【作者】 娄群峰；

【导师】 陈劲枫；

【作者基本信息】 南京农业大学 ， 蔬菜学， 2004， 博士

【摘要】 黄瓜（Cucumis sativus L．）是世界上重要的蔬菜作物，其性别表现具有多种类型其中全雌性在黄瓜的杂交制种中具有巨大的优势。选育强雌或全雌性品种是目前黄瓜育种的主要目标之一。同时，由于黄瓜性别表现的多样性，以黄瓜为模式植物所阐述的性别分化机理对于丰富植物发育生物学理论具有重要的意义。本文基于各种生态类型的黄瓜材料，建立F₂、BC₁以及BC₂代等黄瓜性型分离群体，在阐述全雌性遗传规律的基础上，利用RAPD和AFLP分子标记技术研究获得了与黄瓜全雌性位点紧密连锁的标记：从中国黄瓜中克隆出了ACC合酶基因，分析了该基因在植株不同部位及雌雄花芽中的表达特点，并研究了乙烯对该基因表达的诱导效应；同时利用同工酶技术研究了黄瓜性别诱导中体内3种同工酶的变化特点，分析了同工酶与黄瓜植株性别表现的关系。具体内容分述如下： 1、利用不同生态类型的全雌性与弱雌性黄瓜正反交配组获得F₁代，并建立F₂、BC₁和BC₂代性型分离群体，分析了黄瓜性型的遗传规律以及光温环境的变化对不同生态型黄瓜性型遗传特点的影响。结果表明，黄瓜的全雌性由一对核基因控制，全雌对弱雌为显性。当全雌性位点处于纯合状态时，全雌性状表现稳定，不易受到光温等环境因素的影响。而当全雌性位点处于杂合状态时，其全雌性状的表现与遗传背景有一定的关系，部分生态类型表现稳定全雌，而部分生态类型则易受光温等环境因素的影响，全雌性表现出不完全显性特征，植株基部出现少量雄花，但总体的雌性表现均很强。可见，黄瓜的全雌性除了主效基因控制外，可能还存在某些修饰基因的调控，这些修饰基因可能受到光温的信号调节而起作用。 2、以日本全雌性黄瓜与中国华南型弱雌黄瓜杂交后多代自交的分离后代，利用RAPD技术对黄瓜性别特异基因进行分子标记连锁研究。共选用300个10碱基的随机引物，按分离群体分组分析法（BSA）进行多态性筛选，获得5个在全雌和弱雌基因池（gene p001）间表现多态性的引物．单株检测表明，引物B11具有全雌特异性，在大多数检测的全雌性单株中均可扩增出一条约1000bp的特征带。而在雌雄同株的单株中则未见。利用MAPMAKER（Version 3.0）软件计算得到该标记与全雌性位点连锁距离在12.2cM。将该全雌性特异的片段命名为B_11-1000。 3、另外，以全雌品种“戴多星”自交系和弱雌品种“北京截头”多代自交系为双亲杂黄瓜全雌性基因分子标记及ACC合醉基因的克隆与表达研究交获得F:，然后得到F:性型分离群体.利用BSA法构建全雌和弱雌两个基因池，筛选了64对AFLP选择性引物Ec。班.NN+Msel.NNN组合，获得在全雌和弱雌基因池间表现多态的引物组合共5对，分别为A刀CAT:42、AG/CAA265、八T/CTC260、毛叼CT阮3、TG/CAC234.经F:代性型分离群体单株验证，发现仅EcoRI.TG+Msel一CAC引物组合在全雌单株中穗定扩增出一条分子量为234bP的特异带，以MApMAKER（ version 3.0）软件分析，该标记与全雌性位点的连锁距离在6.7cM.命名该连锁标记为TG/CAC234.将该特异条带回收、克隆、测序，设计特异SCAR引物，再对凡代单株基因组DNA进行扩增，仅在全雌单株中扩增出一条分子量为1“bP的特异带，表明已成功地将与黄瓜全雌性连锁的AFLP标记转化为操作简便、表现稳定的SCAR标记，该标记命名为SAI“. 4、根据Trebltsh（1 997）公布的与黄瓜雌性基因F位点表现共分离的ACC合酶基因C万刁CS了序列，设计了特异引物，通过PCR克隆方法克隆获得了中国弱雌性黄瓜中的全长为1 024bP的Acc合酶序列.BLAsT分析表明，本研究所获得的ACc合酶基因与全长为1 025bP的c，刁C万了基因同源程度在99%以上。表明不同生态型黄瓜中ACC合酶基因序列保守性很强.序列分析表明该ACC合酶基因包含6个开放阅读框.PCR检测发现，该基因不具有性型特异性，在所研究的欧洲型和中国黄瓜材料中，无论全雌或雌雄同花的黄瓜基因组中均能检测到该基因的存在. 5、利用Rl’- PCR技术研究了ACC合酶基因C琴注C国了基因在黄瓜全株上下不同部位的表达特征.结果表明，在植株的根、茎、叶、雄花芽和雌花芽中均能检测到C琴刁C’S1基因的转录，但是在不同部位其表达程度存在明显的差异.ACC合酶在根、茎、叶、雄花芽和雌花芽中的表达程度呈递增的趋势.其中在根和茎中仅检测到痕量的表达，而在叶、雄花芽和雌花芽中的表达量相对较高，雌花芽中表达童最高.同时研究了乙烯利处理黄瓜弱雌株茎尖后，茎尖中C琴才C万I基因表达的变化特点.结果发现，处理后24小时该基因被诱导增强表达. 6、同时还比较了Trizol试剂盒、异硫氛酸脉和CTAB法用于黄瓜花芽总RNA的提取效果，结果表明，改良的Cl’AB法较其它两种方法能更有效地提取黄瓜花芽总RNA.该方法能有效去除黄瓜花芽中酚类和多糖类物质的干扰.电泳检测能看到整齐的285、1 85和55三条带型，经紫外分光光度计分析，260从280比值在1 .9一2.0之间，提取率在80一100卿1 oomg左右.利用该法所提的总RNA进行DDPq’- pcR能得到清晰的扩增带型. 7、以雄花的诱导剂稍酸银和雌花诱导剂乙烯利分别处理黄瓜植株茎尖，比较?更多还原

【Abstract】 Cucumber ( Curcumas saliva L.) is an important vegetable crop. Its sex phenotypes includes several types, and the genoecy plays an important role in cucumber heterosis breeding. Gynoecy breeding is one of the main breeding aim in cucumber breeding. Researches about its sex determination have an important value for the basic theoretics of plant development biology. Based on different ecological type materials of cucumber, F1, F2, BC1 and BC2 population were constructed, and the inheritance law of sex was study; RAPD and AFLP markers linked to gynoecious loci were attained, these markeres could be utilized for marker assist selecting (MAS) of cucumber gynoecious lines; ACC synthase gene was cloned by the method of PCR technique, and its expression level was studied in different organs including root, stem, leaf, male flower bud and female flower bud, and its expression was studied also when female flowers were induced with ethyphon; Isozyme technique was employed to explored the relationship between isozyme change characterization and sex expression. The details were described as follows:1. F1,F2, BC1 and BC2 populations from cross of gynoecious and monoecious cucumber of differental ecological types were employed to analyse the inheritance character of cucumber sex, and the effects of photoperiod and tempreture on sex phenotype and its inheritance. The results showed that the inheritance of gynoecious in cucumber was controlled by one dominant gene, and gynoecious was dormant on monoecious. When the loci of gynoecious was in homozygous, the gynoecious phenotype was stable, and was not effected by the photoperiod and tempareture. But if the loci of gynoecious was in heterozygous, the expression of gynoecious might be relative with the genetic background. Some ecospecies had stable gynoecious phenotype, but other had sub-gynoecious phenotype with few male flower in early days in few plants according to the growing season. Accordingly, the gynoecious in cucumber was controlled mainly by one key gene, and there was also other genes that could modify gynoecious expression by the signal of photoperiod and tempreture.2. The sex separate progeny from the cross between Japanese gynoecious and Chinese cucmber were employed to study the molecular markers linked to the sex determination genes of cucumber with RAPD technology. With the method of BSA, 300 randomlyselected 10bp primers were used in PCR amplification of the DNA pools from gynoecious and monoecious cucumber, respectively, and polymorphism were found in five primers in three replications. Those polymorphic primers were used to detect the individual samples with different sex phenotype, a band of 1000 bp was amplified specifically in DNA from the gynoecious plants tested. This band is absent in the monoecious plants and marked as B11-1000- 12.2 cM of the genetic distance to gynoecious loci was computered with a software of MAPMAKER(version 3.0)3. hi the present study, selfed gynoecious cucumber ’Delta star’ and monoecious cucumber ’Beijing Jietou’ were used as parents to make F1 and then selfed to get F2 sex segregated population. Gynoecious and monoecious DNA pools were developed separately using bulked segregant analysis (BSA). Amplified fragment length polymorphism (AFLP) technique with 64 primer combinations were employed to find the polymorphisms between these DNA pools, and five primer combination of AA/CAT142, AG/CAA265, AT/CTC260, TA/CTG263 and TG/CAC234 were found polymorphism between both gene pools. These markers were testified with individual DNA of the F2 population. Only the primer combination of TG+CAC was found to amplify a specific fragment of 234bp in the individual gynoecious plants. Linkage analysis using the software of MAPMAKER (version 3.0) indicated its genetic distance to the gynoecious loci was 6.7 cM, and this AFLP marker was designed as TG/CAC234. This band was collected and sequenced to synthesize a sequence-characterized amplified region (SCAR) primer. The primer was used to amplify the individual DNAs of t更多还原