【作者】 杨爽；

【导师】 陈彦惠；

【作者基本信息】 河南农业大学 ， 作物遗传育种， 2010， 硕士

【摘要】 热带、亚热带玉米种质具有丰富的遗传变异,对其改良并利用可以拓宽中国玉米种质遗传基础、提升育种水平。然而,光周期敏感性严重限制了热带、亚热带种质在温带的利用。因此,分离、克隆并研究玉米光周期敏感相关基因并揭示光周期敏感的遗传特性和内在分子机制,对热带、亚热带玉米种质的有效利用具有重要的理论和实践意义。本研究以光周期敏感自交系CML288和光周期钝感自交系黄早4为材料,利用同源克隆的方法克隆了与拟南芥CO、水稻OsHd1同源的玉米ZmCOL基因;利用实时荧光定量PCR法研究了ZmCOL基因的昼夜表达模式,分析了不同发育时期叶片和茎尖中ZmCOL基因的表达情况及ZmCOL基因与ZmGI和ZmFT基因之间的表达关系;构建了ZmCOL基因全长及保守结构域的35S融合表达载体,对ZmCOL基因全长及保守结构域进行了亚细胞定位研究;构建了ZmCOL基因的过量表达载体,通过农杆菌浸染花序法转化拟南芥,得到了转基因一代植株;通过玉米光周期敏感相关性状,对21份玉米自交系的光周期敏感性进行了初步鉴定,并结合这21份材料中ZmCOL基因的序列变异,分析了ZmCOL基因序列变异与玉米光周期敏感性状的相关性。主要研究结果如下:1.利用同源克隆等方法,成功克隆了玉米类ZmCOL基因。该基因序列含有2个外显子和1个内含子,CDS序列中含有1197/1188bp的开放性阅读框,推测编码一条含有398/395个氨基酸的肽链。系统进化树分析发现,ZmCOL与水稻OsHd1位于同一进化分枝,其亲缘关系最近,同源性达87.10%。蛋白结构预测显示其蛋白含有CO/Hd1基因典型的BBOX和CCT功能域,表明ZmCOL基因是CO/Hd1的同源基因。在光敏感材料CML288和光钝感材料黄早4的ZmCOL基因的ORF中有一连续九个碱基的插入/缺失变异,该变异引起了ZmCOL蛋白序列的差异,推测该变异与玉米自交系的光周期敏感程度相关。2.应用实时荧光定量PCR研究了ZmCOL基因在CML288和黄早4长短日照两种处理条件下的昼夜节律表达规律及其在不同部位、不同的发育阶段的表达规律,分析了ZmCOL基因与拟南芥GI和FT的在玉米中的同源基因ZmGI、ZmFT的表达关系。结果表明:在长短日照处理条件下,与拟南芥CO和水稻OsHd1一样,两种玉米自交系中ZmCOL基因表达均表现出昼夜节律性。ZmCOL基因在黄早4和CML288的叶片和茎尖中均有表达,但在玉米的不同发育时期,ZmCOL基因的表达量呈现出高低不同的动态变化,其在茎尖和叶片中的表达高峰时期与玉米光周期诱导敏感期一致,表明ZmCOL基因可能在玉米的光周期诱导敏感期发挥作用,在该时期ZmCOL基因在不同敏感程度的自交系中的主要表达部位有所不同,在光钝感材料中ZmCOL基因在叶片的表达量较高,而在光敏感材料中ZmCOL基因在茎尖的表达量较高,但ZmCOL基因在叶片中的表达量最终决定了玉米自交系材料能否正常完成光周期诱导敏感期的光周期诱导。基因间的表达关系研究表明,ZmGI基因位于ZmCOL基因的上游,调控ZmCOL基因的昼夜表达节律;ZmCOL基因位于ZmFT的上游,在玉米发育的前期及光周期诱导敏感期发挥作用;而下游基因ZmFT,在玉米发育的后期即花发育敏感时期起调控作用。3.成功构建了玉米ZmCOL基因的35S融合表达载体并通过基因枪轰击,使其在洋葱表皮瞬时表达,结果表明,ZmCOL是一个核定位蛋白,其保守结构域CCT是该基因唯一的一个核定位信号,具有核定位功能;构建了过量表达载体3301-221-ZmCOL,且将该基因转入野生型拟南芥,对阳性候选转基因的PCR鉴定表明,已经成功将该基因转入拟南芥。4.通过分析21份不同的玉米自交系的光周期敏感性及表型性状与ZmCOL基因序列变异的相关性,结果表明,不同种质类群的玉米自交系光周期敏感程度不同,其中热带种质的自交系光周期敏感性最强;表型性状的动态聚类将不同玉米自交系光周期敏感程度划分为Ⅰ级、Ⅱ级、Ⅲ级和Ⅳ级四个等级,随着等级的升高,其含有的玉米自交系的光周期敏感性越强;ZmCOL基因序列变异位点2+(523-531)与光周期敏感相关性状密切相关,与抽雄、散粉、吐丝、株高、穗位及叶片数的相关性均达显著或极显著水平;而其它位点的变异与光周期敏感性状之间的相关性不显著。更多还原

【Abstract】 Tropical and subtropical maize germplasm contain abundant hereditary variation, which can broaden the genetic base of Chinese maize germplasm to enhance the breeding level by improving and using of these germplasm. However, photoperiod sensitivity severely limits the use of tropical and subtropical germplasm in temperate zone. In the result, isolating, cloning and analyzing the function of photoperiod-sensitive genes is an important way to research the molecular mechanism of maize’s photoperiod-sensitivity.We cloned the ZmCOL gene by the method of orthologus gene cloning in a photoperiod-sensitive tropical maize (Zea mays L.) inbred line CML288 and a photoperiod-insensitive temperate inbred line HuangZao4, which was the cognate of CO gene(Arabidopsis) and OsHd1 gene(rice). Then we studied the circadian rhythm expression pattern of ZmCOL, analyzed the expression pattern of ZmCOL in shoot apical meristems (SAM) and leaves, and researched the relationship among ZmCOL, ZmGI, and ZmFT, in order to investigate the function and mechanism of ZmCOL during the photoperiod induction. We constructed several 35S fusions between GUS and ZmCOL or conserved domain of ZmCOL to test whether ZmCOL L protein is localized to the nucleus, and which domain of ZmCOL is sufficient to localize GUS to the nucleus. Then we constructed the over expression vectors of ZmCOL, transformed it into the Arabidopsis with Floral Dip method, and obtained several transformants. We identified the photoperiod sensitivity of 21 maize inbred lines by photoperiod-sensitive related traits of maize, and investigated the relationship between sequent variations of ZmCOLs and photoperiod-sensitive related traits. The main results are as follows:1. We had cloned the maize ZmCOL gene through orthologus gene cloning. ZmCOL gene sequence has two exons and one introns, and contained a predicated 1197/1188bp open reading frame in the CDS sequence, which could encode a peptide with 398/395 amino acids. By phylogenetic analysis, we found that the ZmCOL and the rice OsHd1 are in the same evolutionary branch with the protein sequence sharing 87.10% homology to rice OsHd1. The predicated protein that especially containing CO/Hd1 gene family is composed of a zinc finger B-box and a CCT domain. That is to say, ZmCOL gene belonged to the member of CO/Hd1 gene family. There is a continuous nine base insertion / deletion mutation variation in the ORF of ZmCOL between photoperiod-sensitive tropical maize inbred line CML288 and photoperiod-insensitive temperate inbred line Haungzao4, and it caused three amino acids differences of ZmCOL protein sequence, suggesting that maybe the variation is closely related to photoperiod sensitivity.2. We used fluorescence quantitative RT-PCR to research the circadian rhythm expression pattern of ZmCOL under long day and short day conditions in CML288 and HuangZao4, analysesed the expression pattern of ZmCOL in shoot apical meristems (SAM) and leaves, and researched the relationship among ZmCOL, ZmGI, and ZmFT. The results showed that similar to it’s Arabidopsis and rice homologs(CO and OsHd1), ZmCOL gene exhibited diurnal expression patterns while ZmCOL gene expressed both in SAM and leaf under long day and short day conditions in CML288 and HuangZao4; in different development stages of maize, however, the expression of ZmCOL genes showed different levels or changes. And the peak period of ZmCOL gene expression in SAM and leaves were notably similar to the phase of induction photoperiod-sensitivity, indicating that ZmCOL gene may be joined the regulation of photoperiod, and functional importance in the phase of induction photoperiod-sensitive. And in this period, the main expression parts of ZmCOL is different in different inbred lines: in the photoperiod-insensitive material ZmCOL gene expression higher in leaves, in the photoperiod-sensitive material ZmCOL expression higher in SAM. But the expression level of ZmCOL gene in leaves finally decide whether the maize inbred lines can complete the photoperiod-induction in the phase of induction photoperiod-sensitivity. Analysis by the expression of ZmGI, ZmCOL and ZmFT showed that, ZmGI gene located the upstream of ZmCOL gene, regulating the circadian rhythms expression of ZmCOL gene; ZmCOL gene located the upstream of ZmFT gene, and ZmCOL gene played an important role in the phase of induction photoperiod-sensitivity, while the downstream gene ZmFT functional were of importance in the later phase of corn growth, important to the floral development.3. We had constructed several 35S fusions between GUS and ZmCOL or conserved domain of ZmCOL, a transient expression assay in onion bulb epidermal cells by gene gun bombardment, the result showed that: ZmCOL can localize GUS to the nucleus of the cells, suggesting that CO is a nuclear protein, and the CCT domain is an NLS of ZmCOL, it is sufficient to target GUS to the nucleus. And we constructed the over expression vectors of ZmCOL, transforming it into the Arabidopsis with Floral Dip method. The result of PCR showed that we have got several transformant plants.4. We analyzed the photoperiod-sensitivity related phenotypic traits of 21 maize inbred lines and studied the relationship between sequent variations of ZmCOLs and photoperiod-sensitivity related traits, the results showed that: different maize germplasm groups have different photoperiod sensitivity, specifically, inbred lines of tropical germplasm have the strongest photoperiod sensitivity. Dynamic cluster of phenotypic characteristics divided different maize inbred lines into different sensitive grades:Ⅰ,Ⅱ,ⅢandⅣ. For maize inbred lines, the higher of the level, the stronger of the photoperiod-sensitivity. The analysis of sequence indicated that only the sequence variation of 2+(523-531)was significantly associated with the photoperiod-sensitivity related traits, however, the relationship between variation among the other six sequence variations and photoperiod-sensitive related traits were not significant.更多还原