【作者】 吴连成；

【导师】 陈彦惠；

【作者基本信息】 河南农业大学 ， 作物遗传育种， 2008， 博士

【摘要】 热带、亚热带玉米种质具有丰富的遗传变异,是拓宽我国玉米种质遗传基础,提升育种水平的有效途径。然而,玉米的光周期反应是利用热带、亚热带种质的主要障碍。本研究以热带玉米自交系CML288和温带自交系黄早4为材料,采用长短日照相互挪移和激光扫描共聚焦显微镜观察等方法,研究了玉米光周期敏感时期及其对生长发育的影响,并在此基础上采用同源克隆等方法成功克隆与光周期反应和花发育相关的9个基因,分析这些基因在不同光周期条件下的表达规律,初步探讨了光周期敏感性和开花转换的分子机理。主要研究结果如下:1.将玉米的顶端分生组织形态、叶片数和生育期指标相结合,提出了研究玉米光周期敏感时期的方法,将玉米雄穗的光周期敏感时期分为光周期成花诱导期和早期花发育的敏感期。结果显示,在短日照条件下,热带自交系CML288的光周期成花诱导期和早期花发育的敏感期分别在第4～7片展开叶和第6～9片展开叶之间;温带自交系黄早4雄穗的光周期成花诱导期和早期花发育的敏感期分别在第4～6片展开叶和第6～8片展开叶之间。在长日照条件下,CML288雄穗的光周期诱导期和早期花发育的敏感期分别在4～14片展开叶和第14～22片展开叶之间;黄早4雄穗的光周期诱导期和早期花发育的敏感期分别在第4～8片展开叶和第8～11片展开叶之间。2.将玉米的叶片数、吐丝期和ASI值相结合,提出了研究玉米雌穗光周期敏感期的方法,确定了玉米雌穗的光周期敏感期。结果显示,在短日照条件下,黄早4和CML288雌穗的光周期敏感期分别在第5片展开叶展开的第2天到第9片叶展开的第3天,第6片展开叶展开的第2天到第10片叶展开的第3天;在长日照条件下,黄早4和CML288雌穗的光周期敏感期分别在第8～12片展开叶和第14～18片展开叶之间。3.光周期不仅影响玉米的成花诱导,同时还影响玉米雌雄穗的发育。两个自交系长日照条件的雄穗长度及雄穗分枝数要明显长于或多于短日照条件。在雄穗早期发育的光周期敏感期,长短日照相互挪移会造成雄穗分枝数和雄穗长度发生变化,而其它时期不会发生变化。热带自交系CML288从长日照向短日照挪移,第14片叶到19片叶之间的雌穗发育,表现出从发育完全正常到不发育的一个渐变过程,19片叶后雌穗不能发育。反之,从短日照移到长日照,第8片叶到12片叶之间的雌穗发育,表现出从不发育到发育完全正常的渐变过程,8片叶之前挪移雌穗也不能发育。4.采用同源克隆和RACE结合的方法,从两个自交系中分别克隆出了ZmGI、ZmHd1、ZmFT1(ZCN8)、ZmFT2(ZCN18)、ZmTFL1(ZCN1)、ZmTFL3(ZCN3)、ZmFD(ZmDLF1)、ZmLFY(ZFL)、ZmAP1(ZAP1)等9个基因的cDNA和gDNA序列。序列分析结果显示,在短日照和长日照植物中,光周期调控开花和花发育的遗传途径具有保守性,遗传途径的各个组成基因及其同源物都具有本基因的功能域,相互间的cDNA序列具有较高的同源性。但这9个基因的保守性还存在差别,并且单双子叶植物之内的差别小于单双子叶植物之间的差别。ZmGI基因的cDNA与水稻的OsGI、拟南芥的GI等基因cDNA的同源性分别为83%、63%:ZmGI蛋白的氨基酸序列与水稻、拟南芥的同源性分别达91%、69%。ZmHd1(CO)基因的氨基酸序列与单子叶植物水稻、双子叶植物拟南芥对应的氨基酸序列同源性分别达到75%、46%。ZCN1和ZCN3(玉米FT/TFL1家族成员)与水稻的RCN1、RCN3和CEN-like基因的同源性分别达到94%、92%和84%;与拟南芥(Arabidopsis thaliana)TFL1家族基因的TFL1、BFT和ACT的同源性分别达到73%、71%和65%。ZmFD(ZmDLF1)与水稻的Os-DLF1同源性达42%,与拟南芥FD的同源性达25%。ZmLFY2与水稻的RFL同源性达81%,与拟南芥的LFY基因的同源性达52%。ZmAP1氨基酸与水稻的OsMADS15同源性达87%。ZmM4、ZmM15和水稻的OsMADS14的同源性达76%,与拟南芥的FUL同源性达56%,与拟南芥的AP1、CAL的同源性为54%。5.在光周期敏感材料CML288与光周期钝感材料黄早4中,ZmFT2、ZmLFY2和ZmAP的氨基酸存在差别,而ZmTFL1、ZmTFL3、ZmFT1和ZmFD则没有差异。CML288的ZmFT2比黄早4的ZmFT2多7个氨基酸;CML288的ZmLFY2比黄早的多2个氨基酸,他们之间还有3氨基酸的置换;CML288的ZmAP1比黄早的缺2个氨基酸,他们之间还有5氨基酸的置换。此外在距ZmAP1基因的终止密码TAA外14个bp处,光周期敏感材料CML288与光周期钝感材料黄早4相比少了30个bp。6.光周期调控ZmGI、ZmHd1和ZmFT1的表达。在短日条件下,ZmGI、ZmHd1和ZmFT1在叶片中的表达存在着明显的昼夜节律表达,他们的表达峰值分别在光期、暗期和黎明时分。在长日照条件下,ZmGI和ZmHd1的变化不大,但ZmFT1的表达基本上检测不到。对于不同发育时期的叶片中的表达量来说,在短日照条件下,ZmGI、ZmHd1的高量表达时期与黄早4和CML288的光周期成花诱导期的完成相吻合;ZmFT1的高量表达时期与黄早4和CML288的成花诱导和早期花发育的光周期敏感时期相吻合。7.ZmFT1、ZmLFY、ZmAP1和ZmTFL1影响玉米的成花转换。在短日照条件下,ZmFT1的mRNA表达水平在营养生长阶段不断升高,并在开花转换前后达到最大量。在长日照条件下,ZmFT1的表达量下降,并且生育期也相应推迟。在营养生长阶段,ZmLFY在顶端叶分生组织处低量表达,在开花转换时随着ZmFT1高量表达而相继出现高量表达。ZmAP1在ZmFT1和ZmLFY出现高量表达后迅速出现高量表达。而ZmTFL1主要在茎顶端分生组织处,在开花转换前后也出现高量表达。这些结果显示,ZmFT1可能上调ZmLFY和ZmAP1的表达进而促进开花,而ZmTFL1的功能可能是维持花序的生长进而形成叶片或分枝。更多还原

【Abstract】 An efficient way for increasing the maize breeding level in China is to broaden Chinese maize germplasm with tropical and subtropical germplasms which contain abundant hereditary variation.To unveil the mechanisms of floral transition in maize under different photoperiods and overcome the photoperiod sensitivity which severely restricts the use of tropical and subtropical germplasms,this study observes the effects of photoperiod on maize and determines the photoperiod-sensitive phases in maize.We clone some key genes related to maize photoperiod sensitivity and flower development,analyze their function,and studied the effects of photoperiod on maize by gene regulatory network.The main results are as follows:1.Combining the phenotypes of stem apical meristem,leaf number and flowering time,we determine the photoperiod sensitive phase in maize tassel,and divide the photoperiod sensitive phase into photoperiod-sensitive inductive phase and photoperiod-sensitive flower development phase.Under Short-Day（SD） condition, the photoperiod-sensitive inductive phase and photoperiod-sensitive flower development phase of Huangzao 4 are during the expansion of the 4th～6th full leaf and 6th～8th full leaf;the photoperiod-sensitive inductive phase and photoperiod-sensitive flower development phase of CML288 are during the expansion of the 4th～7th full leaf and 7th～9th full leaf.Under Long-Day（LD） condition,the photoperiod-sensitive inductive phase and photoperiod-sensitive flower development phase of Huangzao 4 and CML288 are respectively prolonged one time and five times. The results indicate that CML288 is more sensitive to photoperiod than Huangzao 4.2.Leaf number,days to silking and ASI are used to determine photoperiods ensitivity phase in maize ear development.The results indicate that the ear photoperiod-sensitive phase of Huangzao 4 and CML288 are respectively during the second day of the 5th fully expanded leaf to the third day of the 9th fully expanded leaf and during the second of the 6th fully expanded leaf to the third day of the 10th fully expanded leaf under SD condition.Under LD condition,the time is during 8th～ 12th and 14th～18th 1 fully expanded eaf separately.3.Photoperiod affects the development of the tassel and ear during the photoperiod sensitive phase.The results show that Huangzao 4 and CML288 appear to be more vigorous,the length of tassel and the number of tassel tiller increase under LD.When the plants are transferred reciprocal between long-day（LD）(15hd^-1) and short-day conditions（SD）(9hd^-1) during the photoperiod-sensitive flower development phase,the length of tassel and the number of tassel tiller are affected. The maize ear development of CML288 is severely affected by transfer between long-day（LD）(15hd^-1) and short-day conditions（SD）.Transferred from LD to SD during the period during the 14th fully expanded leaf to the 18th fully expanded leaf, the fecundity of ear is decreased,and the ear does not develop after the 19th fully expanded leaf.Transferred from SD to LD,the ear does not develop before the second day of 8th fully expanded leaf,the fecundity of ear is increased during the period from the 8th fully expanded leaf to the 11th fully expanded,leaf and ear developed normal after the 11th leaf fully expanded.4.ZmGI,ZmHd1（CO）,ZmFT1（ZCNS）,ZmFT2（ZCN18）,ZmTFL1（ZCN1）, ZmTFL3（ZCN3）,ZmFD,ZmLFY and ZAP1 were isolated,or cloned,and analyzed. The result suggest that the component of the genetic network which controls flowering time and floral transition of plant in response to day length are conserved in monocots and dicots or in SD plants and LD plants.But the conservations of different gene in monocots or dicots are diversities.The results of homology analysis with its ortholog of rice and Arabidopsis are that ZmGI is 83%and 63%,ZmHd1 is 75%and 46%,ZmTFL3 is 94%and 73%,ZmFD is 42%and 25%,ZmLFY is 81%and 52%. The homology of amino acid of ZmGI between maize with rice and Arabidopsis is 91%and 69%.The homology of amino acid of ZAP1 between maize with Arabidopsis is 87%.5.The gene sequences are diversities in photoperiod-sensitive maize and photoperiod-insensitive maize.Seven amino acids are absented between the 66^th and the 74^th amino acids of ZmFT2 putative protein in Huangzao 4 compared with ZmFT2 in CML288.Two amino acids are defaulted and three amino acids are changed in putative protein of ZmLFY2 in Huangzao 4 compared with ZmLFY2 in CML288. Two amino acids are defaulted and five amino acids are changed in putative protein of ZmAP1 in CML288 compared with ZmAP1 in Huangzao 4.Moreover,30bp are lost between the 14^th and the 45^th bp outside the stop codon of ZmAP1 in CML288. 6.The results of RT-PCR about maize leaves and stem apical suggest that the expression patterns of ZmGI,ZmHd1 and ZmFT1 are regulated by photoperiod. Under SD conditions,the expression of ZmGI,ZmHd1 and ZmFT1 in maize leaf have diurnal fluctuation,peaking in light,in dark and before dawn respectively.Under LD, the expression of ZmGI and ZmHd1 are upregulated slightly,ZmFT1 is downregulated,while the expression of ZmFT1 in CML288 can not be detected.For the expression patterns of those genes in leaves during different development stages, the high expression periods of ZmGI and ZmHd1 in Huangzao 4 and CML288 are consistent with their photoperiod-sensitive inductive phase,and ZmFT1 is consistent with its photoperiod-sensitive phase.7.ZmFT1,ZmLFY,ZmAP1 and ZmTFL1 regulate the floral transition.FT mRNA levels in leaves during vegetative growth in SD reach its maximum around the floral transition.FT expression is reduced in LD which is consistent with the delay of flowering time.ZmLFY expression is detected in stem apical and reach maximum followed with the maximum expression of ZmFT1.ZmAP1 expression sharply increases after the maximum expression of ZmFT1 and ZmLFY.ZmTFL1 expression is mainly in shoots apical and is upregulated around the floral transition.These results suggest that ZmLFY and ZmAP1 are upregulated by ZmFT1,and then ZmLFY and ZmAP1 together initiate the transition from the vegetative meristem to inflorescence meristem.However,ZmTFL1 maintains the shoot apical meristem in indeterminate stage to develop leaves and branches.ZmFT1 is relative to photoperiod-sensitivity in maize.更多还原