象草4CL基因片段的克隆及RNAi表达载体构建

摘要/Abstract

摘要： 象草是热带和亚热带地区广泛栽培的多年生资源植物。为探讨华南象草4-香豆酸:CoA 连接酶(4CL)基因下调表达对木质素合成的影响,本研究设计1对特异引物,PCR扩增获得一段长为374 bp的干扰片段。通过TOPO克隆,将该片段克隆到载体pCR^/GW/TOPO^,构建了入门克隆载体pENTR-4CL;再经过LR反应使pE-NTR-4CL上的干扰片段进入目的载体pCB2004B上,形成表达载体pCB2004B-4CL,最后通过冻融法将其转入到根癌农杆菌EHA105中。菌液PCR结果表明RNAi质粒已成功转入农杆菌,为进一步利用RNAi技术研究4CL基因的功能奠定了基础。

Abstract: Pennisetum purpureum is a perennial resource plant and widely cultivated in tropical and sub-tropical regions. A pair of specific primers was designed to assess the effect of down-regulated expression of the 4-coumarate: CoA ligase (4CL) gene on lignin composition of P. purpureum cv. Huanan, using the 4CL gene sequence of P. purpureum cv. Mott (accession No: GU997597.1). An interference fragment of 374 bp was obtained by PCR amplification with the above primers and it was cloned into the pCR^/GW/TOPO^ vector by TOPO cloning to obtain pENTER-4CL. The corresponding fragment on destination vector pCB2004B was then replaced by the interference fragment of pENTR-4CL by LR reaction to form the RNAi (RNA interference) expression vector pCB2004B-4CL. The Agrobacterium-mediated transformation of the expression vector was carried out using the freeze-thaw method, and the expected fragments appeared in the PCR products using bacterial liquid as a template. This study was a basis for research on the 4CL function of P. purpureum by RNAi technology.

中图分类号:

S816
S543.9

霍松,陈慧,朱琼华,解新明 . 象草4CL基因片段的克隆及RNAi表达载体构建[J]. 草业学报, 2012, 21(1): 296-311.

HUO Song, CHEN Hui, ZHU Qiong-hua, XIE Xin-ming. Cloning of a 4CL gene fragment and construction of a RNAi expression vector of Pennisetum purpureum[J]. Acta Prataculturae Sinica, 2012, 21(1): 296-311.

参考文献

Xie X M, Zhou F, Zhang X Q, et al. Genetic variability and relationships between MT-1 elephant grass and its closely related cultivars assessed by SRAP markers. Journal of Genetics, 2009, 88(3): 281-290.
Farrell G, Simons S A, Hillocks R J. Aspects of the biology of Ustilago kamerunensis, a smut pathogen of Napier grass (Pennisetum purpureum). Phytopathology, 2001, 149: 739-744.
解新明, 赵燕慧, 霍松, 等. 象草不同品种木质素合成关键酶活性的动态变化. 草地学报, 2010, 18(4): 523-527.
张向前, 周峰, 解新明. MT-1象草及其近缘品种的外稃微型态特征. 草业学报, 2010, 19(4): 159-165.
Zamore P D, Tuschl T, Sharp P A, et al. RNAi: double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals. Cell, 2000, 101: 25-33.
Fire A, Xu S, Montgomery M K, et al. Potent and specific genetic interference by double- stranded RNA in Caenorhabditis elegans. Nature, 1998, 391: 806-811.
Boudet A M. A new view of lignification. Trends in Plant Science, 1998, 3: 67-71.
Whetten R, Sederoff R. Lignin biosynthesis. The Plant Cell, 1995, 7: 1001-1003.
范丙友, 陆海, 蒋湘宁. 维管植物4-香豆酸: 辅酶A连接酶(4CL)研究进展. 林业科学, 2007, 43(2): 96-103.
Zhao Y, Kung S D, Dube S K. Nucleotide sequence of rice 4-coumarate: CoA ligase gene 4-CL.1. Nucleic Acids Research, 1990, 18(20): 6144.
Heath R, McInnes R, Lidgett A, et al. Isolation and characterisation of three 4-coumarate: CoA-ligase homologue cDNAs from perennial ryegrass (Lolium perenne). Journal of Plant Physiology, 2002, 159(7): 773-779.
黄胜雄, 胡尚连, 孙霞, 等. 木质素生物合成酶4CL基因的遗传进化分析. 西北农林科技大学学报(自然科学版), 2008, 36(10): 199-206.
马建, 魏益凡, 厉志, 等. 植物RNA干扰表达载体构建方法的研究. 安徽农业科学, 2009, 37(18): 8364-8366.
徐化学, 熊建华, 傅彬英. 应用Gateway技术构建水稻OsDAD1基因的RNA干涉载体. 分子植物育种, 2007, 5(1): 133-136.
张茹, 李金花, 柴兆祥, 等. Chi基因的克隆及转基因马铃薯植株的获得. 草业学报, 2009, 18(6): 51-58.
Helliwell C, Waterhouse P. Constructs and methods for high-throughput gene silencing in plants. Methods, 2003, 30(4): 289-295.
许德晖, 黄辰, 刘利英, 等. 高效siRNA设计的研究进展. 遗传, 2006, 28(11): 1457-1461.
Wesley S V, Helliwell C A, Smith N A, et al. Construct design for efficient, effective and high-throughput gene silencing in plants. The Plant Journal, 2001, 27(6): 581-590.
李丽文, 朱延明, 李杰, 等. RNAi 技术在植物功能基因组学中的研究进展. 东北农业大学学报, 2007, 38(1): 119-124.
Lei Z Y, Zhao P, Cao M J, et al. High-throughput binary vectors for plant gene function analysis. Journal of Integrative Plant Biology, 2007, 49(4): 556-567.
赵燕慧, 解新明. 象草不同品种木质素含量的动态变化研究. 见: 黄必志, 毕玉芬. 中国草学会牧草育种委员会第七届代表大会论文集. 昆明:云南科技出版社, 2009: 149-158.
贾小霞, 张金文, 王汉宁, 等. 抗真菌和抗除草剂基因多价植物表达载体构建及对烟草遗传转化的研究. 草业学报, 2009, 18(1): 86-93.

[1]	张前兵,艾尼娃尔·艾合买提,于磊,鲁为华,常青. 绿洲区不同灌溉方式及灌溉量对苜蓿田土壤盐分运移的影响[J]. 草业学报, 2014, 23(6): 69-77.
[2]	王绍飞,罗永聪,张新全,黄琳凯,马啸,刘恋. 14个多花黑麦草品种(系)在川西南地区生产性能综合评价[J]. 草业学报, 2014, 23(6): 87-94.
[3]	王勇,原现军,郭刚,闻爱友,王坚,肖慎华,余成群,巴桑,邵涛. 西藏不同饲草全混合日粮发酵品质和有氧稳定性的研究[J]. 草业学报, 2014, 23(6): 95-102.
[4]	王鸿泽,王之盛,康坤,邹华围,申俊华,胡瑞. 玉米粉和乳酸菌对甘薯蔓、酒糟及稻草混合青贮品质的影响[J]. 草业学报, 2014, 23(6): 103-110.
[5]	覃方锉,赵桂琴,焦婷,韩永杰,侯建杰,宋旭东. 含水量及添加剂对燕麦捆裹青贮品质的影响[J]. 草业学报, 2014, 23(6): 119-125.
[6]	邱小燕,原现军,郭刚,闻爱友,余成群,巴桑,邵涛. 添加糖蜜和乙酸对西藏发酵全混合日粮青贮发酵品质及有氧稳定性影响[J]. 草业学报, 2014, 23(6): 111-118.
[7]	史传奇,刘玫,王臣,张欣欣,程薪宇. 东北野豌豆族植物叶形态结构的研究及其分类学意义[J]. 草业学报, 2014, 23(6): 157-166.
[8]	韩宝贺,朱宏. 镉胁迫对白三叶的富集能力、叶片显微结构及其生理特性的影响[J]. 草业学报, 2014, 23(6): 167-175.
[9]	刘会杰,李胜,马绍英,张品南,时振振,杨晓明. H₂O₂胁迫下豌豆初生根及抗氧化酶系统对外源Ca²⁺的响应[J]. 草业学报, 2014, 23(6): 189-197.
[10]	张军,宋丽莉,郭东林,郭长虹,束永俊. MADS-box基因家族在蒺藜苜蓿的全基因组分析[J]. 草业学报, 2014, 23(6): 233-241.
[11]	康俊梅,张铁军,王梦颖,张怡,杨青川. 紫花苜蓿QTL与全基因组选择研究进展及其应用[J]. 草业学报, 2014, 23(6): 304-312.
[12]	李君临,张新全,玉柱,郭旭生,孟祥坤,罗燕,闫艳红. 含水量和乳酸菌添加剂对多花黑麦草青贮品质的影响[J]. 草业学报, 2014, 23(6): 342-348.
[13]	漆婧华,张峰,王莺,孙国钧. 黄土高原半干旱区覆膜玉米农田氮变化动态研究[J]. 草业学报, 2014, 23(5): 13-23.
[14]	田晨霞,张咏梅,王凯,张万. 紫花苜蓿组织解剖结构对NaHCO₃盐碱胁迫的响应[J]. 草业学报, 2014, 23(5): 133-142.
[15]	孙红,于应文,马向丽,牟晓明,廖加法. 黔西北岩溶区九种灌木综合营养价值评价[J]. 草业学报, 2014, 23(5): 99-106.