卷叶贝母HDS基因的克隆与表达分析

Cloning and Expression Analysis of HDS Gene in Fritillaria cirrhosae

为克隆卷叶贝母中编码生物碱合成相关的关键酶4-羟基-3-甲基-2-丁烯基焦磷酸合酶(4-hydroxy-3-methyl-2-butenyl pyrophosphate synthase,HDS)的开放阅读框,运用生物信息学和荧光定量手段对该基因进行分析。基于转录组测序结果,本研究首先通过PCR技术克隆获得卷叶贝母HDS基因(FcHDS)开放阅读框序列,并运用生物信息学方法对该基因进行分析,预测其编码蛋白的结构与功能,其次利用荧光定量PCR(RT-qPCR)检测Fc HDS基因在野生鳞茎和再生鳞茎(通过激素组合刺激获得的组织培养物)中的表达情况。经研究发现FcHDS ORF片段长度为2223 bp,编码740个氨基酸,并与NCBI上公布的油棕、凤梨、烟草、水稻等植物HDS蛋白具有较高的同源性;二级、三级结构预测发现FcHDS蛋白主要由α-螺旋构成;RT-qPCR与总生物碱含量测定实验表明FcHDS基因的表达水平与先前研究的总生物碱含量的变化趋势一致,均为再生鳞茎高于野生鳞茎,而且在根茎叶中都有表达,说明不具有组织特异性。FcHDS蛋白质特征区及同源性等生物信息学分析结合激素组合响应模式的分析结果证明FcHDS可能是一个有生物学功能的蛋白质,其表达受激素组合诱导表达,本研究为卷叶贝母HDS基因功能研究打下基础,同时为利用基因工程手段提高卷叶贝母中生物碱含量提供了理论基础。

To clone the ORF of 4-hydroxy-3-methyl-2-butenyl pyrophosphate synthase(HDS),a key enzyme related to alkaloid synthesis in Fritillaria cirrhosa synthase,using bioinformatics and fluorescence quantitative methods to analyze the gene.Based on the results of transcriptome sequencing,this study first cloned the ORF sequence of F.cirrhosa vulgaris HDS gene(FcHDS)by PCR technology,and analyzed the gene using bioinformatics methods to predict its code.The structure and function of the protein,and secondly,fluorescence quantitative PCR(RT-q PCR)was used to detect the expression of FcHDS gene in wild bulbs and regenerated bulbs(tissue cultures obtained through hormone combination stimulation).The study found that the FcHDS ORF fragment is 2223 bp in length,encoding 740 amino acids,and has high homology with the HDS proteins of oil palm,pineapple,tobacco,rice and other plants published on NCBI;secondary and tertiary structure prediction It is found that FcHDS protein is mainly composed of α-helix;RT-qPCR and total alkaloid content determination experiments show that the expression level of FcHDS gene is consistent with the change trend of total alkaloid content in the previous study.Both regenerated bulbs are higher than wild bulbs,and in rhizomes.It is expressed in the leaves,indicating that it is not tissue-specific.The bioinformatics analysis of the characteristic regions and homology of FcHDS protein combined with the combination of hormone response patterns proves that FcHDS may be a protein with biological functions.Its expression is induced by the combination of hormones.This study is Fritillaria vulgaris HDS gene The functional research lays the foundation and at the same time provides a theoretical basis for the use of genetic engineering to increase the alkaloid content in F.cirrhosa.