摘要
构建p53基因RNA干涉DNA片段的目的,是将其应用于植物悬浮培养中的细胞周期调节.依据拟南芥p53基因与其他高等植物具有高度保守区域的特点,合成其两个区域的核苷酸片段(Ⅰ和Ⅱ)及其相应的反向片段(Ⅰ′和Ⅱ′),并在Ⅱ和Ⅱ′端部分别引入内含子两个端部核苷酸序列.在相互连接后进行PCR选择扩增,其产物再与克隆载体连接并经过蓝白斑筛选获得重组DNA;在电泳和核苷酸测序鉴定后表明,最终得到了Ⅰ–5′–内含子–Ⅱ′–Ⅱ–内含子–3′–Ⅰ′序列的重组DNA片段.该片段两端含多克隆位点,通过插入植物的表达载体进入细胞基因组,在细胞中其转录产物将形成发夹结构,经胞内酶切后可以形成短的双链RNA片段,将具有干涉p53基因表达的功能.
The purpose which some DNA regions of p53 gene were constructed into RNA interfere segments was applicable to regulation of the cell cycles during the suspension culture of plant cells. According to the highly conservative regions between Arabidopsis thaliana p53 gene and genes of other higher plants,two regions of both nucleotide segments （ Ⅰ and Ⅱ） and the opposite sequence segments（Ⅰ＇and Ⅱ＇）of p53 gene were linked together in this research. In addition,two terminal sequenecs of an intron were added into the terminals of sequence Ⅱ and Ⅱ＇ respectively. The recombination DNA segments of Ⅰ-5＇ intron-Ⅱ＇-Ⅱ-intron 3＇-Ⅰ＇were gained by linking of Ⅰ and Ⅱ＇, Ⅱ and Ⅰ＇ ,screening of PCR, cloning and filtering between blue and white spots,and identifying of the electrophoresis and the nucleotide sequencing of the recombination DNAs in turn. Because of two terminals of the recombination segment contains polyclone sites,it can be transferred into the expressed vehicles and inserted into genomes of some plants easily,and it＇s transcription products will form some hairpin structures in the cells. When the hairpin structures are cut into short double strands RNA segments by endoenzymes in the cells ,they can interfere with expression of p53 gene.
出处
《天津科技大学学报》
CAS
2010年第3期19-22,26,共5页
Journal of Tianjin University of Science & Technology
基金
天津市应用基础及前沿技术研究计划项目(08JCZDTC15300)
关键词
拟南芥
P53基因
保守序列
RNAi片段
Arabidopsis thaliana
p53 gene
conservative sequences
RNAi segments
