大白菜BrTNL3基因的异常表达与CMS7311雄性不育发生的关系解析

Cytoplasmic male sterility (CMS) not only show great potential in protecting the hybrid seed and new varieties, but also provide a valuable model for discovering the interaction between nucleus and cytoplasm. So far, the molecular mechanism related to the formation of male sterility in CMS was still unknown. In the previous research when we identified genes that differentially expressed during flower bud development between cytoplasmic male-sterile plant and male-fertile plant in heading Chinese cabbage (Brassica rapa L. ssp. pekinensis), we found that the expression of TIR-NBS-LRR 3 (BrTNL3) was not detected in all samples of male-fertile buds, but showed a significantly rising tendency during male-sterile bud development, which suggested over-expression of the BcTNL3 gene could lead to the male sterility in CMS7311. In this study, we plan to clone the BrTNL3 gene from CMS7311; Then, detect the fertility modification of the CMS7311 by down-regulated its expression in heading Chinese cabbage by RNAi method. Meanwhile, we also want to clarify the effect of the over-expressing BrTNL3 gene on plant morphogenesis in Arabidopsis. Our aim was to reveal therelationship between the abnormal expressed BrTNL3 gene and the formation of male sterility in CMS7311 of heading Chinese cabbage.

植物细胞质雄性不育系不仅在杂交制种及新品种保护方面具有巨大的应用潜力，也为揭示细胞核和细胞质间相互作用提供一个很有价值的研究模型，但迄今为止有关植物细胞质雄性不育发生的分子机制仍不清楚。课题申请者之前在筛选鉴定大白菜CMS7311不育发生相关基因时发现TIR-NBS-LRR 3（BrTNL3）基因只在雄性不育株花蕾中表达，且其表达量随着雄性不育的发生呈现快速上升趋势，推测该基因的异常表达可能与大白菜CMS7311雄性不育的发生相关。本研究计划克隆BrTNL3基因全长，并构建植物RNAi载体进行大白菜CMS7311的遗传转化，研究该基因下调表达所导致的大白菜CMS7311育性变化；同时借助拟南芥过表达阐明BrTNL3基因的异常表达对植株形态发生的影响。通过上述正、反义转基因研究揭示大白菜BrTNL3的异常表达与CMS7311雄性不育发生的关系，具有一定的理论研究价值。

pol型细胞质雄性不育（CMS）是已经在十字花科作物中广泛应用的不育源，但是其雄性不育发生的分子机制仍不清楚。已有研究表明植物NBS-LRR结构类基因除了参与抗病信号转导之外，在植物对逆境适应中也起重要作用，其过量表达可导致多种多样的形态学缺陷，甚至死亡现象。申请者前期筛选鉴定大白菜CMS7311不育发生相关差异表达基因时发现BrTNL3只在雄性不育株花蕾中表达，且其表达量随着雄性不育的发生呈现出快速上升趋势，推测该基因异常表达可能与大白菜CMS7311雄性不育的发生相关。本项目利用CRISPR/Cas9技术构建植物表达载体pBSE401-hSpCas9-Bol022784，敲除油菜体内BrTNL3同源基因Bol022784，获得转基因T1代单株，测序分析表明靶位点多发生单碱基突变，暂未发现显著表型变化。由于BrTNL3全长过长，且体内表达量极低，项目组试图通过RACE、文库筛选、重叠PCR等技术克隆基因全长，目前仅获得含有BrTNL3预测全长的基因组片段6151bp，并完成植物过表达载体的构建，正在进行拟南芥遗传转化。植物富含甘氨酸蛋白可促进花粉发育，其基因表达异常时造成雄蕊异常，申请者前期也发现一个BrGRP17基因在不育株花蕾中上调表达。本项目通过SMART结合DSN技术构建大白菜pol型雄性不育花蕾均一化全长cDNA文库之后，进一步克隆白菜BrGRP基因完整ORF（Bra004066），构建表达载体pCambia2301-35s-BrGRP，进行拟南芥和油菜遗传转化，最终获得拟南芥转基因T3代纯合株系和油菜T2代，表型均无显著变异，激素敏感性及盐胁迫等处理表明：转BrGRP基因拟南芥对ABA和NaCl比较敏感。此外，本项目还进行了白菜花发育数字化表达谱分析及BrLFY基因表达定量分析等研究工作。以上研究为深入探讨大白菜pol型雄性不育发生的分子机制提供一定的理论支持。