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作物学报 ›› 2011, Vol. 37 ›› Issue (11): 1926-1934.doi: 10.3724/SP.J.1006.2011.01926

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

马铃薯块茎糖基转移酶基因的克隆及其RNAi载体的构建

王旺田1,2，张金文1,2,*，王蒂1,2,*，张俊莲1,2，司怀军1,2，陶士珩3   

  1. 1 甘肃农业大学农学院, 甘肃兰州 730070; 2 甘肃省作物遗传改良与种质创新重点实验室, 甘肃兰州 730070; 3 西北农林科技大学生命科学学院, 陕西杨凌 712100
  • 收稿日期:2011-04-27 修回日期:2011-07-15 出版日期:2011-11-12 网络出版日期:2011-09-06
  • 通讯作者: 张金文, E-mail: jwzhang305@163.com, Tel: 0931-7631167; 王蒂, E-mail: wangd@gsau.edu.cn, Tel: 0931-7631167
  • 基金资助:

    本研究由国家重点基础研究发展计划(973计划)项目(2010CB134404), 甘肃省自然科学基金项目(0710RJZA088)和教育部博士生国内访学项目(Z107-020901)资助。

Cloning of Rhamnosyl Transferase Gene and Construction of Its RNAi Vector in Potato

WANG Wang-Tian1,2,ZHANG Jin-Wen1,2,*,WANG Di1,2,*,ZHANG Jun-Lian1,2,SI Huai-Jun1,2,TAO Shi-Heng3   

  1. 1 College of Agronomy, Gansu Agricultural University, Lanzhou 730070, China; 2 Gansu Key Laboratory of Crop Genetic & Germplasm Enhancement, Lanzhou 730070, China; 3 College of Life Sciences, Northwest A&F University, Yanglin 712100, China
  • Received:2011-04-27 Revised:2011-07-15 Published:2011-11-12 Published online:2011-09-06
  • Contact: 张金文, E-mail: jwzhang305@163.com, Tel: 0931-7631167; 王蒂, E-mail: wangd@gsau.edu.cn, Tel: 0931-7631167

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摘要： 为探讨鼠李糖基转移酶(rhamnosyltransferase, sgt3)基因与类固醇糖苷生物碱(steroidal glycoalkaloid, SGAs)合成关系, 揭示sgt3基因在SGAs合成中的作用, 本研究根据GenBanK No：DQ266437保守区设计特异引物, 通过RT-PCR技术获得马铃薯块茎sgt3相似基因；采用生物信息学相关软件分析, 预测sgt3相似基因cDNA序列编码的蛋白质结构和功能, 构建基于sgt3基因的植物干扰表达载体。结果表明, 克隆的sgt3相似基因与报道的sgt3基因序列相似性达到99.54%, 其ORF长1 500 bp, 编码505个氨基酸残基, 具有UDPG糖基转移酶保守结构域及许多重要功能位点；三级结构预测表明, 该氨基酸同糖基转移酶单体结构模型相似, 为糖基转移酶家族成员, 表明可能具有合成类固醇糖苷生物碱功能, 基因序列已注册到GenBank, 序列登录号为HM188447。以此为靶序列, 构建由Actin启动子和CIPP启动子驱动的与sgt1和sgt2基因相似度高的含有sgt3基因序列的干扰表达载体, 将为糖苷生物碱的合成、代谢的进一步研究及低糖苷生物碱转基因马铃薯品种的培育奠定基础。

关键词: 马铃薯, 鼠李糖基转移酶, 基因克隆, 序列分析, RNAi载体

Abstract: Steroidal glycoalkaloids (SGAs) are potentially harmful metabolites found in potatoes and other Solanaceous plants. SGAs accumulation affects food quality and safety, we hope to use molecular biology methods to reduce SGAs content and assist breeding efforts to ensure food safety, develop new and improved varieties of potatoes. To investigate the relationship between the gene expression of the rhamnosyltransferase (sgt3) and the biosynthesis of steroidal glycoalkaloid (SGAs), and reveal the role of sgt3 gene in biosynthetsis of potato steroidal glycoalkaloid, similar gene cDNA sequence of the potato sgt3 was obtained from the total RNA of potato tubers by reverse transcription polymerase chain reaction (RT-PCR) using specific primers synthesized according to the conserved domain of GenBanK No: DQ266437 sequence. The protein function and structure of the similar sgt3 gene cDNA sequence were predicted and analyzed by related software of bioinformatics technology. Interference expression vector based on sgt3 gene was constructed. The Blast result showed that the gene sequence shared a high level of similarity with the sgt3 gene in GenBank (accession No: DQ266437) and its homology was 99.54%, and similarity of the amino acid sequence was 99%. The full-length of cDNA was 1 500 bp, which contained 505 amino acids, UDPG glycosyltransferase conserved domain and many important functional sites. The 3D structure of protein was predicted by homology comparative modeling in Swiss-Model, the results showed that the 3D structure of SGT3 was highly similar to that of the glycosyltransferase, so it was inferred that SGT3 should be a member of glycosyltransferase superfamily that has function of steroidal glycoalkaloid. Sgt3 similar gene obtained here was rhamnosyl transferase gene, and its sequence was submitted with GenBank No: HM188447. The RNA interference transformation expression vector in which sgt3 gene was regulated by Actin and CIPP promoters was constructed, which will lay a solid foundation for the synthesis of alkaloids glycosides, further research of metabolism and cultivation of transgenic potato varieties with low indican alkaloids character.

Key words: Potato, sgt3 gene, Gene cloning, Sequence analysis, RNA interference vector

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