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Acta Agron Sin ›› 2011, Vol. 37 ›› Issue (11): 1926-1934.doi: 10.3724/SP.J.1006.2011.01926

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

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 Online:2011-11-12 Published: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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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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