菊芋HtCCR1基因的克隆与表达分析
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引用本文:张新业,苏彦苹,乔 洁,王聪艳,李文静.菊芋HtCCR1基因的克隆与表达分析[J].西北植物学报,2019,39(11):1919~1928
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作者单位
张新业1,2,3,苏彦苹1,2,乔 洁1,王聪艳1,2,李文静1,2,3* (1 廊坊师范学院 生命科学学院, 河北廊坊 0650002 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) 
基金项目:河北省高等学校科学技术研究项目(BJ2018044);
中文摘要:肉桂酰辅酶A还原酶(cinnamoyl CoA reductase, CCR)是木质素合成代谢的关键酶。该研究以菊芋(Helianthus tuberosus L.)‘廊芋8号’为材料,克隆到1个菊芋的CCR基因,命名为HtCCR1(GenBank登录号为MN205540),其开放阅读框(ORF)长975 bp,编码324个氨基酸,其中含有FR_SDR_e保守结构域。系统进化分析表明,HtCCR1与向日葵CCR蛋白(XP_021989763.1)共聚于一支,二者亲缘关系最近。实时定量PCR分析表明,HtCCR1基因在菊芋茎和叶中的表达量显著高于在根和块茎中;盐(150 mmol·L-1 NaCl)胁迫处理6、12和24 h后,处理组HtCCR1基因的表达量均显著高于对照组;干旱(20% PEG6000)胁迫6和12 h后,处理组HtCCR1基因的表达较对照组均显著上调。成功构建pET 28a HtCCR1原核表达载体,转化大肠杆菌BL21(DE3)并诱导出了符合预期大小的蛋白, 表明HtCCR1重组蛋白已成功表达。该研究结果为进一步研究HtCCR1基因的功能及利用基因工程手段调节菊芋中木质素的生物合成奠定了基础。
中文关键词:菊芋  肉桂酰辅酶A还原酶(CCR)  原核表达  荧光定量PCR  胁迫处理
 
Cloning and Expression Analysis of HtCCR1 Gene from Helianthus tuberosus L.
Abstract:Cinnamoyl CoA reductase (CCR) is the key enzyme and plays an important role in the biosynthesis of lignin monomer. In this study, one CCR gene—HtCCR1 (GenBank accession number MN205540) was cloned from the Helianthus tuberosus variety ‘Lang Yu 8’. The open reading frame (ORF) of HtCCR1 was 975 bp, which encoded 324 amino acids. The HtCCR1 protein harbored the FR_SDR_e conserved domain. Phylogenetic analysis showed that HtCCR1 was closely related to the Helianthus annuus CCR protein (XP_021989763.1). The gene expression pattern and responses to salt and drought stresses of HtCCR1 were detected through qRT PCR. The result showed that HtCCR1 was continuously expressed in root, stem, leaf and tuber, and the expression in stem and leaf was the strongest; the expression of HtCCR1 in treatment group was significantly higher than that in control group at 6, 12 and 24 h after salt treatment; the transcription of HtCCR1 was significantly up regulated after 6 and 12 h of drought treatment. The prokaryotic expression vector of HtCCR1 (pET 28a HtCCR1) was constructed and transformed into Escherichia coli BL21 (DE3), and after IPTG induction, the recombinant protein of the expected size was expressed. These results provided a basis for the further study on function of HtCCR1 and the regulation of lignin biosynthesis in Helianthus tuberosus using genetic engineering.
keywords:Helianthus tuberosus L.  cinnamoyl CoA reductase (CCR)  procaryotic expression  qRT PCR  stress treatment
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