时结合前期转录组数据对候选片段内的基因进行了功能预测，其中Zm00001d039141（参与蛋白质磷酸化及调节酶活性等）、Zm00001d039102（参与氧化还原过程）、Zm00001d039016（参与细胞内蛋白质转运及信号转导等）、Zm00001d039090（参与蛋白质进入细胞核）、Zm00001d039142（与光合作用相关）和Zm00001d039052（参与碳水化合物的跨膜转运）一共6个基因作为候选基因进行了遗传转化载体的构建，并进行转基因验证。

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