马铃薯块茎糖基转移酶基因的克隆及其RNAi载体的构建

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长为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sgtl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.%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 cDN A 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 ac-cording 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 vec-tor 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 C1PP 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.