咖啡酰辅酶A-O-甲基转移酶是木质素生物合成过程中的关键酶之一。该研究以慈竹嫩茎为材料,采用RT-PCR及RACE方法,克隆CCoAOMT基因,并对其进行生物信息学分析。结果表明:1个慈竹CCoAOMT基因cDNA全长序列被成功克隆,该序列全长为1 080 bp,含有1个777 bp的读码框,编码了1个包含258个氨基酸的蛋白质,分子质量约为28.861 ku,等电点为5.33, 二级结构中α-螺旋占42.25%,β-转角占8.91%,延伸链占15.89%、无规则卷曲占32.95%。对氨基酸多序列比对发现,慈竹与绿竹的CCoAOMT1基因亲缘关系最近,但慈竹CCoAOMT基因编码的氨基酸与绿竹相比,在第10位点上发生了缺失,5个位点发生了变异。慈竹、绿竹、毛竹、玉米CCoAOMT基因编码的氨基酸中都含有1个相对保守的无序化区域。NaCCoAOMT1基因组织表达结果表明,其在未展开叶、笋上部和中部、茎的上部和中部的表达量较高。笔者将克隆并获得的慈竹木质素合成酶CCoAOMT基因全长序列(GenBank注册号为JF742462)命名为NaCCoAOMT1,分析认为其可能与慈竹木质素生物合成有关。
Abstract
Caffeoyl-coA O-methyltransferase(CCoAOMT)is a key enzyme in lignin biosynthesis. A gene of CCoAOMT family with the cDNA length of 1 080 bp was cloned by RT-PCR and RACE method from young stems of Neosinocalamus affinis and bioinformatics analyzed. The full-length sequence of the CCoAOMT gene from N. affinis was successfully cloned in this studies. The results showed that the gene contained an open reading frame of 777 bp, which encoded a polypeptide of 258 amino acids, the molecular weight of the predicted protein was 28.861 ku, its theoretical PI was 5.33, the gene’s secondary structure consist of α-helix, β-turn, extended strand and random coil; the proportions were 42.25%,8.91%, 32.95% and 15.89%, respectively. The BLASTp results indicated that N. affinis had the highest identity with Bambusa oldhamii. There is a base deletion at the tenth site, and has five variable sites compared with CCoAOMT of B. oldhamii. One relative conserved disordered domain was found in the CCoAOMT gene from N. affinis, B. oldhamii, Phyllostachys edulis and Zea mays. The expression of NaCCoAOMT1 showed that the gene highly expressed in young leaves, upper and middle parts of shoots and stem. This is the first time that NaCCoAOMT1 was cloned in N. affinis. The sequence of NaCCoAOMT1 has been submitted to GenBank with the accession number JF742462.
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基金
收稿日期:2011-08-04 修回日期:2012-02-01
基金项目:四川省“十二五”重点攻关项目(2011YZGG-10); 四川省应用基础研究基金项目(05JY029-101); 四川主要丛生竹定向培育技术项目(10zx1102)
第一作者:吴晓宇,硕士生。*通信作者:胡尚连,教授,博士。E-mail: hushanglian@126.com。
引文格式:吴晓宇, 胡尚连, 曹颖,等. 慈竹CCoAOMT基因的克隆及生物信息学分析[J]. 南京林业大学学报:自然科学版,2012,36(3):17-22.
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