【作者】 原丽华；

【导师】 朱立煌； 蔡润；

【作者基本信息】 上海交通大学 ， 生物医学工程， 2010， 博士

【摘要】 分枝多少,长势强弱是黄瓜重要的农艺性状。对于培育腌渍品种和鲜食品种而言,两者对侧枝性状的要求是不同的。一般来说对于培育腌渍品种来说,多数选用有限生长型,一次采收。分枝的多少、长势的强弱就成了决定单株产量的最直接因素了。而对于培育鲜食品种来说,尤其是国内黄瓜一般采用搭架栽培,要求在整个生长季内不断采收,这就需要维持主茎旺盛的生命力,防止早衰。而侧枝过多,势必会削弱主茎的生长势,还会造成空间密闭给病害发生创造了有利条件,而人工去除则费时费力,所以国内倾向于栽培少分枝或无分枝类型。了解清楚侧枝的发育机理不仅有助于育种学家因地制宜的培育不同侧枝类型的黄瓜品种,另一方面也能丰富分枝发育机制的基础研究。但长期以来,对黄瓜侧枝的研究都限于对侧枝性状的遗传学分析,一般认为,多侧枝性状至少受到5个加性因子控制,且较容易受到环境和群体结构的影响。但这些研究都止步于对侧枝性状的QTL定位,没有鉴定控制侧枝形成的关键基因,更未能对控制黄瓜分枝的分子机理进行探究。这类型的研究无法对黄瓜分枝机理做出深入地探讨。基于此种原因,我们希望从本实验室收集的不同分枝类型的黄瓜品系入手,根据拟南芥等植物物研究侧枝发育的方法对黄瓜分枝的机理做进一步的研究。首先根据分枝多少将我们实验室收集的黄瓜育种资源分成三种不同的分枝类型:多侧枝,S06是这一类中的代表,表现为在春秋两季栽培时叶腋内都会萌发大量的侧枝,一般是每节都有一个侧枝;少侧枝,这一类的代表为S94,表现为侧枝数量较少,每十节有不超过4个侧枝,但是侧枝较长;无侧枝类型,这一类的代表是S61和S92,S61一般没有侧枝长出,但是栽培后期温度升高后在较低节位会有少于两条的侧枝萌发;而S92在叶腋处会形成侧枝花打顶(侧枝变成有限生长型),多数节位的侧枝在不到1CM就开始变成有限生长型,但是较低节位会有一到两个长到8-10CM开始打顶。在对这些侧枝的表型进行初步分类的基础上,我们参照了在拟南芥和豌豆上对侧枝产生的分析途径,进行了嫁接试验,以期通过这个研究长距离信号转导的方法将它们进一步分类。结果发现一部分多分枝品系的接穗被少侧枝品系的砧木改变,说明这些黄瓜侧枝后期发育受到一种嫁接转移信号的调控,而多侧枝的形成原因恰恰是这种抑制信号缺失的结果。而在用无侧枝S61做砧木的嫁接组合中,它不能改变多侧枝接穗法表型,而S92则可以。该结果暗示S61无侧枝的原因可能是侧芽起始障碍,而S92无侧枝的原因可能涉及多个方面。为了能有更加有力的证据,我们先后作了打顶试验和侧芽的细胞学检查,结果显示:S06、S94和s92在顶端生长受抑制时,侧芽的生长都有不同程度的促进;而在顶端生长抑制解除时,S61的叶腋处依然没有侧枝长出。细胞学检查的结果也显示在S06、S94和S92的叶腋处有侧芽形成,而在S61的叶腋处只有花芽原基。以上的实验说明在多侧枝的黄瓜品系中多侧枝的原因是抑制信号的缺失;而无侧枝S61的形成原因则是侧芽原基起始障碍。进一步通过对S92、S61和S06的遗传分析,确定了S61的无侧枝性状受一个隐性单基因的控制,而S92的无侧枝性状显然受到不止一个基因的控制。考虑到S61无侧枝的表型与番茄、拟南芥、水稻等植物中的侧枝抑制突变体(ls/las/moc1)的表型类似,可以推测在黄瓜中应该存在它们的同源基因。我们根据番茄和拟南芥的LATERAL SUPPRESSOR基因的保守序列,利用RACE技术克隆了它们在黄瓜中的同源基因,CLS(EU503217),蛋白同源关系分析显示属于GRAS转录因子家族,在进化关系上与其它物种GRAS家族中的侧枝抑制基因归于同一个群。利用信号肽(NLS)预测软件对CLS蛋白序列进行分析但是没有发现核定位信号序列,于是构建了CLS:GFP融合蛋白进行瞬时表达试验,结果显示CLS蛋白定位在细胞核里,推测作为转录因子行使功能。Southern分析显示在黄瓜基因组中只有一个CLS基因的拷贝,在将CLS基因的cDNA序列转化入拟南芥ls突变体后发现,CLS基因的表达可以挽救拟南芥ls的突变体表型,这表明CLS是具有与LS/LAS相同功能的直系基因。半定量RT-PCR显示CLS基因的表达部位与拟南芥,番茄类似,为了更详细的了解CLS基因的时空表达,我们选择不同分枝类型的品系,选取不同播种时期的顶芽FAA固定后作RNA原位杂交,结果显示在有侧枝的黄瓜植株中CLS的表达部位与拟南芥LAS的表达部位类似,转录本都集中在来源于顶端分生组织的侧生分生组织区域,而且在新老分生组织的边界区都能清楚地看见CLS基因的转录信号。说明它在黄瓜侧芽起始过程中发挥了重要作用,是侧生分生组织起始的标志基因。值得注意的是,在无侧枝的S61植株的侧生分生组织区域却检测不到CLS的转录信号,说明S61无侧枝的原因与CLS基因的表达缺陷有关。随后,我们根据CLS基因在S61与S06序列上的差异设计得CAPS标记,在S61与S06的分离群体中检测该标记的分离,也没有发现它与无侧枝性状的共分离。考虑到控制S61无侧枝性状的是一个隐性单基因,说明在黄瓜中存在另一个基因在上游调控着CLS基因的表达,此基因的正常表达是CLS基因转录的前提。综上所述,我们认为黄瓜侧枝的发育也可以分解为由不同基因调控的侧芽原基的发生、侧芽形成和侧芽后期生长等过程。在侧芽原基的起始过程中,CLS发挥重要的作用,像拟南芥的LS一样,是侧生分生组织起始的标志基因。同时,黄瓜的后期发育也受到一种可以嫁接转移物质的调控,根据MAX依赖的信号通路在单子叶和双子叶植物中的保守性,我们推测黄瓜中的这个可以嫁接转移的信号物质可能也是类胡罗卜素的衍生物。更多还原

【Abstract】 Commercial cucumber (Cucumis sativus L.; 2n=2x=14) has been of culinary importance to humans for millennia. With the increased requirement in cucumber-like vegetables, yield has been a focus of cucumber breeders for over 50 years. The lateral branch number is an important agronomic trait for cucumber production, to which, though, pickling (processing) and fresh-eaten cucumber cultivars have different requirements. Therefore, studies on the genetic and molecular mechanisms of cucumber lateral branching will be of help to cucumber breeders to create special cucumber plant architectures. In the past years, however, the studies on cucumber branching have been restricted on the processing cucumber types, whereas, these studies were only focused on the number, locations and effects of the detectable QTLs for possible maker-assisted selection (MAS) of an appropriate branch trait in cucumber breeding and not involved in the branching mechanisms of cucumber.Based on this situation, to explore the possible branching mechanisms in cucumber materials in our laboratory, which could be readily classified into three types in terms of branch numbers, i.e. multiple branching (multi-branching), fewer branching and non-branching types. The grafting and decaption experiments, bud examination and genetic analyses on these cucumber lines suggested that the non-branching phenotype of S61 is caused by a defect in lateral bud formation and controlled by a single recessive gene. In order to isolate a Lateral Suppressor (LS)-homologous gene, Cucumber Lateral Suppressor (CLS), we used the homo-based cloning strategy according to the conserved structures of the lateral suppressor genes, LS, LAS and MOC1. Homology analysis showed that the cDNA encoded protein is a member of the plant-specific GRAS family proteins which contains the characteristic sequence motifs of the GRAS family. The phylogenetic analysis showed that CLS together with the known LATERAL SUPPRESSORs in tomato, rice, Arabidopsis, carrot, forms a distinct cluster, implying that CLS may have a function similar to LS/LAS/MOC1. Because no conventional nuclear localization signal (NLS) domain was predicted in CLS by in silico analysis with the predication program, the instantaneous expression was permored. Nuclear targeting of CLS is consistent with its predicted transcription regulatory role. Southern blotting experiment showed that only one copy of CLS exists in cucumber genome. Interestingly, the CLS could LAS. Next, semi-quantitative RT-PCR analysis showed that CLS could be detected in all tested tissues except the internode. Moreover, RNA in situ hybridization revealed that the pattern of CLS transcript accumulation in axillary meristems was similar to that of LAS in Arabidopsis, suggesting that CLS plays an important role in cucumber lateral meristem initiation. Remarkably, the CLS transcript signal could not be detected in the axils of leaf primordia in the non-branching line, S61, indicating that the CLS expression defect is responsible to its non-branching phenotype. However, no nucleotide changes was found in the ORF region of the CLS from S61 except for the three nucleotide changes in 3’UTR and some other alterations in the intron region away from the splicing boundaries. Based on these changes, a CAPS marker was designed. But the recessive phenotype wasn’t co-segration with the CAPS marker in the 06/61 F2 population.All the results suggest that the lateral branching development in cucumber can also be divided into two major processes. One is the lateral bud inination and the other is the outgrowth of the lateral buds. CLS plays a key role in cucumber axillary meristem initiation. The CLS expression defect is responsible to the branchless phenotype of S61.更多还原