【作者】 邓明华；

【导师】 邹学校； 朱海山；

【作者基本信息】 中南大学 ， 植物学， 2011， 博士

【摘要】 利用胞质雄性不育系(CMS)做母本是辣椒杂种优势利用的重要途径。但辣椒CMS的基础理论研究十分薄弱。本研究以湖南省蔬菜研究所选育的辣椒CMS-9704A、保持系(M-9704B)、恢复系(R-9701)和Fl为试材,研究了辣椒CMS的分子生理机制。辣椒属包含有5个栽培种和20-30个野生种,对于该属种间的亲缘关系,系统学家的观点不一。本研究采用辣椒11个植物种为试材,探讨了辣椒属内种间的亲缘关系。结果如下：1.在花粉败育阶段,CMS-9704A花蕾的超氧阴离子(O2-),过氧化氢(H2O2)和丙二醛(MDA)的含量高于M-9704B。CMS-9704A花蕾的活性氧清除酶过氧化物酶(POD),超氧化物歧化酶(SOD)和过氧化氢酶(CAT)的活性也高于M-9704B。Pod,Mn-Sod,Cat1,Cat2和Cat3基因的表达量与酶活性在两系之间的变化一致。因此,CMS-9704A的不育性可能与CMS的花蕾长期处于由活性氧异常积累引起的慢性过氧化有关。2.在花粉败育的高峰期(小孢子单核靠边期),CMS-9704A花蕾的抗坏血酸(ASA),谷胱甘肽(GSH)、总ASA含量低于M-9704B,ASA/ (ASA+DHA)的比值在两系之间差异不显著；CMS-9704AGSH,总GSH含量和GSH-(GSH+GSSG)比值低于M-9704B,氧化性谷胱甘肽(GSSH)含量高于M-9704B;CMS-9704A的抗坏血酸过氧化物酶(APX),谷胱甘肽过氧化物酶(GPX),谷胱甘肽还原酶(GR)酶活性和表达水平低于M-9704B。因此,CMS-9704A花蕾活性氧的异常积累与不育系非酶促抗氧化系统的异常有关。3.在小孢子败育过程中,CMS-9704A的苹果酸脱氢酶(Mdh),NAPD-苹果酸酶(NADP-Me),NADP-以柠檬酸脱氢酶(NADP-Icdh)和6-磷酸葡萄糖脱氢酶(G6pdh)的表达均出现下调,而Ndpk的变化不明显。因此,由于Mdh,NADP-Me,NADP-Icdh和G6pdh的表达受到抑制,使三酸循环和磷酸戊糖途径受阻,导致能量供应不足,与CMS-9704A雄性不育有一定关系。4.对CMS-9704A,M-9704B及R-9701线粒体DNA进行了RAPD分析,共使用500条随机引物,其中有67引物在“三系”之间都得到了扩增产物,19条引物扩增结果在“三系”之间表现出了遗传多样性。在CMS-9704A与M-9704B的线粒体DNA之间也发现了明显的差异,在CMS-9704A中获得特异片段9条。5.从CMS-9704A中扩增到ATP合成酶亚基6(Atp6)和细胞色素C氧化酶亚基2(CoxⅡ)两个基因的部分序列Atp6-706和CoxⅡ-708,生物信息学分析发现分离的Atp6-706和CoxⅡ-708片段与GenBank中的茄科作物线粒体CoxⅡ和Atp6基因的相似性高达95%以上；在M-9704B和R-9701中均未能扩增到任何序列,说明辣椒CMS-9704A的CoxⅡ和Atp6基因与M-9704B和R-9701在线粒体DNA水平上存在差异,这种结构上的变化暗示可能与辣椒CMS相关。6.从CMS-9704A叶片DNA中扩增到一个与已经报道的与辣椒CMS基因ORF456同源的基因ORF168, ORF168的DNA序列与ORF456相比在第150个密码子处产生了一个碱基“C”的缺失,形成编码168个氨基酸的新序列。ORF168在CMS-9704A中稳定表达；核恢复基因对ORF168在F1的表达无影响,推测核恢复基因可能通过转录后剪接或翻译/翻译后修饰的途径对ORF168的转录产生影响。7.辣椒(Capsicum annuum L.) 6个变种的花粉呈矩圆形、椭圆形或圆形；极面观均为三裂片圆形；具三萌发沟,沟长达两极,沟的末端在极面上不连接形成合沟；花粉外壁纹饰在扫描电子显微镜下以刺-颗粒状复合纹饰为主。变种间在花粉形状和外壁纹饰方面有一定差异。根据其特征可以对辣椒进行分类。8.在辣椒5个栽培种之间,其亲缘关系的远近为C.annuum, C. frutescens, C. chinese, C. baccatum, C. pubesens。中国云南南部与美洲的C.frutescens辣椒资源具有较大的差异。对5份中国辣椒资源研究发现：C. frutescens种质辣椒素和二氢辣椒素含量要高于C. annuum种质。不同的辣椒种质之间辣椒素和二氢辣椒素含量及其比值的遗传多样性丰富。更多还原

【Abstract】 Producing Hybrid seeds using cytoplasmic male sterility (CMS) as female parent is a very important way to utilize heterosis in pepper. However, basic researches of cytoplasmic male sterility mechanism in pepper are still poor. In order to enrich the understanding of cytoplasmic male sterility, quicken the usefulness of CMS-9704A, the maintainer (M-9704B), restorer (R-9701) and F1 as experiment materials, the relationship of CMS-9704A with the metabolism of ROS was investigated, some molecular marks were established and a novel gene ORF168 associated with CMS was cloned and analyzed.Capsicum is a medium-sized genus with 5 cultivated spices and 20-30 wild spices. Different opinions on the circumscription of Capsicum and relationships among different species also occur. Studies on molecular phylogeny on Capsicum have not been reported until now. In this studyⅠfocus on Capsicum’s molecular phylogeny based on palynology and DNA barcoding. It is really helpful for us to solve taxonomic problems of higher taxa in Capsicum, improve study level and technique in Capsicum. The followings were our experiment results.1. At the abortion stage (pollen mother cell meiosis stage), anthers of CMS-9704A had higher contents of O2-, H2O2 and MDA than those of the maintainer. Simultaneously, there were higher activities of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) in scavenging ROS in the anthers of the CMS line than in the maintainer. Abundance of Pod, Mn-Sod, Cat1, Cat2 and Cat3 mRNA in anthers of CMS-9704A corresponded with the activities of these three genes. Therefore, it is possible that the sterility in CMS-9704A is related to the abortion of microspores, induced by chronic oxidative stress caused by an abnormal increase in ROS, and also by membrane lipid peroxidation in the mitochondria in the early stage of pollen development.2. At the abortion peak (uninucleate microspore stage), anthers of CMS-9704A had lower contents of ASA, DHA, Total ASA, GSH and Total GSH than those of the maintainer 9704B, except for that of GSSH. The ratio of ASA/(ASA+DHA) between the anthers of CMS-9704A and maintainer 9704B was litter, while the ratio of GSH/(GSH+GSSG) in anthers of CMS-9704A was lower than that of the maintainer. There were lower activities and e xpression level of APX, GPX, and GR in the anthers of the CMS line than in the maintainer. Therefore, the excessive ROS accumulation in the anthers of CMS-9704A is related with the lower contents of non enzymatic antioxidant system.3. The transcription of Mdh, NADP-Me, NADP-Icdh and G6pdh were remarkably down-regulated during the period of microspores abortion, except for that of Ndpk. Therefore, it was deduced that the reaction of tricarboxylic acid cycle and pentose phosphate pathway weakened due to the decreased transcription of Mdh, NADP-Me, NADP-Icdh and G6pdh genes, the energy was seriously in short supply during the course of pollen development, Hence insufficient energy production might cause male sterility of pollen in some extent.4. The mitochondrial DNA of cytoplasmic male sterility line, its maintainer line and restorer line of Pepper were studied by RAPD analysis. All of lines had obtained amplified products through 67 out of total 500 random primers. Hey emerged polymorphism in three lines through 19 primers. Some obvious differences in mitochondrial DNA were detected between CMS and its maintainer line, and 9 special bands in CMS line were found. The relationship of those different bands in CMS to cytoplasmic male sterility needs farther study.5. The CoxⅡand Atp6 gene fragments, CoxⅡ-708 and Atp6-706 (GenBank accession numbers:FJ986190 and FJ986191), associated with cytoplasmic male sterility (CMS) were cloned from the mitochondrial DNA of a pepper (Capsicum annuum L.) cytoplasmic male sterile line using PCR. These fragments did not have complete encoding region but showed the homology of 95% with CoxⅡand Atp6 genes from the referred Solanaceae in GenBank. No band was cloned from the mitochondrial DNA of maintainer and restorer lines. The result suggested a close relationship of pepper CMS with the variation in DNA sequence of CoxⅡand Atp6 gene in CMS line.6. A DNA, homologous to ORF456 verified associated with CMS in chili pepper, named ORF168, was cloned from leaves of pepper 9704A. Results revealed that the ORF168 had one nucleotide deleted in codon 150 (nt450delC) and consists of a 507-bp single-coding exon which encodes 168 amino acid compared with the original sequence data of ORF456. The RT-PCR results showed that the ORF168 gene was transcribed in buds and leaves at all developmental stage of 9704A (CMS) and hybrid F1, while no band was detected in the maintainer line. The nuclear restorer gene may restore the CMS fertility by post-transcriptional processing or translational/post-translational modification, but did not interfere with transcription of ORF168 genes directly.7. Morphological studies of pollen of 6 varieties of Capsicum annuum L. in China were reported in this paper. The result showed that the pollen shapes were rectangular, or elliptic, or suborbicular from the equatorial view and three-lobed in polar view; the aperture was three-colpate, which extended to polar area and no converaged; the ornamentation of the exine was spinule-granular under SEM. There were some differences in pollen shape and the ornamentation of the exine among different varieties. The pollen shape and the ornamentation of the exine used in this experiment could be classified. This classification was comparable to the traditional subspecies classification.8. The results from most parsimonious trees showed that the relationships of the 5 cultivated species are C. annuum, C. frutescens, C. chinese, C. baccatum, C.pubesens. Our results also showed that the C. frutescens from China and NGRL have great differences, which provided us with new evidence to the arguments that the south of Yunnan has an important role in the origin and development of pepper.Capsaicin and dihydrocapsaicin content in pericarp placenta and total fruit of 5 different varieties of China were detected. The results showed that the germplasm belonging to C. frutescens have the higher capsaicin and dihydrocapsaicin content than those in the germplasm belonging to C. annuum. There are higher genetic diversity of capsaicin and dihydrocapsaicin content and the ratio of capsaicin/dihydrocapsaicin among the different pepper varieties.更多还原