玉米隐花色素基因CRY1a的克隆及功能分析

发布时间：2018-05-18 07:26

  本文选题：玉米 + 隐花色素　； 参考：《山西大学》2016年硕士论文

【摘要】：玉米(Zea may)不但是重要的粮食作物,同时也是高光效的C4模式植物。隐花色素(cryptochrome,CRY)是高等植物主要的蓝光受体,与光形态建成、开花起始、种子休眠、生物产量等性状的发育密切相关。模式植物拟南芥的隐花色素研究已经较为深入,在单子叶植物中除了对水稻、小麦、高粱、大麦的CRY1a进行研究外,玉米CRY1a一直未被研究。鉴于隐花色素参与调控植物生长发育的重要作用,开展对玉米隐花色素的研究迫在眉睫。本研究利用同源克隆的方法克隆、测序验证得到玉米B73两个ZmCRY1a基因的的编码区cDNA序列;采用实时荧光定量PCR(qRT-PCR)检测ZmCRY1a在不同器官、光质、光转换及长日照、短日照条件下的表达水平;利用相关生物信息学软件分析ZmCRY1as氨基酸序列及蛋白质功能结构域,并对ZmCRY1a和其他植物CRY1a蛋白的同源性进行分析。结果发现,ZmCRY1as与AtCRY1和OsCRY1a蛋白结构类似,包含1个DNA photolyase结构域、1个FAD binding 7结构域、1个Crytochrome C结构域;氨基酸序列同源性分析发现,ZmCRY1as与水稻OsCRY1a同源性最高,与拟南芥、大豆等双子叶植物的CRY1的同源性较低;ZmCRY1as在成株期叶片中表达量最高:ZwCRY1a1、ZmCRY1a2在叶中的表达量分别是其根中的52.1倍、15.3倍;相对于黑暗下,二者在各种持续光质中的表达丰度均较高,尤其蓝光和远红光条件下,分别达到黑暗中ZmCRY1a1表达量的9.5和9.0、6.3和8.3倍;尽管是作为编码蓝光受体的基因,ZmCRY1a1和ZmCRY1a2基因的表达却能强烈地响应远红光和红光刺激,其转录最大峰值分别达到各自黑暗时的4.8和3.9、22.8和14.5倍;同样二者也能响应光周期处理。长日照条件下,ZmCRY1a1与ZmCRY1a2的转录存在差异,表现为ZmCRY1a1的转录在一个光周期内共出现5个峰值,最高峰位于光照12 h,而ZwCRY1a2的转录只有4个峰值,最高峰位于进入黑暗2 h时;短日照条件下,两个ZmCRY1a的表达出现了极其相似的模式,均在进入黑暗后出现两个最高峰,分别位于一个光周期的第18 h和第22 h时,而在光照期间虽然有峰值出现,但峰值较低。同时把ZmCRY1a1和ZmCRY1a2基因构建到玉米过表达载体pBCXUN中,转化到农杆菌菌株GV3101,并侵染拟南芥,收获到T0代种子。得出的结论是:玉米ZmCRY1as与拟南芥AtCRY1a和水稻OsCRY1a具有相似的结构;ZmCRY1a在叶中表达量最高,并且能响应各种光质处理,特别是远红光;ZmCRY1as也能响应长日照和短日照条件处理。
[Abstract]:Zea maya is not only an important food crop, but also a C4 model plant with high light efficiency. Cryptochrome CRY is the main blue light receptor in higher plants, which is closely related to the development of light morphogenesis, flowering initiation, seed dormancy and biological yield. The study of cryptic pigment in Arabidopsis thaliana has been carried out deeply. Except for the study of CRY1a of rice, wheat, sorghum and barley in monocotyledonous plants, maize CRY1a has not been studied. In view of the important role of cryptosin in regulating the growth and development of plants, it is urgent to carry out the research on maize cryptic pigment. In this study, the coding region cDNA sequence of two ZmCRY1a genes of maize B73 was cloned by homologous cloning and sequenced, and ZmCRY1a was detected by real-time fluorescence quantitative PCR qRT-PCRR in different organs, light quality, light conversion and long sunlight. The ZmCRY1as amino acid sequence and protein functional domain were analyzed by bioinformatics software, and the homology of ZmCRY1a and other plant CRY1a proteins was analyzed. The results showed that ZmCRY1as had similar structure to AtCRY1 and OsCRY1a protein, including one DNA photolyase domain, one FAD binding 7 domain and one Crytochrome C domain, and the amino acid sequence homology analysis showed that ZmCRY1as had the highest homology with rice OsCRY1a and had the highest homology with Arabidopsis thaliana. The homology of CRY1 in dicotyledonous plants, such as soybean, was lower. The highest expression of ZmCRY1as in adult leaves was: ZwCRY1a1, ZmCRY1a2 in leaves was 52.1 times or 15.3 times higher than that in roots, and the expression abundance of ZmCRY1as and ZmCRY1a2 in all kinds of persistent light quality was higher than that in dark. Especially in blue light and far red light, the expression of ZmCRY1a1 was 9.5 times and 9.0 times higher than that in dark, and the expression of ZmCRY1a1 and ZmCRY1a2 genes, which encode blue light receptor, could respond strongly to the stimulation of far red light and red light, although the expression of ZmCRY1a1 and ZmCRY1a2 genes as blue light receptor could strongly respond to the stimulation of far red light and red light, respectively. The maximum transcriptional peaks were 4.8 and 3.9 ~ 22.8 and 14.5 times of their dark ones, respectively, and they could also respond to photoperiod processing. The transcription of ZmCRY1a1 was different from that of ZmCRY1a2 under long sunlight, which showed that the transcription of ZmCRY1a1 appeared five peaks in a single photoperiod, the highest peak was at 12 h of light, while the peak of ZwCRY1a2 was only 4, and the highest peak occurred at 2 h into darkness. Under the condition of short sunlight, the expression of ZmCRY1a appeared very similar pattern, both of them appeared two peaks after dark, at the 18th and 22nd hour of one photoperiod, but the peak appeared in the period of illumination. But the peak value is lower. At the same time, ZmCRY1a1 and ZmCRY1a2 genes were constructed into maize overexpression vector pBCXUN, transformed into Agrobacterium tumefaciens GV3101, infected Arabidopsis thaliana and harvested T0 seeds. It is concluded that maize ZmCRY1as has similar structure to Arabidopsis thaliana AtCRY1a and rice OsCRY1a. ZmCRY1a has the highest expression in leaves and can respond to all kinds of light quality treatments, especially to long sunlight and short sunlight.
【学位授予单位】：山西大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：Q943.2

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