Molecular Cloning of GhCAD6 Gene and Construction of Its Sense,Anti-sense and RNAi Expression Vectors

doi:10.7668/hbnxb.2009.06.005

Abstract

Abstract: 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.

Key words: Gossypium hirsuturm L., Cinnamyl alcohol dehydrogenase, Expression vectors, RNAi

CLC Number:

NI Zhi-yong, MA Wen-jing, LU Meng, WANG Juan, LI Bo, FAN Ling. Molecular Cloning of GhCAD6 Gene and Construction of Its Sense,Anti-sense and RNAi Expression Vectors[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2009, 24(6): 20-26. doi: 10.7668/hbnxb.2009.06.005.

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References

[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.

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