杨树木质素合成酶c3h基因的克隆及其序列分析

Evolution Analysis of c3h cDNA Fragment from Poplar

根据CYP98A3(GenBank登录号为AY064170)cDNA序列保守区设计引物,从正在分化的2年生‘欧美杨107’次生木质部提取的总RNA经RT-PCR扩增出一基因片段,然后与pMD20-T载体连接。重组质粒经限制性内切酶酶切、特异引物PCR扩增和测序鉴定。结果表明,扩增片段长度为994bp,其中包含一个长为495bp的开放阅读框,编码的氨基酸序列(登录号为CAP47423)与NCBI中AY064170的CYP98A3编码氨基酸序列的相似性为91%,初步断定其为CYP98A3家族中的一员,其cNDA序列GenBank登录号为AM920690。参照国内外已发表的部分植物的c3h基因序列,构建了c3h的遗传进化树,分析了不同植物c3h的遗传进化关系。

Coumarate-3-hydroxylase is one of important lignin synthetases. Based on cDNA sequence conserved domain of CYP98A3 （accession number AY064170）,a pair of primers were designed and used to amplify a fragment of c3h gene by RT-PCR from poplar（Populus × euramericana cv. ＇74/76 ＇）developing second xylem RNA,then cloned into pMD20-T vector,identified by restriction enzyme cutting,PCR amplification and sequencing. The sequence of the amplified DNA fragment was 994 bp in size and contained a 495 bp ORF（open reading frame）. Alignment with amino acid sequence of c3h retrieved from NCBI （accession number. AY064170）showed that the similarity was 91%. Therefore, the cloned DNA fragment could primarily classified as a member of the CYP98A3 family. It was submitted to GenBank,the accession number was AM920690. Phylogenetic tree was constructed based on c3h genes from GenBank,and the evolution was also analyzed.