兰花远缘杂交后代高频发生2n雄配子的分子机理

The germplasm resources with high frequent production of 2n gametes are the key to create polyploid orchid resources and further develop polyploid orchid cultivars. But the molecular mechanisms of 2n gametogenesis in orchid is still unclear. Through continuous observation of three years, the resources with stable high frequency producing 2n male gametes were obtained from the combination of Cymbidium sinense × Cymbidium lancifolium, and the cytological mechanism of 2n male gametes was also clarified. In this study, the hybrid offsprings with high and low frequency of 2n male gametes formation are employed to investigate molecular mechanisms of 2n gametogenesis ability. Anthers from two materials are subjected to performing de novo transcriptome assembly and gene expression analysis using Illumina HiSeqTM2000 sequencing technology. Through comparative analysis, the changes of transcription group are clarified, and at the same time, the candidate genes related of 2n male gametogenesis are identified by using of the technologies of RT-PCR and real time PCR. Primers are designed according to the cDNA sequence of candidate gene, and full length DNA sequences are obtained by using of electronic cloning method. The functions of the candidate genes were identified by examining the production of 2n male gametes in pollinias of transgenic plantlet of Cymbidium ensifolium in vitro using genetic transformation experiment of over-expression vector and interference vector, with the method of Agrobacterium-mediated transformation rhizomes.The results will lay the foundation for future investigation on Cymbidium polyploid breeding and evolution, also has the vital significance to the development of Chinese orchid industry.

高频产生2n配子的种质资源是有效创建兰花多倍体资源，开展兰花多倍体育种的关键，但兰花2n配子发生的分子机理仍是空白。本课题组通过连续3年的观察从墨兰与兔耳兰的杂交后代中获得了稳定高频发生2n雄配子的资源，并对其2n雄配子发生的细胞学机理进行了研究。在此基础上，本研究对高、低频发生2n雄配子兰花杂交后代进行转录组测序，通过数据和序列的比较分析，获得兰花高频发生2n雄配子的候选基因；采用RT-PCR和real time PCR技术，分析各候选基因在高、低频发生2n雄配子兰花中的表达，确定目的基因；构建目的基因的过量表达载体和RNA干涉载体，用农杆菌介导法转化建兰根状茎，获得转基因植株；对转基因建兰植株进行试管花诱导，观察花粉块2n雄配子的发生，验证目的基因的功能。这一研究为国兰多倍体育种和多倍体进化研究奠定基础,对国兰产业的发展具有重要的意义。

以金嘴墨兰×兔耳兰的杂交后代株系中高频发生2n雄配子的67-80（JT-H）和低频发生2n雄配子的67-109（JT-L）开花植株为材料，取其不同发育阶段花药混合后进行转录组测序，通过生物信息学分析，获得了兰花高频发生2n雄配子的13个候选基因：SDS、NPK11、NPK12、NPK13、NACK11、NACK12、MAP2K2、MAP2K91、MAP2K92、MAP3K1、MAP3K3、MAP3K7和CDC1；采用qRT-PCR技术，分析了13个候选基因在67-80和67-109不同发育阶段花粉块中的表达差异，明确了与兰花远缘杂交后代高频发生2n雄配子密切相关的目的基因SDS，通过RACE技术获得了兰花SDS的同源基因，命名为CslSDS（登录号：KY873304）；对目的基因进行PCR扩增、测序得到CslSDS基因序列全长2122 bp，含有一个1749 bp的开放阅读框（ORF），编码582个氨基酸，67-80和67-109的SDS编码区和非编码区的总长度为3069 bp，其中含有6个内含子和7个外显子；建立了金嘴墨兰×银针杂交后代14-16-16根状茎的试管花诱导体系；构建了目的基因SDS的过量表达载体，并用农杆菌介导法转化了14-16-16的根状茎，以期获得转基因植株，为进一步验证目的基因的功能奠定基础。这一研究为国兰多倍体育种和多倍体进化研究具有重要的意义。