| 华山松大小蠹2个甲羟戊酸路径基因的克隆与表达 |
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| 引用本文： | 王媛媛,余佳敏,代鲁鲁,陈辉.华山松大小蠹2个甲羟戊酸路径基因的克隆与表达[J].西北林学院学报,2020,35(1):140-149. |
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| 作者姓名： | 王媛媛 余佳敏 代鲁鲁 陈辉 |
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| 作者单位： | （1.华南农业大学 林学与风景园林学院，广东 广州 510642；2.西北农林科技大学 林学院，陕西 杨陵 712100） |
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| 基金项目： | 陕西省自然科学基础研究重大项目(2017ZDJC-03) |
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| 摘 要： | 香叶基二磷酸合酶/法尼基焦磷酸（G/FPPS）和异戊二烯焦磷酸异构酶（IDI）是单萜和倍半萜生物合成途径分支点的关键酶。通过克隆华山松大小蠹DaG/FPPS和DaIDI基因的全长序列，预测基因和编码蛋白的性质、结构和功能，并对序列特征和不同发育时期（幼虫、蛹、初羽化期成虫和扩散期成虫）以及不同组织（头、前中肠、后中肠、后肠和剩余躯体）DaG/FPPS和DaIDI表达的研究，旨在揭示DaG/FPPS和DaIDI在华山松大小蠹信息化学物质合成中的分子调控机制。采用RT-PCR和RACE克隆华山松大小蠹DaG/FPPS和DaIDI基因，DNAStar、Blast、ExpasyProtscale、SignalP 4.1、PSORT和TargetP等多种生物信息学方法对基因全长cDNA序列、基本理化性质、系统进化关系、信号肽和蛋白亚细胞定位等进行分析。用Real-time PCR检测DaG/FPPS和DaIDI在华山松大小蠹不同发育时期和不同组织中的表达。结果表明，克隆获得华山松大小蠹香叶基二磷酸合酶/法尼基焦磷酸DaG/FPPS和异戊二烯焦磷酸异构酶DaIDI全长cDNA序列，分别命名为DaG/FPPS和DaIDI，DaG/FPPS编码的氨基酸序列与山松大小蠹DpF/GPPS相似度最高达95%，氨基酸序列含有一个RALS 四肽基序、7 个异戊烯基转移酶保守区（Ⅰ-Ⅶ）和2个典型的DD**D的天冬氨酸富含区；DaIDI编码的氨基酸序列与山松大小蠹IDI相似度最高，达到92%。Expasy Protscale结果表明DaG/FPPS蛋白疏水性氨基酸明显多于亲水性氨基酸，推测其为疏水性蛋白，而DaIDI蛋白亲水性氨基酸数量大于疏水性氨基酸，故推测DaIDI为亲水性蛋白，有较好的水溶性。信号肽预测结果表明，DaG/FPPS和DaIDI均不含信号肽，TargetP结果表明，DaG/FPPS和DaIDI蛋白含线粒体靶向肽，结合PSORT结果推测DaG/FPPS和DaIDI均定位于线粒体。华山松大小蠹DaG/FPPS和DaIDI在不同发育时期和不同组织中均有表达，其中完全羽化期与扩散期成虫保持一致，该阶段表达量最高，不同组织中前中肠表达量最高。
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| 关 键 词： | 华山松大小蠹 DaG/FPPSDaG/FPPS DaIDIDaIDI 生物信息学分析 转录水平 |
Cloning and Expression of Two Mevalonate Pathway Genes of Dendroctonus armandi |
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| WANG Yuan-yuan,YU Jia-min,DAI Lu-lu,CHEN Hui.Cloning and Expression of Two Mevalonate Pathway Genes of Dendroctonus armandi[J].Journal of Northwest Forestry University,2020,35(1):140-149. |
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| Authors: | WANG Yuan-yuan YU Jia-min DAI Lu-lu CHEN Hui |
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| Institution: | （1.College of Forestry and Landscape Architecture,South China Agricultural University,Guangzhou 510642,Guangdong,China; 2.Northwest A&F University Forestry college,Yangling 712100,Shaanxi,China） |
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| Abstract: | Geranyl/farneyl pyrophosphate (G/FPPS) and isoprene pyrophosphate isomerase (IDI) play important roles in deciding the biosynthetic pathway of monoterpene and sesquiterpene.The research cloned the full length of DaG/FPPS and DaIDI genes of Dendroctonus armandi to predict the properties,structures and functions of the genes and coding properties,followed by bioinformatic analysis and expression analysis of the genes in different developmental stages (larvae,pupae,teneral adult and emerged adult) and different tissues (head,anterior midgut,posterior midgut,hindgut and carcass) for demonstrating the molecular mechanism of D.armandi DaG/FPPS and DaIDI in synthesis semiochemical production.RT-PCR and RACE were employed to clone complete cDNA of DaG/FPPS and DaIDI in D.armandi.The full-length cDNA,physical and chemical characteristics,phylogenetic relationship,signal peptide,subcellular localization of DaG/FPPS and DaIDI in D.armandi were analyzed by a variety of bioinformatics softwares such as DNAStar,Blast,ExpasyProtscale,SignalP 4.1,PSORT,TargetP et al.Real-time PCR was utilized to examine the DaG/FPPS and DaIDI expression pattern in different developmental stages and different tissues.Two novel cDNAs were isolated from D.armandi,one of them encoding geranyl/farnesyl diphosphate synthase was designated DaG/FPPS,the other one encoding isoprene pyrophosphate isomerase was designated DaIDI.Comparison of the deduced amino acid sequence with other insect showed that DaG/FPPS shared 95% identity with DpF/GPPS of Dendroctonus ponderosae,the amino sequence had a RALS,seven isopentenyl transfers conservative regions and two aspartic acid rich regions; and comparison of the deduced amino acid sequence with other insect showed that DaIDI shared 92% identity with IDI of D.ponderosae.The results of Expasy Protscale showed that the DaG/FPPS protein had more hydrophobic amino acid than hydrophilic amino acid,so we guessed it was a hydrophobic protein.While the DaIDI protein had more hydrophilic amino acid than hydrophobic amino acid,so we guessed it was a hydrophilic protein with good water solubility.DaG/FPPS and DaIDI didn‘t have signal peptides,and they had mitochondrial targeting peptides.From analyzing the data of PSORT and TargetP,we could guess that DaG/FPPS and DaIDI were located at mitochondrial.Gene expression pattern in different developmental stages and tissues illustrated that DaG/FPPS and DaIDI were expressed in all developmental stages and tissues,the complete emergence stage was consistent with the diffusion stage,and the highest expression level was in this stage,and the highest expression level was in the anterior midgut of different tissues. |
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