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Effects of RNAi Silencing of SSIII Gene on Phosphorus Content and Characteristics of Starch in Potato Tubers |
DU Hong-hui, YANG Tao, MA Cong-yu, FENG Dan, ZHANG Ning, SI Huai-jun, WANG Di |
1.Gansu Provincial Key Laboratory of Aridland Crop Science, Gansu Key Laboratory of Crop Genetic and Germplasm Enhancement,Lanzhou 730070, P.R.China
2.College of Life Science and Technology, Gansu Agricultural University, Lanzhou 730070, P.R.China
3.Biological Research Institute, Gansu Academy of Sciences, Lanzhou 730000, P.R.China |
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摘要 The sense and antisense fragments of the soluble starch synthase (SSIII) gene and the intron fragment of somatic embryogenesis receptor-like kinase (SERK1) gene were cloned from potato using PCR techniques. The RNAi plant expression vectors pBI-SSIII-RNAi and pBIC-SSIII-RNAi were constructed which containing fusion fragment of “sense fragment-intron-antisense fragment” driven by the constitutive expression promoter CaMV 35S and the tuber-specific expression promoter CIPP, respectively. The putative transgenic plants of potato cultivars Kexin-1 and Kexin-4 were obtained using Agrobacterium-mediated transformation method. PCR assay showed that the interference fragment of SSIII gene was integrated into potato genome. The RT-PCR analysis showed that the expression of SSIII gene was repressed apparently on the transcription level. Starch granules of the transgenic potato plants were different in morphology and became cracked in starch granule centre compared with the non-transgenic control plants. The amylose content of starch was increased by 2.68-29.05%, amylopectin to amylose ratio of starch had declined significantly, and the phosphorus content of the starch of the transgenic plants was reduced 9.94-58.36% compared with control plants. The results could provide certain foundation for improvement of potato starch quality.
Abstract The sense and antisense fragments of the soluble starch synthase (SSIII) gene and the intron fragment of somatic embryogenesis receptor-like kinase (SERK1) gene were cloned from potato using PCR techniques. The RNAi plant expression vectors pBI-SSIII-RNAi and pBIC-SSIII-RNAi were constructed which containing fusion fragment of “sense fragment-intron-antisense fragment” driven by the constitutive expression promoter CaMV 35S and the tuber-specific expression promoter CIPP, respectively. The putative transgenic plants of potato cultivars Kexin-1 and Kexin-4 were obtained using Agrobacterium-mediated transformation method. PCR assay showed that the interference fragment of SSIII gene was integrated into potato genome. The RT-PCR analysis showed that the expression of SSIII gene was repressed apparently on the transcription level. Starch granules of the transgenic potato plants were different in morphology and became cracked in starch granule centre compared with the non-transgenic control plants. The amylose content of starch was increased by 2.68-29.05%, amylopectin to amylose ratio of starch had declined significantly, and the phosphorus content of the starch of the transgenic plants was reduced 9.94-58.36% compared with control plants. The results could provide certain foundation for improvement of potato starch quality.
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Received: 20 July 2011
Accepted:
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Fund: This work was supported by the National High-Technology R&D Program of China (2006AA100107) and the National Natural Science Foundation of China (30871573, 31160298). |
Corresponding Authors:
Correspondence SI Huai-jun, Tel: +86-931-7631077, E-mail: hjsi@gsau.edu.cn; WANG Di, Tel: +86-931-7632826, E-mail: wangd@gsau.edu.cn
E-mail: hjsi@gsau.edu.cn
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About author: DU Hong-hui, Mobile: 18009325320, E-mail: duhonghui2008@163.com; |
Cite this article:
DU Hong-hui, YANG Tao, MA Cong-yu, FENG Dan, ZHANG Ning, SI Huai-jun, WANG Di.
2012.
Effects of RNAi Silencing of SSIII Gene on Phosphorus Content and Characteristics of Starch in Potato Tubers. Journal of Integrative Agriculture, 12(12): 1985-1992.
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[1]Abel G J W, Springer F, Willmitzer L, Kossmann J. 1996.Cloning and functional analysis of a cDNA encoding anovel 139 kDa starch synthase from potato (Solanumtuberosum L.). The Plant Journal, 10, 981-991.[2]Andersson M, Melander M, Pojmark P, Larsson H, BülowL, Hofvander P. 2006. Targeted gene suppression byRNA interference: an efficient method for productionof high-amylose potato lines. Journal of Biotechnology,123, 137-148.[3]Buleon A, Colonna P, Planchot V, Ball S. 1998. Starchgranules: structure and biosynthesis. InternationalJournal of Biological Macromolecules, 23, 85-112.[4]Chen Y Q. 2002. Methods and Technologies of BiochemistryExperiment. High Eduction Press, Beijing. pp. 175-178.(in Chinese)[5]Craig J, Lloyd J R, Tomlinson K, Barber L, Edwards A,Wang T L, Martin C, Hedley C L, Smith A M. 1998.Mutations in the gene encoding starch synthase IIprofoundly alter amylopectin structure in pea embryos.The Plant Cell, 10, 413-426.[6]Edwards K, Johnstone C, Thompson C. 1991. A simple andrapid method for the preparation of plant genomic DNAfor PCR analysis. Nucleic Acids Research, 19, 1349.Edwards A, Marshall J, Sidebottom C, Visser R G F, Smith AM, Martin C. 1995. Biochemical and molecularcharacterization of a novel starch synthase from potatotubers. The Plant Journal, 8, 283-294.[7]Edwards A, Fulton D C, Hylton C M, Jobling S A, Gidley M,Rossner U, Martin C, Smith A M. 1999. A combinedreduction in activity of starch synthase II and III ofpotato has novel effects on the starch of tubers. ThePlant Journal, 17, 251-261.[8]Fontaine T, D′Hulst C, Maddelein M L, Routier F, Pépin TM, Decq A, Wieruszeski J M, Delrue B, van-denKoornhuyse N, Bossu J P. 1993. Toward anunderstanding of the biogenesis of the starch granule.Evidence that Chlamydomonas soluble starch synthaseII controls the synthesis of intermediate size glucansof amylopectin. Journal of Biological Chemistry, 268,16223-16230.[9]Gao M, Wanat J, Stinard P S, James M G, Myers AM. 1998.Characterization of dull1, a maize gene coding for anovel starch synthase. The Plant Cell, 10, 399-412.[10]Guo X M, Zhang X D, Liang R Q, Chen X Q, Yang F P, LiuQ, Song X Y. 2008. Increase of maize amylose contentthrough RNA interference. Journal of AgriculturalBiotechnology, 16, 658-661. (in Chinese)[11]Jobling S A, Westcott R J, Tayal A, Jeffcoat R, Schwall G P.2002. Production of a freeze-thaw-stable potato starchby antisense inhibition of three starch synthase genes.Nature Biotechnology, 20, 295-299.[12]Lloyd J R, Landschütze V, Kossmann J. 1999. Simultaneousantisense inhibition of two starch-synthase isoformsin potato tubers leads to accumulation of grosslymodified amylopectin. Biochemical Journal, 338, 515-521.[13]Lorberth R, Ritte G, Willmitzer L, Kossmann J. 1998.Inhibition of a starch-granule-bound protein leads tomodified starch and repression of cold sweetening.Nature Biotechnology, 16, 473-477.[14]Marshall J, Sidebottom C, Debet M, Martin C, Smith A M,Edwards A. 1996. Identification of the major starchsynthase in the soluble fraction of potato tubers. ThePlant Cell, 8, 1121-1135.[15]Martinl C, Smith AM. 1995. Starch biosynthesis. The PlantCell, 7, 971-985.[16]Shimada T, Otani M, Hamada T, Kim S H. 2006. Increase ofamylose content of sweetpotato starch by RNAinterference of the starch branching enzyme II gene(IbSBEII). Plant Biotechnology, 23, 85-90.[17]Si H J, Xie C H, Liu J. 2003. An efficient protocol forAgrobacterium-mediated transformation of microtuberand the introduction of an antisense class I patatingene into potato. Acta Agronomica Sinica, 29, 801-805.[18]Slattery C J, Halil K I, Okita T W. 2000. Engineering starchfor increased quantity and quality. Trends in PlantScience, 5, 291-298.[19]Swinkels J J M. 1985. Composition and properties ofcommercial native starches. Starch/Stärke, 37, 1-5.[20]Tomlinson K, Craig J, Smith A M. 1998. Major differencesin isoform composition of starch synthase betweenleaves and embryos of pea (Pisum sativum L.). Planta,204, 86-92.[21]Regina A, Bird A, Topping D, Bowden S, Freeman J, BarsbyT, Kosar-Hashemi B, Li Z Y, Rahman S,Morell M. 2006.High-amylose wheat generated by RNA interferenceimproves indices of large-bowel health in rats.Proceedings of the National Academy of Sciences of the United States of America, 103, 3546-3551.[22]Roldán I, Wattebled F, Lucas M M, DelvalléD, Planchot V,Jiménez S, Pérez R, Ball S, D’Hulst C, Mérida A. 2007.The phenotype of soluble starch synthase IV defectivemutants of Arabidopsis thaliana suggests a novelfunction of elongation enzymes in the control of starchgranule formation. The Plant Journal, 49, 492-504.[23]Yang T, Zhang N, Li L, Liu H Y, Si H J, Wang D. 2009.Cloning and bioinformatic analysis of soluble starchsynthase SSIII gene in potato. Molecular PlantBreeding, 7, 545-549. (in Chinese)[24]Yu T F, Xia P. 2005. Characteristic and use of the potatostarch. Chinese Agricultural Science Bulletin, 21, 55-58. (in Chinese)[25]Zhang X, Myers AM, James M G. 2005. Mutations affectingstarch synthaseIII in Arabidopsis alter leaf starchstructure and increase the rate of starch synthesis.Plant Physiology, 138, 663-674. (in Chinese)[26]Zhang X, Szydlowski N, Delvallé D, D’Hulst C, James M G,Myers A M. 2008. Overlapping functions of the starchsynthases SSII and SSIII in amylopectin biosynthesisin Arabidopsis. BMC Plant Biology, 8, 96.Zhang Y C, Tian F. 2007. Research Methods of PotatoExperiments. China Agricultural Science andTechnology Press, Beijing. pp. 185-186. (in Chinese) |
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