刺梨RNA的有效提取及Actin基因片段的克隆  

Extraction of Total RNA from Rosa roxburghii Tratt and the Cloning of Actin Gene Fragment

作　　者：杨玉[1] 安华明[1] 刘明[1] 黄伟[1] 李德燕[1] 王立新[1] 刘厚宇[1] 李顺雨[1] 

机构地区：[1]贵州大学喀斯特山地果树资源研究所,贵州贵阳550025

出　　处：《安徽农业科学》2008年第4期1373-1374,共2页Journal of Anhui Agricultural Sciences

基　　金：国家自然科学基金项目(30660114);贵州省自然科学基金项目(20062038)

摘　　要：[目的]为刺梨的分子生物学研究奠定技术基础。[方法]分别采用改进的CTAB-LiCl法和CTAB法提取刺梨果实中的总RNA和基因组DNA。对刺梨总RNA进行反转录,生成cDNA第1链。根据GenBank上的蔷薇科肌动蛋白(Actin)基因保守序列设计1对跨内含子引物Actin1和Actin2,以cDNA第1链和基因组DNA为模板进行RT-PCR扩增。将含有目的片段的凝胶进行回收纯化,克隆到PMD18-T载体上,转化大肠杆菌TG1感受态细胞,选取带有目的片段的重组质粒进行测序和序列分析。[结果]将目的片段回收后克隆于T载体,可获得298 bpActin基因的cDNA序列。克隆所得核苷酸序列与蔷薇、桃和葡萄的核苷酸序列相似性达83%,与富士苹果、甜菜等的相似性也超过80%。刺梨Actin基因编码的氨基酸序列与梨的相似性最高(达97%),其次是葡萄、花生、大豆、棉花和马铃薯,而与水稻、拟南芥等的相似性也达90%以上。[结论]Actin基因跨内含子,用该Actin基因片段作内标可以减少DNA污染。[Objective]The aim of the research was to lay the technical foundation for the molecular biology research on Rosa roxburghii Tmtt. [Method] Total RNA and genomic DNA were extracted from the fruits of R. roxburghii by the improved CTAB-LiCl method and CrAB method respectively.Reverse transcription was conducted on total RNA from R. roxburghii to generate the 1st chain of cDNA. 1 pairs of cress-intron primers Actinl and Actin2 were designed according to the conserved sequences of Rosaceae Actin gene on GenBank website .With the 1st chain of cDNA and the genomic DNA as templates, RT-PCR amplification was made.The gel contained the target fragment was reclaimed and purified,the target fragment was cloned to PMD18-T vector and transformed into TG1 competent cells of Escherichia coli. The recombinant plasmid contained the target fragment was selected for sequencing and sequence analysis. [Result] The target fragment was reclaimed and cloned into T vector and 298 bp cDNA sequence of Actin gene was obtained.The similarity between the nucleotide sequence obtained from cloning and the nucleotide sequence of Rosa davurica,Prunus persica and Vitis vinifera L.reached 83% ,the similarity between the cloning sequence and that in Fuji apple, sugar beet and so on also exceeded 80%. The sequence silimarity of amino acid coded by Actin gene in R. roxburghii and that in Pyrus communis L. was highest,followed by V. vinifera, Arachis hypogaea L., Glycine max L., Gossypium hirsutum L. and Solanum tuberosum L. ,and the sequence silimarity of amino acid coded by Actin gene in R. roxburghii and that in Oryza sativa L.and Arabidopsis thaliana and so on also reached over 90%. [Conclusion] Actin gene crossed intron and taking this Actin gene fragment as intron could reduce DNA pollution.

关 键 词：剌梨 肌动蛋白 克隆 

分 类 号：Q78[生物学—分子生物学]