甘蓝型油菜硼高效主效QTL qBEC-A3a 基因的克隆与功能研究

Crop yield decrease due to soil boron (B) deficiency is a major agricultural problem in many regions in China. Brassica napus is extremely sensitive to B deficiency, but significant genotypic variations were observed among a wide range of Brassica napus cultivars in response to B deficiency. Based on the mapping of a main-effect B efficiency QTL qBEC-A3a in Brassica napus and the development of near-isogenic lines (NILs) of the QTL, the aim of this project is to cloning the qBEC-A3a gene and characterize its biological functions by using BC4F4 and BC4F4:5 as fine mapping populations. The main researches include: 1) With the fine mapping population and the molecular markers tightly linking to qBEC-A3a, the substitution lines and co-segregation molecular markers happened in the target chromosome region would be selected for fine mapping the qBEC-A3a. And then the candidate genes would be obtained by combining the fine mapping with the gene expression profile analysis from the NILs and their parents responding to B deficiency. 2) To isolate the full-length sequence of the candidate genes and then construct the vectors with over- or RNAi expressions, which will be used to transform Brassica napus, Arabidopsis ecotype Col-0 and mutants of bor1, nip5;1, nip6;1 for conducting the complementary experiments of gene functions. The transgenic plants will be analyzed in gene expression, uptake, transport and utilization of B. 3) to analyze the promoter sequence of the candidate genes and characterize its expressions in tissue, cellular and subcellular levels in both Brassica napus and Arabidopsis. The study will elucidate the underlying molecular mechanism of B efficiency in Brassica napus, and provide B-efficient genes and molecular markers for genetic improvement of B efficiency in Brassica napus, as well as other crops sensitive to B deficiency.

土壤缺硼导致作物减产是我国农业生产中存在的普遍问题。甘蓝型油菜对缺硼敏感，然而不同品种对缺硼的抗性有极显著的基因型差异。本研究在前期定位硼高效主效QTL qBEC-A3a及其近等基因系（NILs）的基础上，利用BC4F4和BC4F4:5家系，克隆qBEC-A3a基因，揭示其硼高效的生物学功能和分子调控机制。主要研究内容为：1）利用上述精细定位群体和与qBEC-A3a紧密连锁的分子标记，在目标区段内寻找交换单株，精细定位qBEC-A3a，同时结合NILs及其亲本的基因表达谱，确定候选基因。2）分离候选基因的全长序列，构建超表达和RNAi载体，进行候选基因的功能互补验证，分析候选基因在转基因株系中的表达、硼的吸收、转运和利用等；3）开展候选基因启动子活性分析、组织和细胞定位等研究。研究结果揭示油菜硼高效的分子机理、为作物硼营养的遗传改良提供硼高效基因和分子标记，有重要的理论和实践意义。

硼是植物必需的微量营养元素。我国耕地土壤缺硼面积大，缺硼是双子叶农作物高产和优质的限制因子之一。甘蓝型油菜需硼多，对缺硼敏感，但不同品种对缺硼的抗性存在极显著的基因型差异。克隆硼高效基因，揭示其抗缺硼的分子调控机理，为作物硼营养性状的遗传改良提供硼高效基因和分子标记，有重要的理论和实践意义。本研究在前期研究的基础上，开展甘蓝型油菜硼高效基因的图位克隆及其生物学功能的分子机制研究，其研究内容主要包括三个方面：1）硼高效主效QTL qBEC-A3a的精细定位与克隆，2）近等基因系对缺硼的反应及其基因表达谱，3）硼高效基因的生物学功能与分子调控机制。研究获得的重要结果包括：1）利用目标QTL的高世代回交群体进行精细定位及其硼高效近等基因系的基因表达谱，成功克隆硼高效候选基因BnaA3.NIP5;1。2）利用候选基因的转基因互补试验和爪蟾卵母细胞Oocytes的异源表达、LA-ICP-MS原位分析硼吸收累积、启动子驱动报告基因GUS/GFP的组织、细胞和亚细胞定位等技术，研究确定BnaA3.NIP5;1基因在高效品种中的表达显著高于低效品种，其编码的BnaA3.NIP5;1蛋白定位在根尖侧根冠细胞靠土壤侧的质膜上，介导硼从土壤向胞内的跨膜吸收，进而促进根系和地上部的生长发育。3）研究发现BnaA3.NIP5;1基因5’UTR区上的CTTTC串联数调控其在高效和低效品种中的差异表达，而应答缺硼上升表达则受上游启动子序列的顺式元件W-box与转录因子BnaA9.WRKY47的结合所激活。4）利用同源序列法克隆硼转运基因BnaC4.BOR1;1c，研究发现在缺硼条件下BnaC4.BOR1;1c在高效品种中的表达显著高于低效品种，它在根中柱、地上部节和花蕾基部等特异表达，促进硼从根向地上部的转运及其向幼嫩组织和花器官的优先分配。相关研究发表论文9篇，其中SCI论文8篇（IF >5的4篇），申请发明专利1项，油菜硼高效近等基因系已转让油菜育种家开展硼高效品种的选育。毕业博士4名，在读博士2名。

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