【作者】 简波；

【导师】 毕玉蓉；

【作者基本信息】 兰州大学 ， 植物学， 2008， 博士

【摘要】 大豆是典型的短日植物,在光周期反应的早期研究中被当作模式植物。与水稻、玉米等短日植物相比,大豆的光周期反应更加敏感,品种间的差异更大,单个品种的适应性狭窄一直阻碍着大豆育种工作的开展。对于光周期反应敏感的晚熟大豆品种米说,短日照不仅是成花诱导的必要条件,而且影响开花、结荚和鼓粒。一旦光周期条件不适,这些品种的生殖生长就会延迟,甚至可从生殖生长状态逆转到营养生长状态。对大豆光周期反应机制的研究将为培育广适应的大豆品种奠定基础。本研究利用RACE技术克隆了四个大豆光周期反应的相关基因,并通过同源比对搜索到七个相关的光周期反应基因,利用本实验窒长期以来建立的短日照(开花)、长日照(持续营养生长)、短日照-长日照(开花逆转)三位一体的实验系统,采用Real-Time PCR技术研究了这11个基因在大豆成花诱导与开花逆转中的表达,并比较了它们在光周期敏感性不同品种中的差异。为了进一步验证与大豆光周期相关的GmSOC1基因的功能,本研究建立了利用发根农杆菌介导的豆科植物百脉根高效快速的转化平台,并将该基因转入百脉根中验证了其功能。具体研究结果如下:1.克隆了四个大豆光周期反应的相关基因GmCOL1、GmCOL2、GmCOL3以及GmSOC1,它们分别编码含311,365,310和211个氨基酸的蛋白。同源比对结果显示,GmCOL1,GmCOL2,GmCOL3与豆科植物苜蓿和豌豆的CO-like基因的同源性最高;GmSOC1与拟南芥AGL20的同源性最高,达到70%,是MADS-box基因家族的一员。2.利用Real-Time PCR技术,通过对10个内标基因的系统分析,我们发现看家基因GmELF1B在大豆光周期反应中较为稳定,可用作大豆光周期相关基因表达研究的内标基因。3.以GmELF1B为内标基因,对克隆的四个大豆光周期相关基因以及通过同源比对筛选到的七个光周期相关的基因在光周期敏感品种ZGDD成花诱导与开花逆转中的表达进行了研究,结果发现,这11个大豆光周期相关基因在其成花诱导过程中的不同光周期条件下均有表达,但是长短日条件下的表达量和表达规律有所不同。它们并非特异地被短日条件诱导,在特定的阶段,长日条件也可以诱导它们的表达。4.所检测的光周期相关基因在光周期敏感性不同的大豆品种的SAM中也均有表达,所不同的是,在光周期钝感品种HH27中这些基因的表达峰值出现的时间普遍比在光周期敏感品种ZGDD中出现的时间早,分子生物学检测结果与HH27在短日条件下开花比ZGDD早的生理现象相符合,但是这些基因在ZGDD中的表达峰值却比HH27中高。5.对于光周期敏感品种ZGDD而言,经一定日数短日诱导后进行长日照处理,可以产生花序逆转和花逆转,甚至整株逆转,植株回复到营养生长的状态。通过研究大豆开花相关基因在ZGDD开花逆转中的表达发现,拟南芥分生组织特异基因LFY和AP1的同源基因GmLFY-like和GmAP1-like在逆转条件下的表达介于短日诱导条件和长日抑制条件之间,说明GmLFY-like和GmAP1-like可能具有维持大豆分生组织特性的功能。而光周期上游基因GmGI-like和PEBP家族基因只在逆转早期与GmLFY-like和GmAP1-like的表达相似,说明它们可能在开花逆转的早期发挥作用。6.与拟南芥中的基因相似,大豆的光周期相关基因也呈现24小时的日变化规律,GmGI-like在长短日条件下的表达与拟南芥在长日下的表达模式相近而与水稻不同;GmCO-like的日变化与水稻Hd1和拟南芥CO均不同,但其在短日条件下的日变化与拟南芥COL1和COL2在长日下的日变化规律相似;大豆PEBP家族基因在短日下的日变化又与水稻短日下的日变化相似,但长日下的日变化却不相同,说明光周划调控大豆相关基因对日长的响应机制可能与长日双子叶植物拟南芥以及短日单子叶植物水稻均不同。7.结合发根农杆菌介导的遗传转化的高效性与百脉根品种"Superroot"简单、快速的再生体系,本研究建立了一个快速、高效的转化平台用于豆科植物基因功能的研究。转基因百脉根植株可在两个半月内获得,分子生物学检测验证了转基因事件的发生。同时,利用双元转化载体pGFPGUSPlus对一些可能影响转化效率的因子进行优化,进而得到92%的高效转化频率。同时,将一个来源于小麦、编码Na~+/H~+逆向转运蛋白的耐盐基因TaNHX2转到百脉根中,并对其耐盐性进行鉴定,从而验证此系统作为基因功能研究平台的实用性。在此基础上,将大豆中克隆的GmSOC1基因转到了百脉根中使其过表达,导致了百脉根提前开花,从而验证了此基因促进开花的功能,同时也说明了此转化平台可以用于逆境生物学与发育生物学的研究。更多还原

【Abstract】 Soybean is a typical short-day plant,being considered as the model plant in the early studies of photoperiodism.Compared with other SD plants,like rice and maize,soybean is more sensitive to photoperiod and the difference among different cultivars is greater than that in rice and maize.The fact that different soybean cultivars can only be planted in limited area due to their sensitivity to photoperiod restricts the extensive cultivation of soybean.For late-maturing soybean,which is sensitive to photoperiod,short-day condition is not only necessary for flower induction,but for blooming, podding and seed maturation.The inapposite photoperiod may result in delayed reproductive phase or even back to vegetative growth.The understanding of photoperiod mechanism involved in soybean may benefit the breeding of extensively adapted soyeban cultivars.In the present study,four photoperiod-related genes were cloned in soybean using RACE,and seven homologues were obtained through BLAST search.Their expression patterns in our exprimental system,which is SD(blooming),LD(persistant vegetative growth),LD-SD(flower reversion),were detected using Real-Time PCR.To further detect the function of GmSOC1,a rapid and highly efficient transforamtion platform was established through Agrobacterium rhizogenes-mediated trnasformation of Lotus corniculatus.Transgenic GmSOC1 L.corniculatus was obtained and its function was investigated. The detailed results were mentioned as follows:1.Four photoperiod-related genes in soybean were cloned,named GmCOL1、GmCOL2、GmCOL3 and GmSOC1,corresponding to proteins of 311,365,310 and 211 amino acids,respectively.The putative amino acid sequence of GmCOL1,GmCOL2 and GmCOL3 showed high similarity with MsCO-like and PsCO-like;the putative amino acid sequence of GmSOC1 showed 70%similarity with AtAGL20,and is a member of MADS-box gene family.2.A suitable internal control makes gene expression results from Real-Time PCR more reliable. We performed a systematic study among ten selected housekeeping genes to validate the most suitable internal control in soybean by Real-time PCR.GmELF1B showed relatively constant expression pattern in the photoperiod response of soybean,and theffore was selected as an internal control in the gene expression studies of photoperiod in soybean.3.The expression patterns of 11 photoperiod-related genes were detected during the flower induction of ZGDD,a photoperiod sensitive cultivar.The results indicated that all of them were expressed under SD or LD conditions and the difference is that they were expressed in different abundance and following different rules.They were not specifically induced by SD condition and LD condition also induced their expression at a given stage.4.The expression patterns of the 11 genes were also detected in the SAM of cultivars with different sensitivity to photoperiod and they were all expressed in HH27,a non-sensitive cultivar and ZGDD,a sensitive cultivar.However,the expression peak in HH27 appeared earlier than that in ZGDD, which coincides with the earlier flowering phenotype of HH27 in SD condition,while the peak value in ZGDD is much higher than that in HH27.5.The flowering process of ZGDD can be reversed when switching from LD to SD condition, resulting in reversed terminal raceme,short terminal raceme,and vegetative terminal.The expression patterns of photoperiod-related genes in the flower reversion of ZGDD were identified and it was found that,GmLFY-like and GmAP1-like,the homologues of meristem identity genes LFY and AP1 showed intermediate expression patterns among SD,LD and SD-LD conditions,indicating that GmLFY-like and GmAP1-like may act as the meristem related genes in soybean.Genes involved in the up-stream photoperiodic pathway like GmGI-like and PEBP family genes expressed similarly with GmLFY-like and GmAP1-like in the early stage of flower reversion indicating they may participate in the early control of flower reversion in soybean.6.The photoperiod-related genes in soyean exhibit a rhythm of 24-h like their homologues in Arabidopsis.In SD and LD condition,GmGI has a similar expression rhythm with that in Arabidopsis in LD and is different from rice.The daily oscillation of GmCO-like genes varies from that in both AtCO and Hd1.However,the daily rhythm of GmCO-like in SD was similar with that of COL1 and COL2 in LD.The PEBP family genes showed a similar rhythm with rice in SD,and that in LD condition were different.All the results may indicate that the day length measurement in soybean is different from that both in Arabidopsis and Rice.7.A rapid and efficient platform for gene function investigation in legumes was developed, combining the simplicity and high efficiency of the Lotus corniculatus cultivar Superroot regeneration system and the availability of Agrobacterium rhizogenes-mediated transformation.Transgenic L. corniculatus plants were obtained in two and a half months and identified by molecular analysis.This system was improved by validating some parameters influencing the transformation frequency,which could reach 92%based on GUS detection.As proof of concept,transgenic Superroot plants overexpressing TaNHX2,which encodes an Na~+/H~+ antiporter,were gained and shown to have increased salt tolerance to salt stress.Transgenic Superroot plants overexpressing GmSOC1 were also obtained and they showed earlier flowering than control,indicating GmSOC1 acted as a floral inducer. It also suggested that this gene function-investigating platform could be used for antibiotic biology and developmental biology.更多还原