芒果4CL基因的克隆及其表达分析

Cloning and Expression Analysis of 4CL Gene from Mango( Mangifera indica)

4-香豆酸:辅酶A连接酶(4CL)是植物类黄酮类化合物和木质素等生物合成途径的一种关键酶,为了研究其在芒果果实中的作用机理,采用RACE方法,从芒果果实克隆得到了一个类黄酮合成相关的4CL基因。该基因全长c DNA序列为1 740 bp,开放阅读框为1 653 bp,编码550个氨基酸,分子量为60.47 k Da,等电点为9.51。对基因组扩增得到了2 318 bp长度的片段分析发现,该基因含有5个内含子,分别在1 013~1 139 bp,1 330~1 415 bp,1 564~1 653 bp,1 722~1 802 bp,1 905~2 000 bp。通过在线软件对其二级结构和三级结构进行了预测,系统发育分析发现该基因编码的蛋白与水曲柳、菘蓝等植物具有较近的亲缘关系。对3种不同着色的芒果品种中的4CL基因的RTPCR进行分析发现:红色的贵妃品种中表达量较高,而绿色的桂七品种中表达量较低。研究表明4CL基因表达芒果果实着色具有密切的相关性。

4-coumarate : coenzyme a ligase( 4CL) is a key enzyme for the plant phenylpropanoid derivatives,flavonoids and lignin biosynthetic pathway. In order to study its mechanism on mango fruit,according to the design of degenerate primers of 4CL gene sequence reported,full-length c DNA sequence of 4CL gene of mango fruit was obtained with 3' RACE and 5' RACE method. The full length c DNA sequence of gene was 1 740 bp,the open reading frame was 1 653 bp,encoding 550 amino acids,the molecular weight of 60. 47 k Da,isoelectric point 9. 51. The2 318 bp length fragment was amplified from genome and analysis,which contained five introns,respectively,in 1 013-1 139 bp,1 330-1 415 bp,1 564-1 653 bp,1 722-1 802 bp,1 905-2 000 bp. The secondary structure and tertiary structure of 4CL protein were predicted by online software. It was found that the gene encoding for the protein had close relationship with ash,Isatis and other plant through phylogenetic analysis. Expression of 4CL gene in different mango varieties showed: the high expression of the Guifei varieties,and the expression of green Guiqi varieties with low volume. The study showed that the 4CL gene expression was closely related to the mango fruit color.