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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

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

[1]Faeth S H, Bush L P, Sullivan T J. Peramine alkaloid variation in neotyphodium-infected Arizona fescue: effects of endophyte and host genotype and environment. J Chem Ecol, 2002, 28: 1511–1526
[2]Ji Y(纪瑛), Lin H-M(蔺海明), Chen Y(陈垣), Zhang S-R(张守润). Nitrogen nutrition effects on character, biomass and alkaloid accumulation of Sophora alopecuroides. Acta Pratac Sin (草业学报), 2008, 17(3): 40–46 (in Chinese with English abstract)
[3]Tang Z-H(唐中华), Yu J-H(于景华), Yang F-J(杨逢建), Zu Y-G(祖元刚). Metabolic biology of plant alkaloids. Chin Bull Bot (植物学通报), 2003, 20(6): 696–702 (in Chinese with English abstract)
[4]Lu S-P(鲁守平), Sui X-X(隋新霞), Sun Q(孙群), Sun B-Q(孙宝启). Biological functions of secondary metabolism of medicinal plants and influences of ecological environment. Nat Prod Res Dev (天然产物研究与开发), 2006, 18(6): 1027–1032 (in Chinese with English abstract)
[5]Bejarano L, Mignolet E, Devaux A, Espinola N, Carraso E, Larondelle Y. Glycoalkaloids in potato tubers: the effect of variety and drought stress on the a-solanine and a-chaconine contents of potatoes. J Sci Food Agric, 2000, 80: 2096–2100
[6]Breton C, Mucha J, Jeanneau C. Structural and functional features of glycosyltransferases. Biochimie, 2001, 83: 713–718
[7]Wang J, Hou B k. Glycosyltransferases: key players involved in the modification of plant secondary metabolites. Front Biol China, 2009, 4: 39–46
[8]Ginzberg I, Tokuhisa J G, Veilleux R E. Potato steroidal glycoalkaloids: biosynthesis and genetic manipulation. Potato Res, 2009, 52: 1–15
[9]McCue K F, Allen P V, Shepherd L V T, Blake A, Malendia M M, Rockhold D R, Novy R G, Stewart D, Davies H V, Belknap W R. Potato glycosterol rhamnosyltransferase, the terminal step in triose side-chain biosynthesis. Phytochemistry, 2007, 68: 327–334
[10]Archana T. RNA interference revolution. Electr J Biotechnol, 2003, 6: 39–49
[11]Hamilton A J, Baulcombe D C. A novel species of small antisense RNA in post-transcripional gene silecng. Science, 1999, 286: 950–952
[12]Wesley S V, Helliwell C A, Smith N A, Wang M B, Rouse D T, Liu Q, Gooding P S, Singh S P, Abbott D, Stoutjesdijk P A, Robinson S P, Gleave A P, Green A G, Waterhouse P M. Construct design for eficient, efective and high-throughput gene silencing in plants. Plant J, 2001, 27: 581–590
[13]Bass B L. Double-stranded RNA as a template for gene silencing. Cell, 2000, 101: 235–238
[14]Baum J A, Bogaert T, Clinton W, Heck G R, Feldmann P, Ilagan O, Johnson S, Plaetinck G, Munyikwa T, Pleau M, Vaughn T, Roberts J. Control of coleopteran insect pests through RNA interference. Nat Biotech, 2007, 25: 1322–1326
[15]Dunoyer P, Himber C, Voinnet O. Induction, suppression and requirement of RNA silencing pathways in virulent Agrobacterium tumefaciens infections. Nat Genet, 2006, 38: 258–263
[16]Borsani O, Zhu J, Verslues P E, Sunkar R, Zhu J K. Endogenous siRNAs derived from a pair of natural cis-antisense transcripts regulate salt tolerance in Arabidopsis. Cell, 2005, 123: 1279–1291
[17]Sunilkumar G, Campbell L M, Puckhaber L, Stipanovic R D, Rathore K S. Engineering cottonseed for use in human nutrition by tissue-specific reduction of toxic gossypol. Proc Natl Acad Sci USA, 2006, 103: 18054–18059
[18]Duan C G, Wang C H, Fang R X, Guo H S. Artificial microRNAs highly accessible to targets confer efficient virus resistance in plants. J Virol, 2008, 82: 11084–11095
[19]Nagamatsu A, Masuta C, Senda M, Matsuural H, Kasai A, Hong J S, Kitamura K, Abe J, Kanazawa A. Functional analysis of soybean genes involved in flavonoid biosynthesis by virus-induced gene silencing. Plant Biotech J, 2007, 5: 778–790
[20]Mattew L. RNAi for plant functional genomics. Compar Funct Genom, 2004, 5: 240–244
[21]Tuttle J R, Idris A M, Brown J K, Haigler C H, Robertson D. Geminivirus-mediated gene silencing from cotton leaf crumple virus is enhanced by low temperature in cotton. Plant Physiol, 2008, 148: 41–50
[22]Fire A, Xu S, Montgomery M K, Kostas S A, Driver S E, Mello C C. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature, 1998, 391: 806–811
[23]Ogita S, Uefuji H, Yamaguchi Y, Koizumi N, Sano H. RNA interference: producing decaffeinated coffee plants. Nature, 2003, 423: 823
[24]Sunilkumar G, Campbell L M, Puckhaber L, Stipanovic R D, Rathore K S. Engineering cottonseed for use in human nutrition by tissue-specific reduction of toxic gossypol. Proc Natl Acad Sci USA, 2006, 103: 18054–18059
[25]He S-L(何水林), Zheng J-G(郑金贵), Wang X-F(王晓峰), Wang Y-H(王燕华), Xu M(许明), Li B-L(李斌莲), Lin M(林明). Plant secondary metabolism: function, regulation and gene engineering. Chin J Appl Environ Biol (应用与环境生物学报), 2002, 8(5): 558–563 (in Chinese with English abstract)
[26]McCue K F, Allen P V, Shepherd L V T, Blake A, Whitworth J, Maccree M M, Rockhold D R, Stewart D, Davies H V, Belknap W R. The primary in vivo steroidal alkaloid glucosyltransferase from potato. Phytochemistry, 2006, 67: 1590–1597
[27]Kita M, Hirata Y, Moriguchi T, Endo-Inagaki T, Matsumoto R, Hasegawa S, Suhayda C G, Omura M. Molecular cloning and characterization of a novel gene encoding limonoid UDP-glucosyltransferase in Citrus. FEBS Lett, 2000, 469: 173–178
[28]Wang W-T(王旺田), Zhang J-W(张金文), Wang D(王蒂), Tao S-H(陶士珩), Ji Y-L(季彦林), Wu B(吴兵). Relation between light qualities and accumulation of steroidal glycoalkaloids as Well as signal molecule in cell in potato tubers. Acta Agron Sin (作物学报), 2010, 36(4): 629–635 (in Chinese with English abstract)
[29]Ma L-B(马凌波), Zhang D-B(张大兵), Shen M-S(沈明山), Chen L(陈亮), Chen M-Z(陈睦传). Cloning and sequencing of the cDNA of UDPG-glucosyltransferase from Stevia rebaudiana. J Xiamen Univ (厦门大学学报), 2002, 41(5): 531–535 (in Chinese with English abstract)
[30]Wesley S V, Helliwell C A, Smith N A,Wang M B, Rouse D T, Liu Q, Gooding P S, Singh S P, Abbott D, Waterhouse P M. Construct design for efficient, effective and high- throughput gene silencing in plants. Plant J, 2001, 27: 581–590
[31]Thomas C L, Jones L, Baulcombe D C, Maule A J. Size constrains for targeting post-transcriptional gene silencing and for RNA-directed methylation in Nicotiana benthamiana using a potato virus X vector. Plant J, 2001, 25: 417–425
[32]Burch-Smith T M, Miler J L. PTGS approaches to large-scale functional genomics in plants. In: Hannon ed. RNAi: a Guide to Gene Silencing. New York: Cold Spring Harbor Laboratory Press, 2003. pp 243–263
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