Scientia Agricultura Sinica ›› 2013, Vol. 46 ›› Issue (18): 3750-3757.doi: 10.3864/j.issn.0578-1752.2013.18.002

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

Prodution of High-Amylopectin Potato Plants by Using ihpRNAi Technology

 LIU  Yu-Hui-12, WANG  Li-3, YANG  Hong-Yu-1, YU  Bin-12, LI  Yuan-Ming-4, ZHANG  Jun-Lian-12, WANG  Di-12   

  1. 1.College of Agronomy, Gansu Agricultural University/Gansu Key Laboratory of Crop Genetic and Germplasm Enhancement, Lanzhou 730070
    2.Gansu Key Laboratory of Aridland Crop Science, Lanzhou 730070
    3.College of Life Sciences and Technology, Gansu Agricultural University, Lanzhou 730070
    4.Gansu Rural Development Research Institute, Lanzhou 730070
  • Received:2013-03-13 Online:2013-09-15 Published:2013-06-03

Abstract: 【Objective】 The objective of this study is to develop transgenic potato (Solanum tuberosum L.) plants with high-amylopectin starch in its tubers.【Method】RNA interference expression vector pBI121g-PgABI driven by Patatin was transformed into elite potato cultivar ‘Gannongshu 2’ by Agrobacterium-mediated transformation. The transgenic plants were identified by PCR, Southern-blotting, semi-quantitative RT-PCR and real-time quantitative PCR and the starch content of transgenic potatoes was determined.【Result】Ten transgenic potato lines were confirmed by PCR and Southern blot analysis that the target gene integrated into the plant genomes. Result of semi-quantitative RT-PCR indicated that the accumulation of mRNAs derived from GBSSI was inhibited significantly in all transgenic lines, which were not detectable in 6 tansgenic lines. Result of real-time quantitative PCR showed that the inhibition ratio was 66.27%-93.53%. There were significant changes of starch content in ten transgenic microtubers,of which the amylopectin content was up to 90.16%-98.84%, 10.31%-20.92% higher than the non-transgenic microtuber. A significant correlation was found between inhibition ratio of mRNA and amylopectin content of transgenic potato plants (r=0.937, P<0.01). 【Conclusion】ihpRNAi technology can be used effectively in the production of high-amylopectin potato or pure-amylopectin potato by silencing endogenesis gene GBSSI.

Key words: potato (Solanum tuberosum L.) , high-amylopectin starch , ihpRNAi , granule-bound starch synthase

[1]于天峰. 马铃薯淀粉的糊化特性、用途及品质改良. 中国马铃薯, 2005, 19(4): 223-225.

Yu T F. Pasting properties, utilization and quality improvement of potato starch. Chinese Potato Journal, 2005, 19(4): 223-225. (in Chinese)

[2]罗发兴, 黄强, 张乐兴. 蜡质马铃薯淀粉的研究开发.食品研究与开发, 2007, 28(7): 148-149.

Luo F X, Huang Q, Zhang L X. The study and development of waxy potato. Food Research and Development, 2007, 28(7): 148-149. (in Chinese)

[3]Baguma Y, Sun C, Ahlandsberg S, Mutisya J, Palmqvist S, Rubaihayo P R, Magambo M J, Egwang T G, Larsson H, Jansson C. Expression patterns of the gene encoding starch branching enzyme II in the storage roots of cassava (Manihot esculenta Crantz). Plant Science, 2003, 164(5): 833-839.

[4]Wang Z Y, Wu Z L, Xing Y Y, Zheng F Q, Guo X L, Zhang W G, Hong M M. Molecular characterization of rice Wx gene. Science in China,SerB, 1992, 35(5): 558.

[5]Hovenkamp-Hermelink J, Jacobsen E, Ponstein A, Visser R, Vos-Scheperkeuter G, Bijmolt E, Vries J N, Witholt B, Feenstra W. Isolation of an amylose-free starch mutant of the potato (Solanum tuberosum L.). Theoretical and Applied Genetics, 1987, 75(1): 217-221.

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

[7]Kuipers A G J, Soppe W J J, Jacobsen E, Visser R G F. Factors affecting the inhibition by antisense RNA of granule-bound starch synthase gene expression in potato. Molecular and General Genetics, 1995, 246(6): 745-755.

[8]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. Molecular and General Genetics, 1991, 225(2): 289-296.

[9]Hofvander P, Persson P T, Tallberg A, Wikström O. Genetically engineering modification of potato to form amylopectin-type starch: Europe, EP 0563189 [P/OL]. 2010-09-22.

[10]Wesley S V, Helliwell C A, Smith N A, Wang M, 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 efficient, effective and highthroughput gene silencing in plants. The Plant Journal, 2001, 27(6): 581-590.

[11]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(6802): 319-320.

[12]Matthew L. RNAi for plant functional genomics. Comparative and Functional Genomics, 2004, 5(3): 240-244.

[13]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. Journal of Experimental Botany, 2010, 61(5): 1469-1482.

[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]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 and Cell Reports, 2007, 26(10): 1801-1807.

[16]刘玉汇, 王丽, 杨宏羽, 余斌, 李元铭, 张俊莲, 王蒂. 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. 作物学报, 2012, 38(7): 1187-1195.

Liu Y H, Wang L, Yang H Y, Yu B, Li Y M, Zhang J L, Wang D. Cloning of granule-bound Starch synthase gene and construction of its RNAi vector in potato tuber. Acta Agronomica Sinica, 2012, 38(7): 1187-1195.(in Chinese)

[17]王关林, 方宏筠. 植物基因工程: 第二版. 北京: 科学出版社, 2002.

Wang G L, Fang H J. Plant Gene Engineering: 2nd ed.. Beijing: Science Press, 2002. (in Chinese)

[18]Si H J, Xie C H, Liu J. An efficient protocol for Agrobacterium-mediated transformation with microtuber and the introduction of an antisense class I patatin gene into potato. Acta Agronomica Sinica, 2003, 29(6): 801-805.

[19]Murray M, Thompson W F. Rapid isolation of high molecular weight plant DNA. Nucleic Acids Research, 1980, 8(19): 4321-4326.

[20]Livak K J, Schmittgen T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2-△△CT method. Methods, 2001, 25(4): 402-408.

[21]Noda T, Tsuda S, Mori M, Takigawa S, Matsuura-Endo C, Saito K, Arachichige Mangalika W H, Hanaoka A, Suzuki Y, Yamauchi H. The effect of harvest dates on the starch properties of various potato cultivars. Food Chemistry, 2004, 86(1): 119-125.

[22]陈毓荃. 生物化学实验方法和技术. 北京: 科学出版社, 2002.

Chen Y Q. Methods and Technologies of Biochemistry Experiment. Beijing: Science Press, 2002. (in Chinese)

[23]Salehuzzaman S, Jacobsen E, Visser R. Isolation and characterization of a cDNA encoding granule-bound starch synthase in cassava (Manihot esculenta Crantz) and its antisense expression in potato. Plant Molecular Biology, 1993, 23(5): 947-962.

[24]Hofvander P, Andersson M, Larsson C, Larsson H. Field performance and starch characteristics of high-amylose potatoes obtained by antisense gene targeting of two branching enzymes. Plant Biotechnology Journal, 2004, 2(4): 311-320.

[25]Stoutjesdijk P A, Singh S P, Liu Q, Hurlstone C J, Waterhouse P A, Green A G. hpRNA-mediated targeting of the Arabidopsis FAD2 gene gives highly efficient and stable silencing. Plant Physiology, 2002, 129(4): 1723-1731.

[26]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. Proceedings of the National Academy of Sciences of the United States of America, 2010, 107(1): 484-489.

[27]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. Journal of Phytopathology, 2010, 158(7/8): 566-568.

[28]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 and Cell Reports, 2011, 30(8): 1383-1391.

[29]Wu L, Bhaskar P, Busse J, Zhang R, Bethke P, Jiang J. Developing cold-chipping potato varieties by silencing the vacuolar invertase gene. Crop Science, 2011, 51(3): 981-990.

[30]Carciofi M, Blennow A, Jensen S L, Shaik S S, Henriksen A, Buléon A, Holm P B, Hebelstrup K H. Concerted suppression of all starch branching enzyme genes in barley produces amylose-only starch granules. BMC Plant Biology, 2012, 12: 223-239.

[31]Sestili F, Janni M, Doherty A, Botticella E, D'Ovidio R, Masci S, Jones H D, Lafiandra D. Increasing the amylose content of durum wheat through silencing of the SBEIIa genes. BMC Plant Biology, 2010, 10: 144-156.

[32]Hakata M, Kuroda M, Miyashita T, Yamaguchi T, Kojima M, Sakakibara H, Mitsui T, Yamakawa H. Suppression of alpha-amylase genes improves quality of rice grain ripened under high temperature. Plant Biotechnology Journal, 2012, 10(9): 1110-1117.

[33]Guan S, Wang P, Liu H. Production of high-amylose maize lines using RNA interference in sbe2a. African Journal of Biotechnology, 2011, 10(68): 15229-15237.

[34]Koehorst-van Putten H J J, Sudarmonowati E, Herman M, Pereira-Bertram I J, Wolters A M, Meima H, de Vetten N, Raemakers C J, Visser R G. Field testing and exploitation of genetically modified cassava with low-amylose or amylose-free starch in Indonesia. Transgenic Research, 2012, 21(1): 39-50.

[35]Du H H, Yang T, Ma C Y, Feng D, Zhang N, Si H J, Wang D. Effects of RNAi silencing of SSIII gene on phosphorus content and characteristics of starch in potato tubers. Journal of Integrative Agriculture, 2012, 11(12): 1985-1992.

[36]郭志鸿, 张金文, 王蒂, 陈正华. 用RNA干扰技术创造高直链淀粉马铃薯材料. 中国农业科学, 2008, 41(2): 494-501.

Guo Z H, Zhang J W, Wang D, Chen Z H. Using RNAi technology to produce high-amylose potato plants. Scientia Agricultura Sinica, 2008, 41(2): 494-501.(in Chinese)
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