Please wait a minute...

中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2012, Vol. 32 Issue (08): 41-48    
研究报告     
芪合酶基因Fm-STS在何首乌毛状根中的过量表达及dsRNA干扰
朱宽鹏1,2, 赵树进1
1. 广州军区广州总医院 广州 510010;
2. 华南理工大学 生物科学与工程学院 广州 510640
Double-stranded RNA-mediated Gene Silencing and Over-expression of FmSTS in Polygonum multiflorum Thunb Hairy Roots
ZHU Kuan-peng1,2, ZHAO Shu-jin 1
1. General Hospital of Guangzhou Military Command, Guangzhou 510010,China;
2. School of Biological Science and Engineering, South China University of Technology, Guangzhou 510640,China
 全文: PDF(1314 KB)   HTML
摘要： 目的:建立一套探究植物基因功能的方法体系,验证由芪合酶基因保守序列通过RACE扩增技术在何首乌中得到的基因Fm-STS的功能。方法:由含CaMV 35S启动子驱动的gfp基因的植物转基因表达基础质粒pBIN-35S-GFP构建过表达质粒pBIN-35S-STS-GFP(阳性)和双链RNA干扰重组质粒pBIN-35S-正向-反向-GFP(阴性),并同空白表达质粒pBIN-35S-GFP(空白)均导入野生型发根农杆菌ATCC15834中,转化何首乌外植体,诱导生成毛状根并培养,对毛状根进行高效液相色谱分析以及实时荧光定量检测。结果:在过表达组、空白组和干扰组中毛状根中发根农杆菌Ri质粒中的rolB基因和外源基因gfp均有表达,高效液相色谱法分析芪合物二苯乙烯苷含量依次为4.67mg/g、2.18mg/g和0.65mg/g,在mRNA水平上测试荧光定量检测基因Fm-STS表达量:RNAi组是空白组的1/433.53,过表达组是空白组的2.41倍。结论:结果表明过量表达与双链RNA干扰相结合在植物基因功能研究中有良好的应用,何首乌中芪合酶Fm-STS是二苯乙烯苷主要的合成酶。
关键词： 过量表达;  dsRNA;  RNA干扰;  何首乌;  毛状根;  荧光定量PCR;  高效液相色谱法    
Abstract: Objective: To construct a method to research the gene FM-STS’ function that the gene come by RACE amplified from the stilbene synthase gene’s consensus sequence in Polygonum multiflorum Thunb. Method: The over-expression vector pBIN-35S-STS-GFP was constructed from the blank plasmids pBIN-35S-GFP and the gene FM-STS sequence and also the interference expression vector pBIN-35S -forward-reverse-GFP was constructed from the blank plasmids and the forward/ reverse direction sequence on account of double stranded RNA.Sent the over-expression plasmid/the interference plasmid and the blank plasmid into wild-type Agrobacterium rhizogenes ATCC15834, then induced hairy roots and cultured them. In the end the hairy roots were analyzed by HPLC and Real-time PCR. Result: The PCR results showed that the gene rolB and gfp are both expressed in hairy roots, and the content of stilbene glucoside in blank group is 2.18mg/g,in the over-expression group is 4.67mg/g and in the interference group is 0.65mg/g, At the mRNA level to detect gene Fm-STS expression level, in the over-expression group is the highest, it’s 3.17 times in the blank group and 101.44 times in the interference group. Conclusion: The method binding both technique that double stranded RNA-mediated gene silencing and over-expression is made good use of researching the gene’ s function, and the gene FM-STS is the key enzyme gene in synthesis stilbene glucoside.
Key words: Over-expression    dsRNA    RNAi    Polygonum multiflorum    Thunb    Stilbene glucoside    Hairy roots    Real Time PCR    HPLC
收稿日期: 2012-04-27 出版日期: 2012-08-25
ZTFLH:  Q94-3  
基金资助: 广东省自然科学基金(10151001002000012)、广东省科技计划(00697862100303016)资助项目
通讯作者: 赵树进     E-mail: gzzsjzhs@163.com
服务　　
把本文推荐给朋友
加入引用管理器
E-mail Alert
RSS
作者相关文章　　
朱宽鹏
赵树进

引用本文:

朱宽鹏, 赵树进. 芪合酶基因Fm-STS在何首乌毛状根中的过量表达及dsRNA干扰[J]. 中国生物工程杂志, 2012, 32(08): 41-48.

ZHU Kuan-peng, ZHAO Shu-jin. Double-stranded RNA-mediated Gene Silencing and Over-expression of FmSTS in Polygonum multiflorum Thunb Hairy Roots. China Biotechnology, 2012, 32(08): 41-48.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/    或    https://manu60.magtech.com.cn/biotech/CN/Y2012/V32/I08/41

[1] 吕丽爽,汤坚,Chi-Tang Ho.何首乌中二苯乙烯苷清除DPPH自由基机理研究.食品与机械,2007,23(5): 93-97. Lv LS, Tang J, Chi-Tang Ho. Chemical studies of the antioxidant mechanism of stilbene glucose from Polygonum multiflorum thunb: radical reaction products of stilbene glucosideswith DPPH. Food and Machinery, 2007, 23(5): 93-97.
[2] Kati Hanhineva,Harri Kokko,Henri Siljanen,et al. Stilbene synthase gene transfer caused alterations in the phenylpropanoid metabolism of transgenic strawberry.Journal of Experimental Botany,2009,60(7): 2093-2106.
[3] 生书晶,赵树进.何首乌中III型PKS基因的克隆和表达.华南理工大学学报(自然科学版),2010,38(5): 144 -148. Sheng S J, Zhao S J. Molecular analysis of a type III polyketide synthase gene in Fallopia multiflora. Journal of South China University of Technology(Natural Science Edition), 2010,38(5):144-148.
[4] Shujing Sheng, Zhongyu Liu, Wei Zhao, et al. Molecular analysis of a type III polyketide synthase gene in Fallopia multiflora. Biologia,2010,65(6): 939-946.
[5] 唐少冰,周冬根. RNA干扰技术及其在植物研究中的应用. 生物技术通报, 2007,5:65-67. Tang S B, Zhou D G. RNA interference and its application in plant research. Biotechnology Bulletin, 2007,5:65-67.
[6] Sarah J Richardson, Abby Willco, David A Hilton. Use of antisera directed against dsRNA to detect viral infections in formalin-fixed paraffin-embedded tissue. Journal of Clinical Virology, 2010,(49):180-185.
[7] Lisa Timmons, Donald L Court, Andrew Fire. Ingestion of bacterially expressed dsRNAs can produce specific and potent genetic interference in Caenorhabditis elegans.Gene, 2001,263:103-112.
[8] Toshiaki Watanabe, Yasushi Totoki, Atsushi Toyoda. Endogenous siRNAs from naturally formed dsRNAs regulate transcripts in mouse oocytes. Nature, 2008,452:539-543.
[9] 王莉,于荣敏.何首乌毛状根的诱导及其培养.生物技术, 2002,9(2):110-114. Wang L, Yu R M. Hairy root induction and culture of Polygonium multif lorum Thunb. Pharmaceutical Biotechnology, 2002,9 (2) :110-114.
[10] 邵利,崔堂兵,赵树进。何首乌根和茎愈伤组织的二苯乙烯苷含量.华南理工大学学报(自然科学版),2010,38(9):147-151. Shao L, Cui T B, Zhao S J. Contents of stilbene glycoside in root and stem callus of Fallopia multiflora. Journal of South China University of Technology (Natural Science Edition), 2010,38(9):147-151.
[11] 邹湘辉,庄东红,吴云影,等. 实时荧光定量PCR检测小鼠脑组织中3-羟基丁酸受体基因PUMA-G的转录表达.广东医学,2010, 31 (1) :58-60. Zou X H, Zhuang D H, Wu Y Y. Real-time PCR detection of mouse brain tissue expression of PUMA-G, 3 -hydroxy acid receptor gene transcription. Guangdong Pharmaceutical, 2010,31(1):58-60.
[12] Hain R, Bieseler B, Kindl H, et al. Expression of a stilbene synthase gene in Nicotiana tabacum results in synthesis of the phytoalexin resveratrol. Plant Mol Biol, 1990, 15:325-335.
[1] 冯昭,李江浩,王佳华. 刺槐核糖体蛋白同源基因RpRPL22在共生结瘤过程中功能研究[J]. 中国生物工程杂志, 2021, 41(7): 10-21.
[2] 程瑜,施琼,安利钦,范梦恬,皇改改,翁亚光. BMP7基因沉默抑制钙盐诱导猪主动脉瓣膜间质细胞成骨分化 *[J]. 中国生物工程杂志, 2019, 39(5): 63-71.
[3] 冯昭,丑敏霞. Rpfan37在刺槐共生结瘤过程中的功能探究 *[J]. 中国生物工程杂志, 2018, 38(5): 47-55.
[4] 庄旻敏,贾晓会,施定基,朱嘉诚,冯思豫,何培民,贾睿. 转基因聚球藻7942中vp28基因表达效率及其光合特性分析[J]. 中国生物工程杂志, 2018, 38(4): 30-37.
[5] 董维鹏,张少华,许祥,燕炯. 下调Fsp27基因表达联合杨梅素干预对3T3-L1细胞脂解的影响[J]. 中国生物工程杂志, 2018, 38(12): 7-13.
[6] 张进,斯丹,杨致邦,熊玉霞,马廉举,李津阳,蒋仁举. 地塞米松降解新基因的探讨[J]. 中国生物工程杂志, 2018, 38(1): 15-24.
[7] 张丽丽, 徐碧玉, 刘菊华, 贾彩红, 张建斌, 金志强. 转香蕉MaASR1基因的拟南芥株系在干旱胁迫条件下的表达谱分析[J]. 中国生物工程杂志, 2017, 37(11): 59-73.
[8] 任爽, 朱鸿亮. Taqman定量PCR技术检测基因编辑番茄中外源基因拷贝数体系的建立[J]. 中国生物工程杂志, 2017, 37(10): 72-80.
[9] 刘怡萱, 边珍, 马红梅. 癌症基因治疗技术进展与展望[J]. 中国生物工程杂志, 2016, 36(5): 106-111.
[10] 胡娜, 刘清, 唐照勇, 汤禾静, 敖澜, 赵紫豪, 方廖琼. siRNA干扰MMP-9和FAK双基因抑制小鼠黑色素瘤生长和在体迁移[J]. 中国生物工程杂志, 2016, 36(5): 34-39.
[11] 祖力皮也·吐尔逊, 曹春宝, 温浩, 丁剑冰, 德力夏提·依米提. 细粒棘球蚴EgG1Y162基因进化分析、表达及鉴定[J]. 中国生物工程杂志, 2016, 36(4): 78-87.
[12] 刘丽, 杨晓慧, 王瑞明. RNA干扰沉默KAT基因对蜜蜂合成10-HDA的影响[J]. 中国生物工程杂志, 2016, 36(4): 63-68.
[13] 赵志武, 王君实, 马敏, 张少华, 燕炯. 下调Perilipin 1基因表达对3T3-L1细胞脂解的影响[J]. 中国生物工程杂志, 2016, 36(3): 17-22.
[14] 韩双, 杨志丽, 陈丽梅. 过量表达拟南芥CAT提高烟草对气体甲醛的吸收和抗性[J]. 中国生物工程杂志, 2015, 35(5): 41-48.
[15] 左海洋, 陈晓丽, 蔡勇, 郝海生, 秦彤, 赵学明, 路永强, 王栋. 基因差异转录表达验证方法研究进展[J]. 中国生物工程杂志, 2015, 35(5): 96-102.
主管：中国科学院 　主办：中国科学院文献情报中心 　中国生物技术发展中心 　中国生物工程学会