| 摘要: |
| 【目的】克隆雷公藤萜类生物合成途径限速酶HMGR的编码基因，并分析雷公藤叶悬浮细胞中该基因在不同质量浓度壳寡糖诱导下的表达水平，为雷公藤萜类生物碱的生物合成提供理论依据。【方法】根据GenBank中报道的限速酶HMGR的编码基因序列合成简并引物，采用RT-PCR、5′-RACE和3′-RACE方法从雷公藤叶片中获得hmgr基因的cDNA全长序列；用生物信息学方法对限速酶HMGR进行蛋白结构及功能分析；用半定量RT-PCR方法比较hmgr基因在不同质量浓度壳寡糖诱导下的表达差异；用HPLC分析不同质量浓度壳寡糖诱导下雷公藤叶悬浮细胞中3种萜类次生代谢物（内酯醇、吉碱和次碱）的含量。【结果】获得了雷公藤中编码限速酶HMGR的基因全长cDNA序列，该序列开放阅读框长为1 860 bp，编码579个氨基酸。生物信息学分析表明，雷公藤hmgr基因编码的蛋白分子质量为61.678 ku，等电点为6.98，蛋白质稳定性参数为41.33，为不稳定蛋白质，氨基酸序列包含2个与NADPH结合的位点（DAMGMNM和VGTVGGGT）及2个与HMG-CoA结合的位点（EMPVGYVQIP和TTEGCLVA）。半定量RT-PCR结果表明，经50 mg/L壳寡糖诱导12 h的雷公藤叶悬浮细胞中hmgr基因表达最强，且在该诱导条件下细胞中的内酯醇、吉碱和次碱的含量也有不同程度的提高。【结论】雷公藤HMGR蛋白可能为甲羟戊酸（MVA）途径中的限速酶，编码该蛋白的hmgr基因可促进雷公藤萜类物质的合成。 |
| 关键词: 雷公藤 HMGR RACE 壳寡糖 |
| DOI： |
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| 基金项目:国家自然科学基金项目（31272110）；公益性行业(农业)科研专项“生物源农药创制与技术集成及产业化开发”(200903052) |
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| Characterization and expression of gene encoding HMGR from Tripterygium wilfordii |
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| Abstract: |
| 【Objective】This study aimed to clone the gene encoding HMGR,the rate-limiting enzyme in the plant terpenoid biosynthetic pathway from Tripterygium wilfordii,analyze its expression in suspension cell induced by chitosan with different concentrations，and improve the understanding of biosynthesis of terpenoid alkaloids in T.wilfordii.【Method】The degenerate primers were synthesized based on the encoding sequence of the rate limiting enzyme HMGR reported in GenBank and full-length hmgr cDNA sequence in the leaves of T.wilfordii was cloned by RT-PCR,5′-RACE and 3′-RACE.Bioinformatics analysis was used to identify the rate-limiting enzyme structure and functions of HMGR.Semi-quantitative RT-PCR was carried out to compare the expression of the hmgr gene in suspension cell and HPLC was used to determine the content of three secondary metabolites (triptolide,wilforgine and wilforine) after being induced by oligosaccharide with different concentrations of oligosaccharide.【Result】A full-length cDNA encoding HMGR with a ORF of 1 860 bp and a total of 579 amino acids was obtained.Bioinformatics analysis revealed that the deduced HMGR had extensive homology with HMGRs of other plants.The molecular weight of the protein was 61.678 ku,the isoelectric point of HMGR was 6.98 and the stability index was 41.33.The protein was unstable.This protein contained two NADP(H)-binding sites (DAMGMNM and VGTVGGGT) and two HMG-CoA-binding sites (EMPVGYVQIP and TTEGCLVA).Gene expression by semi-quantative RT-PCR showed that the highest HMGR expression occured in T.wilfordii induced for 12 hours by oligosaccharide with a concentration of 50 mg/L.This concentration also played significant role in accumulation of triptolide,wilforgine and wilforine.【Conclusion】HMGR in T.wilfordii may be the rate limiting enzyme in MVA pathway and the gene encoding HMGR can stimulate the biosynthesis of terpenoids. |
| Key words: Tripterygium wilfordii HMGR RACE oligosaccharide |
