摘 要

14-3-3蛋白质在植物生长发育中起了重要的调控作用，参与植物细胞内一系列信号转导和代谢过程, 影响植物的生长发育。本课题旨在利用CODEHOP设计兼并引物PCR扩增杉木种子14-3-3蛋白质基因。相较于传统的运用常规引物来扩增目的基因的方法，利用CODEHOP设计兼并引物PCR扩增目的基因则具有很多优越性。传统的方法有效引物利用率低，不利于扩增。与传统引物相比，CODEHOP引物则由两部分组成，包括短的3’简并核心区域引物和长的5’非简并夹板区。利用CODEHOPs PCR方法设计的引物—每条引物在3’简并核心区域都包含不同的序列，而5’非简并夹板区则是均匀的序列。这种方法可以并克服兼并引物和通用引物设计的内在缺陷，具有更高的特异性和灵敏度。对于未测序的物种利用常规的引物设计很难扩增出蛋白质组获得的蛋白质基因。本研究利用CODEHOP设计杉木种子14-3-3蛋白质的简并引物，利用所设计的简并引物进行反转录PCR，从杉木未成熟的种子中扩增出1000 bp左右的产物。将PCR产物构建pMD-19Vector载体上并转化JM109菌感受态细胞中，菌落PCR扩增测序。结果显示利用CODEHOP方法设计简并引物扩增的目标片段的大小，序列与预期的一致。这将为杉木其他蛋白质基因的克隆和研究提供重要的依据。

关键词： CODEHOP；14-3-3蛋白质基因；杉木；PCR；简并引物

Use CODEHOP design degenerate primers PCR amplification fir seed 14-3-3 protein gene

ABSTRACT

14-3-3 proteins play an important regulatory role in plant growth and development, which involved in a series of signal transduction and metabolic processes, affect the growth and development of plants. This paper aims to use CODEHOP to design degenerate primers PCR amplification fir seed 14-3-3 protein gene. Compared to conventional primers to amplify the target gene, a method using degenerate primers CODEHOP PCR to amply the gene has many advantages. The traditional method is lowly effective, and not conducive to amplification. Compared with the conventional primer, CODEHOP primers have two parts, a short 3' degenerate core region and the 5' non-degenerate plate area. CODEHOPs PCR method of the 3' degenerate core region contains a different sequence, while the 5' non-degenerate region has a homogeneous sequence. This method with higher specificity and sensitivity can overcome inherent defects of the combination primers and universal primer design. It’s difficult to amplify the gene sequence based on protein sequence obtained from proteome using the conventional primer design for non-sequencing species. In this study, CODEHOP software is used to design degenerate primers of Chinese fir 14-3-3 proteins, which carry out reverse transcription PCR. The result showed that 1000 bp product was amplified from immature seeds of Chinese fir. The PCR products were cloned to pMD-19 vector, and then transformed into competent JM109 bacterial cells. Colony PCR was used to amplify the 14-3-3 fragment, which were sequenced. The result showed that the size of target fragment consistent with the expected sequence. This result would be provide an new insight for cloning and studying of other proteins.

Key words：CODEHOP; 14-3-3 protein gene; Chinese fir; PCR; degenerate primers

目 录

1 文献综述…………………………………………………………….………………………... 1

1.1 研究14-3-3蛋白质的重要性………………………………………………………….. 1

1.2 CODEHOP的发展以及利用…………………………………………………………….1

1.3小结 …………………………………………………………….………………………..2

参考文献……………………………………………………………………………………… …3

2 正文 …………………………………………………………….………………………...….. 4

2.1前言………………………………………………………………………………………4

2.2 材料与方法………………………………………………………………………………5

2.2.1 材料 ……………………………………………………………………………………………5