结球甘蓝花粉管类钙调蛋白编码基因的发掘及功能鉴定

Abstract：Ca2+ is an element that is crucial for pollen germination(PG) and pollen tube elongation(PTG) in plants, as one of Ca2+ sensor protein, the physiological function of calmodulin-like (CMLs) in calcium signaling system still need to clarify furtherly. According to the international current researches situation and the development of dynamic analysis:There is no SCR that can recognized specifically with stigma in cross-pollen of Brassica oleracea, while it may contain long half-life developmental stage of pre-existing calcium-sensor proteins, the processes of transporting to functional parts are induced precisionly by calcium ion, causing pollen germination, and then causing types of self-compatibility（SC）or self-incompatibility(SI) responses. In this study,therefore,we analyse the transcription sequencing under the condition of pollen germination and pollen tube growth of Brassica oleracea, screening CMLs genes expression changes before and after pollen germination;construct CMLs::GUS expression vectors under the drive of CMLs promtors，and make sure their expression characteristics;Moreover,we construct RNAi and overexpression expression vectors, respectively,transform cabbage young leaves by bombardment methods, and analyze the functions of CMLs gene we get in pollen germination and pollen tube growth, then identify the interactions between CMLs and proteins-related of pollen germination. Through this study,we hope to get the CMLs in process of pollen tube growth, and could elaborate the process of calcium signal regulation system furtherly, then provide important contents that are innovation for the process of the further research of calcium signaling molecular mechanism in pollen tube growth and new references to the research of plant pollination mechanism.

Ca2+对植物的花粉萌发和花粉管伸长起着重要的作用，作为Ca2+感受器之一的类钙调蛋白（CMLs）在钙信号系统中的花粉萌发和花粉管伸长过程的功能还需进一步阐明。因此，本研究拟从甘蓝花粉萌发和花粉管伸长条件下的转录测序分析，筛选得到花粉萌发前后表达变化的CMLs基因；构建CMLs启动子启动下的CMLs：：GUS表达载体，确定CMLs的表达特性；构建RNAi干扰载体及过表达载体，转化甘蓝，对CMLs基因在花粉萌发和花粉管伸长中的功能进行鉴定；并探索与花粉萌发相关蛋白的相互作用。通过本项目期望得到花粉管伸长中的CMLs编码基因，为甘蓝花粉管伸长过程的钙信号传导分子机理的深入研究提供创新性的具有重要科学价值的内容，能够进一步阐述该过程中的钙信号调控网络系统，为植物受粉机理的研究提供新的参考。

Ca2+对植物的花粉萌发和花粉管伸长起着重要的作用，作为Ca2+感受器之一的类钙调蛋白（CMLs）在钙信号系统中的花粉萌发和花粉管伸长过程的功能还需进一步阐明。本研究主要进展如下：.（1）在甘蓝盛花期去雄后人工授粉，收集不同授粉时间的柱头，苯胺蓝染色后荧光显微镜观测花粉原位萌发情况。（2）对授粉0h、4h、12、24h的柱头通过转录组测序分析，发掘花粉萌发中表达显著差异的5个基因CML39、CM41、CML48、CML49、CML50。并根据转录组结果，对差异表达的基因进行qPCR鉴定。（3）通过同源扩增或RACE等方法得到CML39、CM41、CML48、CML49、CML50基因序列。对所得到的CMLs基因及编码蛋白进行生物信息学分析。（4）构建CML48、CML49、CML50原核表达载体，检测原核表达产物CMLs分别在SDS-PAGE的电泳迁移率变化。（5）扩增得到CML48、CML49、CML50上游的启动子序列，并构建表达载体CMLs::GUS，转化根癌农杆菌。（6）对甘蓝幼苗分别进行干旱、盐胁迫、高温、低温、H2O2、ABA、SA、MeJA处理，qPCR检测茎尖和根中CML39、CML48和CML50基因的表达情况。（7）Crispr/cas9基因编辑转化甘蓝植株（8）根据对甘蓝花粉萌发转录组测序的分析及查阅献，除上述的5个CMLs外，另外克隆7个花粉萌发及发育相关基因：CML13、CML15、CML20、CM21、CML24、CML25、CML42。（9）从甘蓝扩增得到花粉萌发相关基因NPG1、NPGR1及NPGR2，对3个基因进行生物信息学分析。同时对3个基因在花粉萌发0h、2h、4h、12、24h时进行qPCR表达分析。（10）酵母双杂交实验表明CML15、CML20、CML39、CML41、CML48、CML49均能与NPG1、NPGR1及NPGR2在体外发生相互作用，其他6个CMLs均不能与NPG1、NPGR1及NPGR2在体外发生相互作用。通过β–半乳糖苷酶活性得到不同CMLs与NPG1、NPGR1及NPGR2间的相互作用强度。（11）对6个CMLs的EF-Hand结构域定点突变，各突变体均不能与NPG1、NPGR1及NPGR2蛋白发生相互作用。通过本项目得到花粉管伸长中的CMLs编码基因，探索甘蓝花粉管伸长过程的钙信号传导分子机理。