甜高粱4-香豆酸辅酶A连接酶基因(4CL)的克隆与鉴定及时空表达分析

Cloning,characterization and spatio-temporal expression analysis of 4-coumarate: CoA ligase genes(4CL) in sweet sorghum

通过对木质素合成途径中关键的限速酶4-香豆酸辅酶A连接酶(4CL,EC6.2.1.12)基因克隆、鉴定与时空表达分析来研究甜高粱4CL基因家族的特性,为今后利用基因工程手段改造甜高粱木质素提供一定的理论依据。搜索NCBI数据库得到高粱4CL基因家族序列,以甜高粱品种‘大力士’的cDNA为模板克隆得到甜高粱4CL家族中各蛋白编码序列(CDS),CDS连入表达载体pET51B,转入大肠杆菌表达菌株(BL21)进行诱导表达,镍柱纯化后进行酶活性分析。实时定量荧光PCR(qPCR)检测甜高粱4CL基因家族各成员在不同时期和不同组织中的相对表达量。结果表明:通过在NCBI数据库中对植物4CL蛋白序列分析,在高粱基因组中,鉴定出7个4CL类似(4CL-like)基因。序列对位排列分析显示:甜高粱7个4CL-like基因在高粱属中非常保守;其氨基酸序列与水稻相对应的Os4CL有90%左右的相似度;它们可分为4CL ClassⅠ和4CL ClassⅡ两类,其中Sb07G007810、Sb04G005210、Sb10G026130、Sb07G022040、Sb03G000610和Sb06G016630属于4CL ClassⅠ,Sb04G031010属于4CL ClassⅡ。酶活性分析表明甜高粱7个4CL蛋白都具有4-香豆酸辅酶A连接酶活力。通过对甜高粱7个4CL基因时空表达分析发现,参与木质素合成的4CL ClassⅠ类基因在组织生长快速期的表达量非常高,而参与类黄酮合成的4CL ClassⅡ类基因在植物受到太阳直接照射的部位表达量显著地增加。在根和茎中,Sb04G005210基因的表达量最高;在叶中,Sb07G007810基因的表达量最高。上述甜高粱7个基因属于真正的4CL基因。

4-coumarate： CoA ligase（4CL,EC6. 2. 1. 12） was the key limiting rate enzyme in lignin biosynthesis of sweet sorghum. To study the characteristics of sweet sorghum 4CL gene family,we cloned 4CL genes and analyzed the function and spatio-temporal expression. This will provide a theoretical basis for improving the lignin biosynthesis in sweet sorghum by genetic engineering. The coding sequences of 4CL genes,which were identified by blasting in NCBI database,were cloned by taking the cDNA of the sweet sorghum variety‘Dalishi＇as the template. cDNAs with full coding regions were subcloned into pET51B vector and fused with a His tag at the C terminus. The plasmids were transformed into Escherichia coli BL21（DE3） strain for recombinant proteins expression. The expressed recombinant proteins were captured by a nickel resin column and used for enzyme activity analysis. Quantitative RTPCR was carried out to detect the relative expression of 4CL genes in different tissues and developmental stages. Seven 4CL-like genes which encode the putative 4CL were identified in sorghum genome. By sequence alignment analysis,these 4CL-like proteins from sweet sorghum were highly conserved in Sorghum genus. The amino acid sequences of these 4CL proteins in sweet sorghum are about 90% similarity with corresponding 4CL proteins in rice. These 4CL proteins were divided into two sub-categories： Class Ⅰ（Sb07G007810,Sb04G005210,Sb10G026130,Sb07G022040,Sb03G000610 and Sb06G016630） and Class Ⅱ（Sb04G031010）. Enzyme activity assays verified that these seven 4CL proteins had 4CL catalytic activities though with different substrate affinities. By analyzing the expression pattern of the these seven 4CL genes in sweet sorghum,we figure out that the 4CL ClassⅠgenes,which areinvolved in lignin biosynthesis,highly expressed in fast-growing stages. The expression level of the 4CL Class Ⅱ gene which participates in flavonoid synthesis was significantly increased in the parts which directly receive solar radiation. Among the 4CL Class Ⅰgenes,the expression level of Sb04G005210 was the highest in root and stem,and that of Sb07G007810 was the highest in leaf. These seven 4CL-like genes really belong to 4CL family in sweet sorghum.