【作者】 牛艳梅；

【导师】 周鹏；

【作者基本信息】 华南热带农业大学 ， 生物化学与分子生物学， 2007， 硕士

【摘要】 番木瓜是一种典型的呼吸跃变型果实,对其进行成熟的调控及机理的研究具有重要的理论和实践意义。Expansin是一类存在于植物细胞壁上,并能快速缓冲张力,使植物细胞壁松驰的蛋白质。众多的研究资料表明,其在果实的成熟过程中起着重要的作用。近年来,关于果实中的expansin基因的研究越来越多,如香蕉、芒果和荔枝,但尚未见番木瓜expansin的研究报道。本研究提取成熟番木瓜果实的RNA,反转录合成cDNA,以cDNA为模板,根据已报道的expansin氨基酸保守序列设计简并引物进行RT-PCR,得到的扩增产物纯化后与pMD19-T载体连接,转化大肠杆菌,挑选阳性克隆进行序列测定,得到一个大小为531bp的基因片段,命名为Cp-EXP1,并登录Genbank,登录号为:DQ321829。经分析其编码177个氨基酸,具有expansin保守结构。通过与Genbank中其他expansin基因序列同源性比较发现,番木瓜Cp-EXP1与甜樱桃Exp1和酸樱桃Exp2的序列同源性最高,均为85.66%,与苹果Md-Exp1同源性为84.67%,与黄瓜Cs-EXP1同源性为79.92%。根据得到的Cp-EXP1基因保守片段设计引物,提取成熟番木瓜果实的RNA进行3’RACE,得到了长为673bp的Cp-EXP1基因3’端,与RT-PCR得到的保守片段拼接得到长度为710bp的序列,经Genscan分析后发现,得到的Cp-EXP1 3’端序列含有一个完整的阅读框,编码206个氨基酸。提取番木瓜不同组织的RNA,分别以expansin简并引物和管家基因引物进行RT-PCR,结果发现该基因在番木瓜的根、茎、叶、花、果实中均有表达。为了准确分析该基因在番木瓜果实中的作用,以不同成熟阶段的番木瓜果实总RNA为模板,进行荧光定量PCR。实验结果表明,Cp-EXP1基因在不同成熟阶段的番木瓜果实中的表达是有差异的,其表达水平受乙烯调控。首次在番木瓜中克隆expansin基因并进行表达分析,对expansin基因与乙烯的生成、果实成熟机理间关系的研究具有一定的理论指导意义,也为研究番木瓜果实的生长发育以及其品种的改良等打下了良好的基础。更多还原

【Abstract】 Papaya is a kind of typical respiration climacteric fruit, which has important theoretical and practical meanings to regulate its ripening and to study the mechanism of ripening. Expansin is a group of cell-wall-localized proteins which inducing extension of cell wall. Expansins have been shown to play an important role in softening of fruits. In recent, expansin have been studied in many fruit, such as banana, mango and litchi, but it has no report about expansin in papaya.PCR degenerate primers were used to amplify cDNA fragments in papaya fruit by RT-PCR, which were designed with reference to the conserved amino acid sequences of known expansins’amino acid sequences, a cDNA fragment of 531 bp was cloned, named as Cp-EXP1. The Cp-EXP1 cDNA encodes a polypeptide of 177 amino acids, has the conserved structure of expansin amino acid sequence. The result of blast suggested that Cp-EXP1 has 85.66% sequence simility with both Pc-Exp1 and Pc-Exp2, 84.67% with Md-Exp1 from apple and 79.92% with Cs-EXP1 from cumcuber.The primers were designed according to the conserved sequence of Cp-EXP1, then the 3’end fragment of Cp-EXP1 by 3’RACE was amplified. A cDNA fragment of 673 bp was cloned. The result of Genscan indicated that the 3’end of Cp-EXP1 included an integrated ORF and there were 206 amino acids.RNAs of the root,stem,leaf,flower and fruit were extracted, and reverse transcripted to cDNA as the templates for expression. The result of RT-PCR showed that Cp-EXP1 expressed in all of the five organs.In order to further analyse the expression in the different stages during the fruit ripening, RNA was extracted from papaya fruit in different mature period, and quantitative analysis of Cp-EXP1 gene expression was carried by using Real Time PCR method. It was suggested that expression of Cp-EXP1 was regulated by ethylene.It was the first time to clone the expansin gene in the papaya fruit and then analyzed the expression at different organs,which had important theoretical meaning to the relationships between expansin gene and the produce of ethylene and the mechanism of fruit ripening, and build a great base of papaya growth research.更多还原