灰毡毛忍冬LmC3H1基因的克隆及表达模式与绿原酸含量相关性分析

Cloning and Expression Patterns of LmC3H1 Gene in Lonicera macranthoides and Its Correlation with Chlorogenic Acid Content

目的:以灰毡毛忍冬为材料,克隆对-香豆酸3-羟化酶(LmC3H1)基因,进行生物信息学和表达模式分析,结合绿原酸含量,研究推测灰毡毛忍冬LmC3H1基因的功能。方法:通过逆转录聚合酶链式反应(RT-PCR)和RACE技术克隆LmC3H1基因的全长cDNA序列,对该序列进行生物信息学分析,并利用实时荧光定量PCR(Real-time PCR)和HPLC分别测定灰毡毛忍冬茎、叶及不同花期花中LmC3H1的相对表达量及绿原酸含量。结果:克隆得LmC3H1(GenBank:MN177695)基因,开放阅读框(ORF)长度为1533 bp,编码510个氨基酸,推测其分子式为C2618H4134N718O727 S22,相对分子质量为58005.32,等电点8.92,为亲水性蛋白,定位于叶绿体中,具有跨膜区域LLLIPAVLFLISLVYPLI,含有细胞色素P450的保守结构域CYTOCHROME_P450(422-433 aa);Real-time PCR结果显示,LmC3H1在灰毡毛忍冬茎、叶及不同花期花有不同程度的表达,其中在花发育阶段,白色花蕾期相对表达量最高,花蕾初期及白色开花期次之;白色花蕾期花与茎、叶比,花的相对表达量最高,叶的最低;HPLC结果显示,从绿白色花蕾期到金黄色开花期绿原酸含量呈上升趋势,金黄色开花期含量最高,不同器官中,花中绿原酸最高,茎最低。结论:克隆得到灰毡毛忍冬LmC3H1基因,推测LmC3H1可能参与灰毡毛忍冬花绿原酸的生物合成。该研究为进一步研究该基因的功能及探究灰毡毛忍冬绿原酸生物合成和调节机制提供了依据,同时为遗传改良灰毡毛忍冬品质奠定了基础。

Objective:To clone p-coumaroyl quinate/shikimate 3'-hydroxylase gene from Lonicera macranthoides,and analyze its bioinformatics and expression patterns with chlorogenic acid content,in order to speculate the functions of LmC3H1 gene from L.macranthoides.Method:The full-length cDNA sequence of LmC3H1 gene was cloned by reverse trascription polymerase chain reaction(RT-PCR)and RACE techniques.The bioinformatics analysis of the gene sequence was carried out by using relevant software.Real-time fluorescence quantification PCR(Real-time PCR)and HPLC were used to determine relative expression of LmC3H1 and content of chlorogenic acid in stems,leaves and flowers of different flowering stages.Result:The LmC3H1(GenBank:MN177695)gene was cloned,and the open reading frame(ORF)of it was 1533 bp in length and encoded 510 amino acids.The molecular formula was C2618H4134N718O727 S22,the relative molecular mass was 58005.32,and the isoelectric point was 8.92.It was a hydrophilic protein located in the chloroplast with a transmembrane region LLLIPAVLFLISLVYPLI,and contained a conserved domain CYTOCHROME_P450(433-422 aa)in cytochrome P450.The results of Real-time PCR showed that LmC3H1 was expressed in different degrees in stems,leaves and different flowering stages of L.macranthoides.In the flower development stage,the relative expression of white bud stage was the highest,followed by flower buds and white flowering stage.The ratio of flower to stem and leaf was the highest,and the relative expression of flower was the highest.The HPLC results showed that the content of chlorogenic acid increased from greenish white to golden yellow in flowering stage and golden yellow flowering stage.Among the different organs,the flower had the highest chlorogenic acid,and the stem showed the lowest.Conclusion:The LmC3H1 gene of L.macranthoides is cloned,suggesting that LmC3H1 might be involved in the biosynthesis of L.macranthoides chlorogenic acid.This study provides a basis for further studying the functions of the gene and exploring the biosynthesis and regulation mechanism of L.macranthoides chlorogenic acid,while laying the foundation for the genetic improvement of L.macranthoides.