菊芋查尔酮合成酶基因的克隆与表达分析  

Cloning and Expression Analysis of Chalcone Synthase Gene from Helianthus tuberosus

作　　者：李文静 孙艳香[1,2] 付亚娟 苏彦苹[1,2] 王聪艳 侯晓强[1] 张新业 LI Wenjing;SUN Yanxiang;FU Yajuan;SU Yanping;WANG Congyan;HOU Xiaoqiang;ZHANG Xinye(College of Life Science,Langfang Normal University,Langfang Hebei 065000,China;Hebei Key Laboratory of Animal Diversity,Langfang Hebei 065000,China;Langfang Key Laboratory of Cell Engineering and Application,Langfang Hebei 065000,China)

机构地区：[1]廊坊师范学院生命科学学院,河北廊坊065000 [2]河北省动物多样性重点实验室,河北廊坊065000 [3]廊坊市细胞工程与应用研究重点实验室,河北廊坊065000

出　　处：《西北农业学报》2020年第4期603-612,共10页Acta Agriculturae Boreali-occidentalis Sinica

基　　金：河北省高等学校科学技术研究项目(BJ2018044);廊坊市科技支撑计划项目(2019012003)。

摘　　要：为揭示菊芋(Helianthus tuberosus L.)查尔酮合成酶(chalcone synthase,CHS)基因的功能,以菊芋‘廊芋8号’叶片为材料,采用同源克隆和RT-PCR技术,克隆得到菊芋CHS基因命名为HtCHS,其编码区全长为1197 bp(GenBank登录号MN124515),编码398个氨基酸。HtCHS的基因组DNA(gDNA)全长为1567 bp,含2个外显子和1个内含子。序列比对和系统进化树分析显示,不同植物中的CHS氨基酸序列具有极高的同源性,菊芋与同属的向日葵CHS氨基酸序列一致性达到99.25%,共聚于一个分支。HtCHS蛋白相对分子质量为43.61 ku,等电点为6.33。HtCHS蛋白具有查尔酮合成酶结构域,属于查尔酮合成酶超家族成员,HtCHS不含信号肽和跨膜区,属于非分泌蛋白。实时荧光定量PCR结果显示,HtCHS在根中表达量最高。与对照相比,150 mmol·L-1 NaCl和20%PEG 6000处理6 h时HtCHS表达显著上调,处理12 h时显著下调。原核诱导表达分析结果显示,成功诱导出与预测蛋白大小一致的目的蛋白。Chalcone synthase(CHS),the first rate-limiting enzyme in the pathway of flavonoid synthesis,catalyzes the condensation of one molecule of p-coumaroyl-CoA and three molecule of malonyl-CoA to produce naringenin chalcone.In this study,we cloned one CHS gene from Helianthus tuberosus‘Langyu No.8’(named as HtCHS).Full length open reading frame(ORF)of HtCHS contains 1197 bp nucleotide(GenBank accession number MN124515).Its genomic DNA length is 1567 bp containing two exons and one intron.HtCHS encodes a protein with 398 amino acids,which shows highest identity(99.25%)with CHS protein from Helianthus annuus HaCHS(GenBank accession number XP_022009667.1).Phylogenetic analysis showed that HtCHS and HaCHS were clustered into the same evolutionary branch.In addition,the molecular mass and pI of HtCHS were 43.61 ku and 6.33,respectively.HtCHS has one chalcone synthase domain,suggesting that it belongs to the chalcone synthase superfamily.No signal peptide and transmembrane region were found in HtCHS,implying that it is a non-secretory protein.qRT-PCR analysis showed that HtCHS is constitutively expressed in all four tissues examined but with highest level in roots.In addition,expression level of HtCHS is significantly induced by treatment with 150 mmol·L-1 NaCl and 20%PEG 6000 for 6 h but downregulated for 12 h.These results will lay a foundation for further functional analysis of HtCHS and increase in flavonoid compound production by genetic engineering in Helianthus tuberosus.

关 键 词：菊芋 查尔酮合成酶 基因克隆 生物信息学分析 原核表达 基因表达分析 

分 类 号：Q75[生物学—分子生物学] Q78