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作物学报 ›› 2012, Vol. 38 ›› Issue (07): 1187-1195.doi: 10.3724/SP.J.1006.2012.01187

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

马铃薯块茎颗粒结合型淀粉合酶基因的克隆及其RNAi载体的构建

刘玉汇1,2，王丽3，杨宏羽1，余斌1,2，李元铭4，张俊莲1,2,*，王蒂1,2,*   

  1. 1 甘肃省作物遗传改良与种质创新重点实验室 / 甘肃农业大学农学院, 甘肃兰州730070；2 甘肃省干旱生境作物学重点实验室, 甘肃兰州730070；3 甘肃农业大学生命科学技术学院, 甘肃兰州730070；4 甘肃农村发展研究院, 甘肃兰州730070
  • 收稿日期:2012-01-09 修回日期:2012-04-15 出版日期:2012-07-12 网络出版日期:2012-05-11
  • 通讯作者: 张俊莲, E-mail: zhangjunlian99@yahoo.com.cn; 王蒂, E-mail: wangd@gsau.edu.cn
  • 基金资助:

    本研究由国家科技支撑计划(2012BAD06B03), 国家现代农业产业技术体系建设项目(CARS-10-P18)和甘肃省重大专项(1102NKDM025)资助。

Cloning of Granule-Bound Starch Synthase Gene and Construction of Its RNAi Vector in Potato Tuber

LIU Yu-Hui1,2,WANG Li3,YANG Hong-Yu1,YU Bin1.2,LI Yuan-Ming4,ZHANG Jun-Lian1,2,*,WANG Di1,2,*   

  1. 1 Gansu Key Laboratory of Crop Genetic & Germplasm Enhancement /College of Agronomy, Gansu Agricultural University, Lanzhou 730070, China; 2 Gansu Key Laboratory of Aridland Crop Science, Lanzhou 730070, China; 3 College of Life Sciences and Technology, Gansu Agricultural University, Lanzhou 730070, China; 4 Gansu Rural Development Research Institute, Lanzhou 730070, China
  • Received:2012-01-09 Revised:2012-04-15 Published:2012-07-12 Published online:2012-05-11
  • Contact: 张俊莲, E-mail: zhangjunlian99@yahoo.com.cn; 王蒂, E-mail: wangd@gsau.edu.cn

PDF

1319

被引次数

7

摘要： GBSSI是马铃薯块茎中控制直链淀粉合成的关键酶, 为培育高支链淀粉含量或纯支链淀粉含量的转基因马铃薯材料, 根据GenBank登录号X58453设计特异引物, 采用RT-PCR技术获得马铃薯块茎GBSSI相似基因, 利用生物信息学相关软件分析, 预测GBSSI相似基因cDNA序列编码的蛋白质结构和功能。结果表明, 克隆的GBSSI相似基因与报道的GBSSI基因序列相似性达到99.78%, 其开放阅读框长1 824 bp, 编码607个氨基酸, 具有许多重要功能位点；三级结构预测结果表明该蛋白具有淀粉合成功能, 基因序列已注册到GenBank, 序列登录号为EU403426。以此基因CDS内542 bp的靶标序列作为干扰区段, 扩增GBSSI的正反向基因片段, 并引入237 bp的内含子序列, 构建由Patatin启动子驱动的具有“正义基因片段gbss A-内含子VP1-ABI3-like protein-反义基因片段gbss B”的植物干扰表达载体pBI121g-PgABI, 将为淀粉合成的进一步研究和高支链淀粉含量或纯支链淀粉含量的马铃薯品种的培育奠定基础。

关键词: 马铃薯, 颗粒结合型淀粉合酶, 基因克隆, 序列分析, RNAi载体

Abstract: There is about 17% starch in potato (Solanum tuberosum L.) tubers. Potato starch granules are composed of two polysaccharides, unbranched amylose (approximately 20% to 25%) and branched amylopectin (approximately 75% to 80%). To develop transgenic potato with high-amylopectin in tubers, we got a cDNA sequence of the potato GBSSI from the total RNA of potato tubers by RT-PCR using specific primers of conserved domain of GenBank Accession Number X58453 sequence.The GBSSI cDNA sequence shared 99.78% similarity with the GBSSI gene in GenBank (accession No. X58453). The full-length of cDNA was 1 824 bp, which contained 607 amino acids, three conserved domains and many important functional sites. The 3D structure of GBSSI was highly similar to that of the glycogen synthase, indicating that GBSSI has a function of starch synthesis. GBSSI similar gene obtained here was granule-bound starch synthase, and its sequence was submitted to GenBank, with the accession number of EU403426. On the basis of a 542 bp RNAi target region from the CDS of GBSSI, the sense and antisense fragments were amplified and separated by a 237 bp intron to construct the RNA interference expression vector pBI121g-PgABI containing “sense gbssA-VP1-ABI3-like protein intron-antisense gbss B” regulated by Patatin promoter, which will lay a solid foundation for the study on synthesis of starch and breeding of transgenic potato with high amylopectin content or pure amylopectin.

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

[1]Yu T-F(于天峰), Xia P(夏平). Characteristic and use of the potato starch. Chin Agric Sci Bull (中国农学通报), 2005, 21(2): 55–58 (in Chinese with English abstract)

[2]Nuessli J, Handschin S, Conde-Petit B, Escher F. Rheology and structure of amylopectin potato starch dispersions without and with emulsifier addition. Starch-Stärke, 2000, 52: 22–27

[3]Wang Z-R(王中荣), Liu X(刘雄). Study on properties and application of high amylose starch. Cereal & Oil (粮食与油脂), 2005, 11: 10–13 (in Chinese with English abstract)

[4]Xie B-X(谢碧霞), Zhong Q-P(钟秋平), Xie T(谢涛), Li A-P(李安平). Properties, application and development strategies of starch. Nonwood For Res (经济林研究), 2004, 22(4): 61–64 (in Chinese with English abstract)

[5]Tsai C Y. The function of the waxy locus in starch synthesis in maize endosperm. Biochem Genet, 1974, 11: 83–96

[6]James M G, Denyer K, Myers A M. Starch synthesis in the cereal endosperm. Curr Opin Plant Biol, 2003, 6: 215–222

[7]Kuipers A G J, Jacobsen E, Visser R G F. Formation and deposition of amylose in the potato tuber starch granule are affected by the reduction of granule-bound starch synthase gene expression. Plant Cell, 1994, 6: 43–52

[8]Kuipers G, Vreem J, Meyer H, Jacobsen E, Feenstra W, Visser R. Field evaluation of antisense RNA mediated inhibition of GBSS gene expression in potato. Euphytica, 1991, 59(1): 83–91

[9]Jiang P-X(蒋培霞), Wang H-S(王海胜), Li Y-X(李艳霞), Ma X-D(马向东). The mechanism and application prospect of RNA interference (RNAi). J Henan Agric Univ (河南农业大学学报), 2004, 38(1): 64–67 (in Chinese with English abstract)

[10]Smith N A, Singh S P, Wang M B, Stoutjesdijk P A, Green A G, Waterhouse P M. Gene expression: total silencing by intron-spliced hairpin RNAs. Nature, 2000, 407: 319–320

[11]Matthew L. RNAi for plant functional genomics. Comp Funct Genom, 2004, 5: 240–244

[12]Regina A, Kosar-Hashemi B, Ling S, Li Z, Rahman S, Morell M. Control of starch branching in barley defined through differential RNAi suppression of starch branching enzyme IIa and IIb. J Exp Bot, 2010, 61: 1469–1482

[13]Otani M, Hamada T, Katayama K, Kitahara K, Kim S-H, Takahata Y, Suganuma T, Shimada T. Inhibition of the gene expression for granule-bound starch synthase I by RNA interference in sweet potato plants. Plant Cell Rep, 2007, 26: 1801–1807

[14]Zhang G, Cheng Z, Zhang X, Guo X, Su N, Jiang L, Mao L, Wan J. Double repression of soluble starch synthase genes SSIIa and SSIIIa in rice (Oryza sativa L.) uncovers interactive effects on the physicochemical properties of starch. Genome, 2011, 54(6): 448–459

[15]Zhang J-L(张俊莲), Wang D(王蒂), Zhang J-W(张金文), Chen Z-H(陈正华). Modification of pBI121 vector and expression vector construction Na+/H+antiporter of Arabidopsis thaliana. Mol Plant Breed (分子植物育种), 2006, 4(6): 811–818 (in Chinese with English abstract)

[16]Sambrook J, Russell D W. Molecular Cloning: A Laboratory Manual, 3rd edn. New York: Cold Spring Harbor Laboratory Press, 2001. pp 305–320

[17]Reynolds A, Leake D, Boese Q, Scaringe S, Marshall W S, Khvorova A. Rational siRNA design for RNA interference. Nat Biotech, 2004, 22: 326–330

[18]Peng J-S(彭佶松), Zhao S-J(赵淑娟), Wu X-J(吴晓俊), Liu D(刘涤), Hu Z-B(胡之璧), Xu Z-A(许政皑). cDNA cloning and structural analysis of granule- bound starch synthase gene of hairy roots of Astragalus membranaceus. Acta Bot Sin (植物学报), 2004, 42(9): 940–945

[19]Dry I, Smith A, Edwards A, Bhattacharyya M, Dunn P, Martin C. Characterization of cDNAs encoding two isoforms of granule-bound starch synthase which show differential expression in developing storage organs of pea and potato. Plant J, 1992, 2: 193–202

[20]Hammond S M, Bernstein E, Beach D, Hannon G J. An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells. Nature, 2000, 404 293–296

[21]Li J-Y(李竞芸), Zhang G-H(张广辉), Wang S(王森). RNA interference and its applications on plant improvement. Mol Plant Breed (分子植物育种), 2007, 5(6): 145–148

[22]Burch-Smith T M, Miller J L, Dinesh-Kumar S P. PTGS approaches to large-scale functional genomics in plants. RNAi: A Guide to Gene Silencing. New York: Cold Spring Harbor Laboratory Press, 2003. pp 243–264

[23]Li J-R(李加瑞), Zhao W(赵伟), Li Q-Z(李全梓), Ye X-G(叶兴国), An B-Y(安宝燕), Li X(李祥), Zhang X-S(张宪省). RNA silencing of waxy gene results in low levels of amylose in the seeds of transgenic wheat (Triticum aestivum L.). Acta Genet Sin (遗传学报), 2005, 32(8): 846–854

[24]Visser R G F, Somhorst I, Kuipers G J, Ruys N J, Feenstra W J, Jacobsen E. Inhibition of the expression of the gene for granule-bound starch synthase in potato by antisense constructs. Mol Gen Genet, 1991, 225: 289–296

[25]Salehuzzaman S N I M, Jacobsen E, Visser R G F. Isolation and characterization of a cDNA encoding granule-bound starch synthase in cassava (Manihot esculenta Crantz) and its antisense expression in potato. Plant Mol Biol, 1993, 23: 947–962

[26]Hofvander P, Andersson M, Larsson C-T, Larsson H. Field performance and starch characteristics of high-amylose potatoes obtained by antisense gene targeting of two branching enzymes. Plant Biotechnol J, 2004, 2: 311–320

[27]Fire A X 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

[28]Ashrafi K, Chang F Y, Watts J L, Fraser A G, Kamath R S, Ahringer J, Ruvkun G. Genome-wide RNAi analysis of Caenorhabditis elegans fat regulatory genes. Nature, 2003, 421: 268–272

[29]Frankish H. Consortium uses RNAi to uncover genes' function. Lancet, 2003, 361: 584

[30]Wang X B, Wu Q, Ito T, Cillo F, Li W X, Chen X, Yu J L, Ding S W. RNAi-mediated viral immunity requires amplification of virus-derived siRNAs in Arabidopsis thaliana . Proc Natl Acad Sci USA, 2010, 107: 484–489

[31]Sun Z N, Song Y Z, Yin G H, Zhu C X, Wen F J. HpRNAs derived from different regions of the NIb gene have different abilities to protect tobacco from infection with potato virus Y. J Phytopathol, 2010, 158: 566–568

[32]Golldack D, Lüking I, Yang O. Plant tolerance to drought and salinity: stress regulating transcription factors and their functional significance in the cellular transcriptional network. Plant Cell Rep, 2011, 30: 1383–1391

[33]Wu L, Bhaskar P, Busse J, Zhang R, Bethke P, Jiang J. Developing cold-chipping potato varieties by silencing the vacuolar invertase gene. Crop Sci, 2011, 51: 981–990
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