马尾松肉桂酰辅酶A还原酶基因(CCR)克隆及分析

对马尾松肉桂酰辅酶A还原酶基因(CCR)进行扩增、克隆和测序(GenBank登录号:EU753854).应用MEGA4.0.2软件对不同树种CCR基因的核苷酸和氨基酸序列进行比对,结果表明:马尾松CCR基因的外显子和内含子数目与火炬松、杨树、银合欢、光皮桦、冈尼桉、柳桉、蓝桉的外显子和内含子数目相同,都有5个外显子和4个内含子.马尾松CCR基因编码区975 bp,编码324个氨基酸.马尾松外显子Ⅰ,Ⅱ,Ⅲ,Ⅳ,Ⅴ分别为133,155,186,353,148 bp,而内含子Ⅰ,Ⅱ,Ⅲ,Ⅳ分别为1 450,216,737,941 bp.马尾松CCR外显子和内含子连接处序列除内含子Ⅱ/外显子Ⅲ出现CTPuCG/外显子Ⅲ,其余遵循外显子/GTPuAG/外显子组成规律.马尾松外显子Ⅱ,Ⅲ,Ⅳ核苷酸数目与桉属、杨属、桦木属和松属树种相同,但外显子Ⅰ和外显子Ⅴ不尽相同.比对的树种间在该基因编码区核苷酸序列和氨基酸序列上相对保守,也存在一定差异.马尾松与火炬松、光皮桦、银合欢、杨树、冈尼桉、柳桉、蓝桉各物种间编码区核苷酸序列相似性大小分别为99.2%,67.8%,68.0%,68.9%,69.5%,69.3%,69.9%,而相应的氨基酸序列间相似性大小分别为99.1%,73.5%,72.5%,74.4%,75.0%,74.4%,75.0%.马尾松与其他13个树种CCR序列构建的系统进化树表明:3个针叶树种形成1个独立分支,而且最早进化.

Cloning and Sequence Analysis of Cinnamoyl-CoA Reductase Gene (CCR) of Pinus massoniana

The cinnamoyl-CoA reductase gene ( CCR) of Pinus massoniana was amplified, cloned and sequenced (CenBank Accession No: EU753854) . The nucleotide and amino acid sequences of CCR gene among different species were compared and analyzed by MEGA4.0.2. The results showed that the number of exons and introns of CCR gene of P. massoniana were the same as those in Pinus taeda , Populus balsamifera, Leucaena leucocephala, Betula luminifera, Eucalyptus gunnii, Eucalyptus saligna, and Eucalyptus globulus. They all had 5 exons and 4 introns. The size of coding sequence of the CCR gene of P.massoniana, encoding 324 amino acids, was 975 bp. The junction sequences of exons and introns of CCR gene of P.massoniana were in accordance with the law of gene composition (exon/GTPuAG/exon) , except Intron Ⅱ /Exon Ⅲ (GTPuGG/Exon Ⅲ ) . The nucleotide number of Exon Ⅱ to Ⅳ of P. massoniana was the same as those of Eucalyptus, Populus , Betula ,and Pinus . The similarities of nucleotide sequences of the coding region of CCR gene between P. massoniana and P. taeda ,B. luminifera, P. balsamifera, L. leucocephala, E. gunnii, E. saligna, and E. globulus were 99.2%, 67.8%,68.0%, 68.9%, 69.5%, 69.3%, 69.9%, respectively, and the similarities of amino-acid sequences were 99.1%,73.5% , 72.5% , 74.4% , 75.0% , 74.4% , 75.0% , respectively. Molecular phylogenetic trees of P. massoniana with other 13 species (or sequences) reconstructed with CCR and the inferred protein sequence indicated that the 3 conifer species formed a separate clade and evolved earlier.