肉桂醇脱氢酶(CAD)催化木质素单体合成的最后一步反应,将肉桂醛还原生成相应的肉桂醇.采用PCR方法从棉花中克隆了GhCAD6基因1 569 bp的基因组序列,序列分析表明GhCAD6基因由5个外显子和4个内含子组成.为了研究GhCAD6基因的功能,构建由棉纤维组织特异性启动子E6驱动的GhCAD6基因正义、反义表达载体;同时克隆了GhCAD6基因的一段41 bp高度保守的NBD区序列,构建了GhCAD6基因的RNAi干扰载体,并将上述载体转入农杆菌菌株LBA4404中.为通过转基因技术深入研究GhCAD6基因在棉纤维发育和木质素代谢途径中的作用创造条件.
倪志勇
,
马文静
,
吕萌
,
王娟
,
李波
,
范玲
. 棉花肉桂醇脱氢酶基因GhCAD6的克隆及正义、反义与RNAi干扰载体的构建[J]. 华北农学报, 2009
, 24(6)
: 20
-26
.
DOI: 10.7668/hbnxb.2009.06.005
Cinnamyl alcohol dehydrogenase (CAD) (EC 1.1.1.195) catalyses the final step in lignin precursor synthesis reducing the cinnamyl aldehydes to the corresponding alcohols in the presence of NADPH.In this paper,we report the molecular cloning a length of 1 569 bp sequence from genomic DNA of GhCAD6 by PCR.The genomic DNA of GhCAD6 contains five exons and four introns.T o investigate the function of GhCAD6 gene,the sense and anti2sense expression vectors of GhCAD6 were constructed,in which the coding region of the gene was placed under the E6 promoter in either sense or anti2sense orientation.In addition,especial 417 bp fragments of GhCAD6 NBD were also cloned,and this fragment was inserted into plant vector to construct RNAi expression vector of GhCAD6.These constructed vectors were then transformed into Agrobacterium LBA4404.These constructed vectors provided an effective tool for the further studyof GhCAD6 gene function.
[1] Fire A,Xu S Q,Montgomery M K,et al.Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans[J].Nature,1998,391(19):806-811.
[2] Smith N A,Singh S P,Wang M B,et al.Total silencing by intron-spliced hairpin RNAs[J].Nature,2000,407:319-320.
[3] Chuang C F,Meyerowitz E M.Specific and heritable genetic interference by double-stranded RNA in Arabidopsis thaliana[J].Proc Natl Acad Sci USA,2000,97:4985-4990.
[4] 康国章,肖向红,王永华,等.小麦淀粉GBSSH基因的克隆、反义和RNAi载体的构建[J].华北农学报,2008,23(1):7-11.
[5] 朱红林,沙爱华,符秀梅,等.转录调控基因GmLEC1的cDNA克隆及其植物表达载体的构建[J].华北农学报,2009,24(1):64-68.
[6] Mansell R L,Gross G G,Stockigt J,et al.Purification and properties of cinnamyl alcohol dehydrogenase from higher plants involved in lignin biosynthesis[J].Phytochemistry,1974,13:2427-2435.
[7] Halpin C,Knight M E,Foxon G A,et al.Manipulation of lignin quality by downregulation of cinnamyl alcohol dehydrogenase[J].Plant J,1994,6:339-350.
[8] Baucher M,Chabbert B,Pilate G,et al.Red xylem and higher lignin extractability by down-regulating a cinnamyl alcohol dehydrogenase in poplar[J].Plant Physiol,1996,112:1479-1490.
[9] Baucher M,Bernard-Vailhé M A,Chabbert B,et al.Down-regulation of cinnamyl alcohol dehydrogenase in transgenic alfalfa (Medicago sativa L.) and the effect on lignin composition and digestibility[J].Plant Mol Biol,1999,39:437-447.
[10] Ralph J,MacKay J J,Hatfield R D,et al.Abnormal lignin in a loblolly pine mutant[J].Science,1997,277:235-239.
[11] Fan L,Shi W J,Hu W R,et al.Molecular and biochemical evidence for phenylpropanoid synthesis and presence of wall-linked phenolics in cotton fibers[J].Journal of Integrative Plant Biology,received.
[12] Paterson A H,Lan Der E S,Hewitt J D.A rapid method for extraction of cotton(Gossypium spp.) genomic DNA suitable for RFLP or PCR analysis[J].Plant Mol Biol Rep,1993,11:122-127.
[13] Mette M F,Aufsatz W,van der Winden J,et al.Transcriptional silencing and promoter methylation triggered by double-stranded RNA[J].EMBO J,2000,19:5194-5520.
[14] Sijen T,Vijn I,Rebocho A,et al.Transcriptional and posttranscriptional gene silencing are mechanistically related[J].Curr Biol,2001,11:436-444.
[15] Chan S W-L,Zilberman D,Xie Z,et al.RNA silencing genes control de novo DNA methylation[J].Science,2004,303:1336.
[16] 黄冰艳,吉万全,郭蔼光,等.转录后基因沉默(PTGS)及其在作物遗传改良中的应用[J].中国生物工程杂志,2005,25(5):1-5.
[17] Wesley S V,Helliwell C A,Smith N A,et al.Construct design for efficient,effective and high-throughput gene silencing in plants[J].Plant J,2001,27:581-590.
[18] 王雷,种康,许智宏.植物功能基因组学研究的有效工具-RNAi技术[J].植物生理学通讯,2003,39(6):705-710.
[19] Neil A S,Surinder P S,Wang M B,et al.Total silencing by intron spliced hairpin RNAs[J].Nature,2001,407:319-320.
[20] Hammond S M,Bernstern E,Beach D.An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells[J].Nature,2000,404(16):293-296.