马铃薯ARF基因RNAi载体的遗传转化及对其试管薯生理特性的影响

doi:10.11686/cyxb20150216

草业学报 ›› 2015, Vol. 24 ›› Issue (2): 142-147.DOI: 10.11686/cyxb20150216

马铃薯ARF基因RNAi载体的遗传转化及对其试管薯生理特性的影响

裴瑞芳^{1, 2}, 刘英^{1, 2}, 刘柏林¹, 张宁², 司怀军^{1, 2*, *}, 王蒂^{1*, *}

1.甘肃省作物遗传改良与种质创新重点实验室,甘肃省干旱生境作物学省部共建国家重点实验室培育基地,甘肃兰州 730070;
2.甘肃农业大学生命科学技术学院,甘肃兰州 730070

收稿日期:2014-02-25 出版日期:2015-02-20 发布日期:2015-02-20
作者简介:裴瑞芳(1990-),女,甘肃天水人,在读硕士。E-mail:peirfsmile@163.com
基金资助:
甘肃省杰出青年基金项目(1308RJDA011)和国家自然科学基金项目(31160298)资助

Genetic transformation using an RNAi vector containing an ARF gene in potato and effects on physiological characteristics of microtuber

PEI Ruifang^{1, 2}, LIU Ying^{1, 2}, LIU Bailin¹, ZHANG Ning², SI Huaijun^{1, 2, *}, WANG Di^{1, *}

1.Gansu Key Laboratory of Crop Genetic and Germplasm Enhancement, Gansu Provincial Key Laboratory of Aridland Crop Science, Lanzhou 730070, China;
2.College of Life Science and Technology, Gansu Agricultural University, Lanzhou 730070, China

Received:2014-02-25 Online:2015-02-20 Published:2015-02-20

摘要/Abstract

摘要： 用含有ADP核糖基化因子(ARF)基因的干扰载体的农杆菌转化马铃薯栽培品种“甘农薯2号”,获得转化植株48株,经卡那霉素抗性筛选和PCR检测证明ARF基因已成功整合到马铃薯基因组中。用实时荧光定量PCR检测表明,转化植株中ARF基因的表达较对照均有不同程度的下降,在转基因株系Z-11和Z-12中,ARF基因表达的干扰程度分别高达92.53%和93.22%。对转基因植株诱导的试管薯的淀粉、可溶性糖、蛋白质、蔗糖和葡萄糖含量测定结果表明,与未转基因的对照相比,转基因植株的淀粉含量提高了4.29%~34.27%,蛋白质含量提高了1.47%~7.35%,可溶性糖含量提高了1.44%~17.39%;蔗糖含量提高了11.53%~53.84%,而葡萄糖含量降低了6.06%~21.21%。

Abstract: Using potato (Solanum tuberosum) cultivar ‘Gannongshu 2’ as donor, 48 transgenic plants were obtained through an Agrobacterium-mediated transformation method using an RNAi vector containing an ADP-ribosylation factors (ARF) gene. Testing of the transformed plants on a culture media containing kanmycin and PCR assay showed that the AFR gene had been successfully integrated into the potato genome. The analysis of real time fluorescence quantitative polymerase chain reaction (qRT-PCR) data showed that the ARF gene expression declined to varying degrees in the transgenic plants compared with the controls, and interference degree reached 92.53% and 93.22% in the transgenic lines Z-11 and Z-12, respectively. The starch content of the transgenic plants increased by 4.29%-34.27%, the protein content by 1.47%-7.35%, the soluble sugar content by 1.44%-17.39%, and the sucrose content by 11.53%-53.84% compared with control plants; however, the glucose content of the transgenic plants decreased by 6.06%-21.21% compared with control plants.

裴瑞芳, 刘英, 刘柏林, 张宁, 司怀军, 王蒂. 马铃薯ARF基因RNAi载体的遗传转化及对其试管薯生理特性的影响[J]. 草业学报, 2015, 24(2): 142-147.

PEI Ruifang, LIU Ying, LIU Bailin, ZHANG Ning, SI Huaijun, WANG Di. Genetic transformation using an RNAi vector containing an ARF gene in potato and effects on physiological characteristics of microtuber[J]. Acta Prataculturae Sinica, 2015, 24(2): 142-147.

参考文献

[1] Moss J, Vaughan M. Structure and function of ARF proteins: Activators of cholera toxin and critical components of intracellular vesicular transport processes. The Journal of Biological Chemistry, 1995, 270: 12327-12330.
[2] Yang Z. Small GTPases: versatile signaling switches in plants. Plant Cell, 2002, 14(suppl): 375-388.
[3] Kahn R A, Gilman A G. Purification of a protein cofactor required for ADP-ribosylation of the stimulatory regulatory component of adenylate cyclase by cholera toxin. The Journal of Biological Chemistry, 1984, 259: 6228-6234.
[4] Wang L, Su H Y, Wang C L, et al . Structure and functional mechanism of the ADP-ribosylation factor. Chinese Journal of Cell Biology, 2007, 29: 675-681.
[5] Brown H A, Gutowsk C, Moomaw C R, et al . ADP-ribosylation factor, a small GTP-dependent regulatory protein, stimulates phospholipase D activity. Cell, 1993, 75: 1137-1144.
[6] Orci L, Palmer D J, Amherdt M, et al . Coated vesicle assembly requires only coatomer and ARF proteins from the cytosol. Nature, 1993, 364: 732-734.
[7] Memon A R, Clark G B, Thomson G. Identification of an ARF type low molecular mass GTP-binding protein in pea ( Pisum sativum ). Biochemical and Biophysical Research Communications, 1993, 193: 809-813.
[8] Hou L, Li J B, Luo X Y, et al . Cloning, expression and characterization of an ADP-ribosylation factor gene from cotton ( Gossypium hirsutum L.). Acta Agronomica Sinica, 2007, 33(8): 1226-1231.
[9] Gan L, Yu X X, Yu Z, et al . A study on main agronomic traits of yield and quality of clone lines of Solanum tuberosum hybrids F1. Acta Prataculturae Sinica, 2013, 22(4):312-318.
[10] Sambrook J, Russell D. Molecular Cloning: a Laboratory Manual (3rd ed)[M]. New York: Cold Spring Harbor Laboratory Press, 2001: 304-331.
[11] 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.
[12] Kong L F, Zhang J Y, Liu Z P, et al . Clonging of a S-adenosyl methionine synthetase gene from Cleistogenes songorica and its expression under drought stress. Acta Prataculturae Sinica, 2013, 22(1):268-275.
[13] Shanghai Society for Plant Physiology. Experimental Manual of Plant Physiology[M]. Shanghai: Shanghai Science and Technology Press, 1985:136-138.
[14] Zhang Y C, Tian F. Experimental and Research Method of Potato[M]. Beijing: China Agriculture Science and Technology Press, 2007: 152-153.
[15] Li H S. Principles and Techniques of Plant Physiological and Biochemical Experiment[M]. Beijing: Higher Education Press, 2000: 184-185.
[16] Li Y W, William G K, John M L, et al . Functional genomic analysis of the ADP-ribosylation factor family of GTPases: phylogeny among divers eukaryotes and function in C. elegans . The Journal of the Federation of American Societies for Experimental Biology, 2004, 18: 1834-1850.
[17] Naramoto S, Kleine-Vehna J, Roberta S, et al . ADP-ribosylation factor machinery mediates endocytosis in plant cells. Proceedings of the National Academy of Sciences of the United States of America, 2010, 107: 21890-21895.
[18] Zuk M, Prescha A, Keüpczynaski J, et al . ADP ribosylation factor regulates metabolism and antioxidant capacity of transgenic potato tuber. Journal of Agricultural and Food Chemistry, 2003, 51: 288-294.
[19] Liu B L, Zhang N, Wen Y K, et al . Identification of differentially expressed genes in potato associated with tuber dormancy release. Molecular Biology Reports, 2012, 39: 11277-11278.
[20] Tan S D, Zhu M Y, Zhang K R, et al . Effects of submergence on the antioxidative enzymes and carbohydrate contents of Paspalum distichum . Acta Prataculturae Sinica, 2013, 22(1):217-224.
[4] 王磊, 宿红艳, 王昌留, 等. ADP 核糖基化因子的结构及其功能机制. 细胞生物学杂志, 2007, 29: 675-681.
[8] 候磊, 李家宝, 罗小英, 等.棉花ADP-ribosylation factor基因(GhARF1)的克隆与表达分析. 作物学报, 2007, 33(8): 1226-1231.
[9] 甘霖, 于肖夏, 于卓, 等. 马铃薯杂种F1无性株系产量品质等主要农艺性状研究. 草业学报, 2013, 22(4): 312-318.
[11] 司怀军, 谢从华, 柳俊.农杆菌介导的马铃薯试管薯遗传转化体系的优化及反义 class I patatin 基因的导入. 作物学报, 2003, 29(6): 801-805.
[12] 孔令芳, 张吉宇, 刘志鹏, 等.无芒隐子草 SAMSl 基因的克隆及干旱胁迫下的表达分析. 草业学报, 2013, 22(1): 268-275.
[13] 上海植物生理学会.植物生理学实验手册[M]. 上海:上海科学技术出版社, 1985: 136-138.
[14] 张永成, 田丰.马铃薯试验研究方法[M]. 北京:中国农业科学技术出版社, 2007: 152-153.
[15] 李合生.植物生理生化实验原理和技术[M]. 北京:高等教育出版社, 2000: 184-185.
[20] 谭淑端, 朱明勇, 张克荣, 等. 水淹对双穗雀稗抗氧化酶活性及碳水化合物含量的影响. 草业学报, 2013, 22(1): 217-224.

马铃薯ARF基因RNAi载体的遗传转化及对其试管薯生理特性的影响

Genetic transformation using an RNAi vector containing an ARF gene in potato and effects on physiological characteristics of microtuber

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