大豆查尔酮还原酶基因CHR1的功能研究

doi:10.3724/SP.J.1005.2014.0707

遗传 ›› 2014, Vol. 36 ›› Issue (7): 707-712.doi: 10.3724/SP.J.1005.2014.0707

大豆查尔酮还原酶基因CHR1的功能研究

吴楠¹, 王丕武¹, 李丹¹, 代力强¹, 郑成忠¹, 卢实¹, 才源¹, 张卓¹, 曲静¹, 夏海丰^{1, 2}

1. 吉林农业大学农学院, 长春 130118;
2. 通化市农业科学研究院, 梅河口 135000

收稿日期:2014-01-07 出版日期:2014-07-20 发布日期:2014-06-23
通讯作者: 王丕武, 教授, 博士生导师, 研究方向：生物技术与作物遗传育种。E-mail: peiwuw@163.com
作者简介:吴楠, 在读硕士研究生, 专业方向：生物技术与作物遗传育种。E-mail:525762426@qq.com
基金资助:
国家自然科学基金项目(编号：31171568)资助

Function of chalcone reductase gene CHR1 in soybean

Nan Wu¹, Piwu Wang¹, Dan Li¹, Liqiang Dai¹, Chengzhong Zheng¹, Shi Lu¹, Yuan Cai¹, Zhuo Zhang¹, Jing Qu¹, Haifeng Xia^{1, 2}

1. College of Agriculture, Jilin Agricultural University, Changchun 130118, China;
2. Tonghua Academy of Agricultural Sciences, Meihekou, Jilin 135000, China

Received:2014-01-07 Online:2014-07-20 Published:2014-06-23

摘要/Abstract

摘要： 为了验证查尔酮还原酶基因CHR1在大豆苷元合成中的作用, 文章克隆了CHR1基因并构建了RNA干扰表达载体pCPB-CHR1-RNAi, 将载体转化受体大豆品种“吉农28”中, 以期抑制CHR1基因的转录。通过农杆菌介导的遗传转化和PCR检测得到4株T₀ 代阳性植株, 13株T₁代阳性植株。Southern blotting结果表明, 功能元件以单拷贝的形式整合到大豆的基因组中。利用实时荧光定量PCR法(Quantitative real-time PCR, qRT-PCR)测定CHR1基因在mRNA水平上的表达量, 结果表明, 转基因大豆植株中CHR1的表达量与未转化的受体大豆相比降低了60%~99%; 高效液相色谱法(High performance liquid chromatography, HPLC)检测到合成大豆苷元过程中的前体物质异甘草素的含量降低了38.7%。该RNA干扰机制在转录水平上抑制了CHR1基因的表达。

关键词: 大豆, 查尔酮还原酶CHR1, 大豆苷元, RNA干扰载体

Abstract: To verify the function of chalcone reductase gene (CHR1) in soybean Daidzein synthesis, CHR1 gene in soybean was cloned, and an RNAi expression vector pCPB-CHR1-RNAi was constructed. Four transformed plants in T₀generation and thirteen transformed plants in T₁ generation of soybean “Jinong28” were obtained by agrobacterium-mediated genetic transformation, in which transcriptions of CHR1 gene were depressed. Southern blotting showed the functional fragment of pCPB-CHR1-RNAi was integrated into the genome of recipient soybean in the form of a single copy. Detection of the transcription of CHR1 gene using quantitative real-time PCR (qRT-PCR) showed that the expression of CHR1 gene in transformed plants decreased 60%-99% compared to the recipient soybean, while the content of isoliquiritigenin, the precursors of daidzein, decreased 38.7%. These results indicate that RNA interference can suppress the transcription of CHR1 gene expression successfully.

Key words: soybean, chalcone reductase CHR1, daidzein, RNA interference vector

吴楠, 王丕武, 李丹, 代力强, 郑成忠, 卢实, 才源, 张卓, 曲静, 夏海丰. 大豆查尔酮还原酶基因CHR1的功能研究[J]. 遗传, 2014, 36(7): 707-712.

Nan Wu, Piwu Wang, Dan Li, Liqiang Dai, Chengzhong Zheng, Shi Lu, Yuan Cai, Zhuo Zhang, Jing Qu, Haifeng Xia. Function of chalcone reductase gene CHR1 in soybean[J]. HEREDITAS(Beijing), 2014, 36(7): 707-712.

参考文献

[1] 宓鹤鸣. 大豆异黄酮的研究进展. 中草药, 2000, 31(1): 61-64.
[2] S, Graham MY, Yu O, Graham TL. RNA interference of soybean isoflavone synthase genes lead to silencing in tissues distal to the transformation site and to enhanced susceptibility to Phytophthora sojae . Plant Physiology , 2005, 137(4): 1345-1353.
[3] VS, Srinivasa Rao C, Sullia SB, Ladha JK, Reddy PM. Metabolic engineering of rice with soybean isoflavone synthase for promoting nodulation gene expression in rhizobia. J Exp Bot , 2006, 57(9): 1957-1969.
[4] EJ, Kim GH. Daidzein causes cell cycle arrest at the G1 and G2/M phases in human breast cancer MCF-7 and MDA-MB-453 cells . Phytomedicine , 2008, 15(9): 683- 690.
[5] 季晖, 陈秀英, 韩景超, 邵卿, 濮祖茂. 大豆苷元磺酸钠对维甲酸诱导大鼠骨质疏松的保护作用. 中国骨质疏松杂志, 2006, 12(6): 623-626.
[6] 张信岳, 郑经伟, 孟倩超, 郑东升. 葛根素和葛根总异黄酮的雌激素样活性. 中药材, 2002, 25(8): 566-568.
[7] R, Schroder G, Schiltz E, Grisebach H, Schroder J. Induced plant responses to pathogen attack analysis and heterologous expression of the key enzyme in the biosynthesis of phytoalexins in soybean. Eur J Biochem , 1991, 196(2): 423-430.
[8] 陈沛, 邢光南, 赵团结, 李艳, 盖钧镒. 大豆查尔酮还原酶基因 GmCHR 的克隆与植物表达载体构建. 大豆科学, 2013, 32(2): 139-142.
[9] 周海涛, 王丕武. 大豆种子特异性启动子的克隆及功能分析. 西北农林科技大学学报, 2009, 37(12): 105-111.
[10] TL, Graham MY, Subramanian S, Yu O. RNAi silencing of genes for elicitation or biosynthesis of 5-deoxyisoflavonoids suppresses race-specific resistance and hypersensitive cell death in Phytophthora sojae infected tissues. Plant Physiology , 2007, 144: 728-740.
[11] CF, Wu JH, He CZ. Induction of chromosomal inversion by integration of T-DNA in the rice genome. J Genet Genomics , 2010, 37(3): 189-196.
[12] QH, Ramm K, Eamens AL, Dennis ES, Upadhyaya NM. Transgene structures suggest that multiple mechanisms are involved in T-DNA integration in plants. Plant Sci , 2006, 171(3): 308-322.
[13] 王丕武, 曲静, 王巍, 王俊玲. BADH/pepB双价基因无选择标记表达载体转化苜蓿的初步研究. 中国草地学报, 2009, 31(4): 20-23.
[14] 方宏筠. 植物基因工程原理与技术. 北京: 科学出版社, 2002: 742-744.
[15] 陈孝平, 余龙江. 实时荧光定量PCR的应用和进展. 激光生物学报, 2005, 14(6): 466-471.
[16] 张延明, 闫玉清, 徐香玲. 利用实时荧光定量PCR技术检测转基因烟草. 哈尔滨师范大学自然科学学报, 2005, 21(1): 69-72.
[17] KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2 - △△CT method. Methods , 2001, 25(4): 402-408.
[18] TD, Livak KJ. Analyzing real-time PCR data by the comparative C(T) method. Nat Protoc , 2008, 3(6): 1101-1108.
[19] 葛文中, 安红波. 高效液相色谱法测定豆粕中染料木素和大豆苷元的含量. 大豆科学, 2006, 25(4): 461-463.
[20] 白志明, 宋宏哲, 田娟娟. 高效液相色谱法测定大豆异黄酮含量的研究. 中国油脂, 2003, 28(11): 82-84.
[21] EK, Austin MB, Bowman ME, Dixon RA, Noel JP. Structural elucidation of chalcone reductase and implications for deoxychalcone biosynthesis. J Biol Chem , 2005, 280(34): 30496-30503.
[22] GY. Isolation, sequence identification and tissue expression profile of two novel soybean ( Glycine max ) genes-vestitone reductase and chalcone reductase. Mol Biol Rep , 2009, 36(7): 1991-1994.
[23] 大豆查尔酮还原酶基因 GmCHR 的克隆及农杆菌介导的大豆遗传转化的研究[学位论文]. 南京: 南京农业大学, 2013.

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大豆查尔酮还原酶基因CHR1的功能研究

Function of chalcone reductase gene CHR1 in soybean

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