芥菜型油菜多室基因Bjmc1的克隆与功能分析

Multiloculus has been proved to be positively correlated with yield of rapeseed. J163-4 in Brassica juncea shows phenotypic specificity and stability of mutilocular trait, and therefore it is an ideal material for the dissection of molecular mechanism of plant multilocuar charactersitic, and has the value in rapeseed breeding.It has been demonstrated that the multiloculus of J163-4 is controlled by two independent recessive nuclear genes, Bjmc1 and Bjmc2, without cytoplasmic effects. This two nuclear genes have been separated by the development of molecular markers for Bjmc1, and the construction of several backcross populations. By sequencing the BAC clone containing the gene Bjmc1, we obtained the molecular marker SC40 co-segregated with the target gene, and resolved the Bjmc1 to a 80-kb region　located on chromosome B07.In the 80-kb region, there were 21 orfs predicted. In addition, we had sequenced the transscriptomes of the biolcular and mutilocular NIL lines in the BC5 population.. Based on the above results, this study propose to clone the Bjmc1 gene, construct genetic complementation test and RNAi vectors for genetic tranformations and to verify the function of this gene and explore the metabolic net work of the target gene by building larger NIL populations containing the gene Bjmc1, analyzing the BAC clone, chromosome walking, gene prediction, comparative sequencing, expression analysis etc. This study will also clarify the temporal and spatial expression patterns and the protein subcellular localization of the target gene Bjmc1, investigate the cytologic fundament of multilocular morphogenesis and formation. All these will reveal the molecular mechanism of multilocular silique formation, would be helpful to provide evidences for genetic improvement of rapeseed yield trait(s) and to enrich and expand theories about plant flower development.

多室性状是业已证明有利于油菜高产的相关性状。芥菜型油菜J163-4多室受两对隐性核基因控制，其独特、一致稳定的表型说明它是研究植物多室性状分子机制的理想材料。前期研究已将两对基因进行了区分，通过含Bjmc1基因的BAC克隆测序，获得与该基因共分离标记，并将其锁定在B07上80kb区域内，该区段含有21个基因。还对BC5群体多室与两室近等基因系转录组进行了测序。在此基础上，本研究拟通过近等基因系大群体、染色体步移、基因预测、比较测序、表达分析等精细定位并图位克隆Bjmc1基因，构建遗传互补测验载体及RNAi载体进行遗传转化以验证其功能；分析目标基因的时空表达特点及表达产物亚细胞定位；明确目标基因相关的代谢调控网络；研究多室表型形态发生与形成的细胞学基础，从而解析油菜多室角果形成的分子机制，为油菜产量性状的遗传改良提供依据，并丰富、发展植物花发育理论。

在前期研究基础上，我们对Bjmc1基因进行了精细定位，最终将其锁定在分子标记C14MC14和SC151之间的1.14cM范围，通过与目标基因共分离的标记SC40开发及其附近区域的分析，确定拟南芥基因CLV1的同源基因为BjMc1的候选基因。比较测序结果表明，在芥菜型油菜中存在两个CLV1基因的同源基因（BjCLV1a和BjCLV1b）。BjCLV1a的DNA序列在两室和三室材料间没有差别，而BjCLV1b基因在三室油菜中的第一个外显子区域有一个4961bp的Copia-LTR逆转录酶转座子（RTE1）插入，打断了BjCLV1b基因的转录。遗传转化实验进一步确定BjCLV1b基因即是芥菜型油菜两室基因BjMc1。RACE实验结果表明，两室基因BjMc1的cDNA长度为3155bp，而三室基因Bjmc1的cDNA长度为2583bp。BjMc1基因编码一个包含987个氨基酸的蛋白质，包括胞外区域和胞内区域，而Bjmc1基因编码的蛋白质由于Copia-LTR RTE1的插入，在Gly782和Glu783之间被打断，形成仅含有胞外区域的782个氨基酸的蛋白质。这说明Bjmc1基因编码的是一个截短了的蛋白质，该截短的蛋白质上不存在突变位点，暗示Bjmc1基因可能具有显性负性效应。qPCR结果表明，在花序发育早期，BjMc1基因的表达量最高；GUS染色结果表明，BjMc1基因在花蕾原基中的表达量最高，而在茎顶端分生组织、根、茎、子叶和莲座叶的维管束中也有一定程度的表达。亚细胞定位结果显示，BjMc1基因表达产物定位在细胞膜上。Mc1的同源基因在陆地植物中广泛存在，但其结构保守。此外，我们还利用RNAi技术验证Bjmc1基因的功能，对Bjmc1基因参与的调控网络、转录组以及多室表型形成的细胞学进行了分析，对控制多室性状的另一个基因Bjmc2进行了精细定位，对Micro RNA影响油菜角果发育亦进行了探讨。这些结果的获得对阐释油菜多室性状的形成机制与油菜遗传改良的应用具有一定的指导价值。