蓖麻种子低温萌发应答基因RcMsc2的功能及表达调控研究

Low temperature in spring, which is most important environmental factor, leads to slow germination of castor seeds in Tongliao city in the Inner Mongolia autonomous region. It is of great theoretical and practical significance to explore the molecular mechanism of seed germination under low temperature and improve the germination ability of castor seeds to low temperature. Our previous transcriptome sequencing analysis showed that the transcript levels of the RcMsc2 gene were significantly up-regulated in castor cotyledon at low temperature (15℃). The RcMsc2 gene is a cell cycle protein gene, and participate in the regulation of cell cycle. It was speculated that the RcMsc2 gene was likely to be the key candidate gene for controlling castor seed germination under low temperature. This study is intended to clone the RcMsc2 gene by RT-PCR technology and study its expression pattern, promoter activity, and subcellular localization of product. The function of RcMsc2 gene was identified by means of over-expression and RNAi technique, including seed cold germination test, physiological index determination, and lipid content determination. To reveal the mechanism of RcMsc2 gene regulating the germination of castor seeds at low temperature, endogenous gene expression of transgenic plantsalso was analyzed. SNP markers were used to analyze the relationship between the polymorphism of the gene and the low temperature germinating ability of castor varieties. This study will provide a theoretical basis for breeding and cultivation of castor resistance to low temperature germination.

内蒙古通辽地区春播蓖麻种子受低温影响萌发缓慢。探索种子低温下萌发的分子机制，提高蓖麻种子耐低温萌发能力，对于提高蓖麻产量和经济效益具有重要理论与实践意义。前期转录组测序分析表明，RcMsc2基因在低温（15℃）萌发的蓖麻子叶中表达量显著上调，该基因编码细胞周期蛋白，参与细胞周期的调控，推测该基因极有可能是控制蓖麻低温萌发的关键候选基因。本研究拟采用RT-PCR技术克隆RcMsc2基因，研究其表达模式、启动子活性、产物亚细胞定位等特征特性；通过过表达和RNAi技术，综合转基因植株的种子低温萌发试验、抗逆生理指标测定、脂类含量测定和内源基因表达分析手段，鉴定RcMsc2基因功能，揭示RcMsc2基因调控蓖麻种子在低温下萌发的机制；对蓖麻材料进行SNP标记，分析该基因多态性与蓖麻品种低温萌发能力的相关性。该研究将为蓖麻抗低温萌发育种和栽培提供理论依据。

在通辽地区，蓖麻播种期的温度不能满足萌发的需要，导致萌发缓慢，有效生育时期缩短，二级分枝的果穗成熟度不够，产量受到明显影响。RcMsc2是细胞周期蛋白编码基因，前期转录组测序分析表明，该基因在低温（15℃）萌发的蓖麻子叶中表达量显著上调，可能是影响蓖麻低温萌发的关键候选基因，然而其调节机制尚不清楚。. 本项目主要研究了以下内容：. ①RcMsc2克隆、载体构建、生物信息学及荧光定量分析；②RcMsc2基因烟草叶片亚细胞定位；③RcMsc2基因过表达载体的烟草受体转化；④转基因烟草低温胁迫下萌发试验；⑤转基因及非转基因烟草转录组测序分析。. 得到了如下重要结果和关键数据：. ①采用qRT-PCR的方法对RcMsc2基因的相对表达量进行分析，结果发现RcMsc2基因的表达具有组织特异性，并且响应低温、干旱等4种非生物胁迫的诱导，能够在蓖麻非生物胁迫响应中行使相应的功能。. ②RcMsc2是一个具有COG蛋白功能保守区的跨膜蛋白。Msc2-GFP载体亚细胞定位分析发现发光位点主要是细胞质和细胞核。. ③通过农杆菌介导法将RcMsc2基因过表达载体成功转入烟草，并获得36株T2代转基因烟草；转基因烟草和野生型烟草种子低温（15℃）萌发试验表明，转基因烟草种子萌发速率显著高于野生型烟草种子萌发速率（P<0.05）；转录组测序表明，以低温胁迫下的常规烟草为对照，低温胁迫后的转基因烟草有4498个基因发生了显著差异性表达，其中有2274个基因表达上调，有2224个基因表达下调；同时，以非低温胁迫下的转基因烟草为对照，胁迫后的转基因烟草总共有10030个显著差异基因，其中上调基因表达数目为6239个，下调基因表达数目为3791个，说明在低温胁迫情况下转基因烟草更好的响应了低温胁迫；低温胁迫24h后的转基因烟草细胞周期代谢途径及DNA复制信号途径显著受到诱导，说明细胞周期和DNA复制相关基因在转基因烟草耐低温胁迫的进程中发挥了重要作用。.该项目对于解析种子耐低温萌发机制提供了参考数据，具有较强的科学意义。