联合调控对中国红豆杉细胞关键酶基因表达的影响

doi:Q786

中国生物工程杂志

2010, Vol. 30

Issue (08): 31-36 DOI: Q786

研究报告

联合调控对中国红豆杉细胞关键酶基因表达的影响

高明波^1,2**,张卫³,李兴泰²,阮成江^1,2,范圣第¹

1.生物化学工程国家民委教育部重点实验室大连 116600
2.大连民族学院生命科学学院大连 116600
3.澳大利亚Flinders 大学海洋分子生物过程及生物产品实验室阿德莱德 SA5042

Expression Profiling of Genes Involved in Taxuyunnanine C Biosynthesis in Cell Suspension Cultures of Taxus chinensis by Repeated Elicitation with a Newly Synthesized Jasmonate,in situ Absorption and Sucrose Feeding

GAO Ming-bo^1,2,ZHANG Wei³,LI Xing-tai²,RUAN Cheng-jiang^1,2,FAN Sheng-di¹

全文: PDF(1205 KB) HTML

摘要：

红豆杉悬浮培养细胞可以持续提供抗癌药物紫杉醇及一些紫杉烷类。在中国红豆杉悬浮培养细胞中，云南紫杉烷C（Tc）是主要的紫杉烷。为了更理性地调控紫杉醇或有用紫杉烷的生产，有必要深入了解其生物合成过程。采用实时定量PCR（Real-time Quantitative PCR，即RQ-PCR）技术考察经调控后紫杉醇及紫杉烷代谢中关键酶基因—TASY，T5αH，TDAT，T10βH，TαH，T14βH表达水平的变化。在细胞培养的第7天和12天，分别以100μM 2,3-二羟丙基茉莉酸（DHPJA）诱导，同时在细胞培养第7天进行20g/L蔗糖饲喂、100g/L XAD-7HP的原位吸附。该联合调控处理使得细胞培养第30天时，Tc产量高达1517±37mg/L，是对照处理的11.1倍，是DHPJA重复诱导联合蔗糖饲喂处理的1.7倍。RQ-PCR结果显示：DHPJA的加入可使6个基因表达水平显著提高，但在12小时后快速下降，需补充DHPJA以再次提高基因表达水平。吸附剂同时引入会延缓基因表达水平的提高速度，但却能维持基因表达处于一个较高的水平，表现为在细胞培养中后期，基因表达水平将显著高于无吸附剂的调控体系。与13α-羟化相对应的TαH基因有所不同，吸附剂的存在更显著地抑制其表达，但仍有维持表达的功能。

关键词： 中国红豆杉; 云南紫杉烷C; 2,3-二羟丙基茉莉酸; XAD-7HP; 实时定量PCR

Abstract:

Taxus suspension cell culture has the potential to provide a sustainable source of anticancer drug paclitaxel (Taxol) and other taxoids. In the cell culture of Taxus chinensis, taxuyunnanine c (Tc) is the primary taxoid. To design a rational strategy for redirecting the precursor fluxes from other taxoids into paclitaxel production, this study employed quantitative real-time-PCR (RQ-PCR) to understand the dynamic profiling of key biosynthetic pathway genes of palcitaxel and taxoids during the culture process. Six genes —TASY, TDAT, T5αH, TαH, TDAT and T10βH were quantified under a process condition of double elicitation by 100μM 2,3-dihydroxylpropanyl jasmonate(DHPJA) on day 7 and day 12, 20g/L sucrose feeding and 100g/L XAD-7HP absorption on day 7 . This process treatment led to a high accumulation of Tc at 1517±37mg/L on day 30, 11.1 folds of that of control and 1.7 folds of that of double elicitation with sucrose feeding. The RQ-PCR results showed that with DHPJA elicitation, the expression of all the six genes was greatly increased, but sharply declined after 12 hours. This rapid decline suggested that the second DHPJA elicitation was required to sustain gene expression. Simultaneous absorbents addition would delay the improvement of gene expression but maintain the gene expression at a higher level, resulting in higher gene expression over treatments without absorbents. TαH expression was somewhat different which was markedly inhibited by XAD-7HP absorption while absorbants still maintained its expression.

Key words: Taxus chinensis Taxuyunnanine C DHPJA XAD-7HP RQ-PCR

收稿日期: 2010-04-29 出版日期: 2010-08-25

基金资助:

国家自然科学基金（20676130）资助项目

通讯作者: 高明波 E-mail: lily@dlnu.edu.cn

	服务
	把本文推荐给朋友
	加入引用管理器
	E-mail Alert
	RSS
	作者相关文章
	高明波
	张卫
	李兴泰
	阮成江
	范圣第

引用本文:

高明波张卫李兴泰阮成江范圣第. 联合调控对中国红豆杉细胞关键酶基因表达的影响[J]. 中国生物工程杂志, 2010, 30(08): 31-36.

GAO Meng-Bei, ZHANG Wei, LI Xin-Tai, RUAN Cheng-Jiang, FAN Ku-Di. Expression Profiling of Genes Involved in Taxuyunnanine C Biosynthesis in Cell Suspension Cultures of Taxus chinensis by Repeated Elicitation with a Newly Synthesized Jasmonate,in situ Absorption and Sucrose Feeding. China Biotechnology, 2010, 30(08): 31-36.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/Q786 或 https://manu60.magtech.com.cn/biotech/CN/Y2010/V30/I08/31

［1］ Baloglu E, Kingston D G I. The taxane diterpenoids. J Nat Prod, 1999, 62: 14481472.
［2］ Itokawa H. Taxoids occuring in the genus Taxus. In: Itokawa H, Lee K H. TaxusThe Genus Taxus. London: Taylor & Francis, 2003.3578.
［3］ Kikuchi Y,Yatagai M. The commercial cultivation of Taxus species and production of taxoids. In: Itokawa H, Lee K H. TaxusThe Genus Taxus. London: Taylor & Francis,2003.151178.
［4］ Takeya K. Plant tissue culture of taxoids. In: Itokawa H, Lee K H. TaxusThe Genus Taxus.London: Taylor & Francis, 2003.134150.
［5］ Zhao Z J，Xu Y F，Qian Z G, et al. Novel fluoro and hydroxylcontaining jasmonate derivatives as highly efficient elicitors in suspension cultures of Taxus chinensis. Bioorganic & Medicinal Chemistry Letters, 2004, 14:47554758.
［6］ Qian Z G，Zhao Z J，Xu Y F, et al. Novel chemically synthesized hydroxylcontaining jasmonates as powerful inducing signals for plant secondary metabolism. Biotechnology and Bioengineering, 2004, 86( 7): 809816.
［7］ Qian Z G, Zhao Z J, Xu Y F, et al. Highly efficient strategy for enhancing taxoid production by repeated elicitation with a newly synthesized jasmonate in fedbatch cultivation of Taxus chinensis cells. Biotechnol Bioeng, 2005, 90(6): 516521.
［8］ Croteau R. Cyclization of geranylgeranyl diphosphate to taxa4(5),11(12)diene is the committed step of Taxol biosynthesis in Pacific yew. J Biol Chem, 1995, 270:86868690.
［9］ Hezari M, Croteau R. Taxol biosynthesis: an update. Planta Med, 1997, 63: 291295.
［10］ Hefner J, Ketchum R E, Croteau R. Cloning and functional expression of a cDNA encoding geranylgeranyl diphosphate synthase from Taxus canadensis and assessment of the role of this prenyltransferase in cells induced for taxol production . Arch Biochem Biophys, 1998, 360(1) :6274.
［11］ Ketchum R E B, Christopher D R, Qiu D Y, et al. Taxus metabolomics: methyl jasmonate preferentially induces production of taxoids oxygenated at C13 in Taxus x media cell cultures. Phytochem, 2003, 62: 901909.
［12］ Croteau R, Ketchum R E B, Long R M, et al. Taxol biosynthesis and molecular genetics. Phytochem Rev, 2006, 5: 7597.
［13］ Jennewein S, Long R M, Williams R M, et al. Cytochrome P450 taxadiene 5ahydroxylase, a mechanistically unusual monooxygenase catalyzing the first oxygenation ste Pof Taxol biosynthesis. Chem Biol, 2004, 11: 379387.
［14］ Jennewein S, Rithner C D, Williams R M, et al. Taxoid metabolism: taxoid 14bhydroxylase is a cytochrome P450dependent monooxygenase. Arch Biochem Biophys, 2003, 413: 262270.
［15］ Croteau R. Administering cultured Taxus cells with early precursors reveals bifurcations in the taxoid biosynthetic pathway. Phytochem, 2007, 68:335341.
［16］高明波，张卫，虞星炬. 原位吸附和茉莉酸甲酯联合使用显著提高中国红豆杉细胞云南紫杉烷c的生产，生物工程学报，2010，26（2）：223229. Gao M B, Zhang W, Yu X J.Chin J Biotech, 2010, 26(2): 223229.
［17］徐德昌，纪巍，程大友,等. TaqMan探针荧光定量PCR检测甜菜低温胁迫相关miRNAs，中国甜菜糖业，2008，4：36. Xu D C, Ji W, Cheng D Y, et al.China Beet & Sugar, 2008, 4: 36.
［18］ Ezekiel N, Camille P D, Susan C R, et al. Expression profiling of genes involved in paclitaxel biosynthesis for targeted metabolic engineering. Metab Eng, 2006, 8:385–394.

[1]	徐勇, 沈翀, 邱兴天, 蔡鹏, 黄敏仁, 余世袁. 热带假丝酵母木糖乙醇发酵相关基因的筛选与分析[J]. 中国生物工程杂志, 2012, 32(11): 61-69.
[2]	徐勇, 沈翀, 邱兴天, 蔡鹏, 黄敏仁, 余世袁. 热带假丝酵母木糖乙醇发酵相关基因的筛选与分析[J]. 中国生物工程杂志, 2012, 32(11): 61-69.
[3]	李媛, 任长虹, 吴永红, 叶巧, 石锦平, 屈武斌, 郑晓飞, 刘虎岐, 张成岗. 多重PCR在质粒拷贝数检测中的应用[J]. 中国生物工程杂志, 2011, 31(06): 93-98.
[4]	金华利, 张富春, 张爱莲, 李轶杰, 王宾. 钠米颗粒介导质粒DNA转染体外真核细胞[J]. 中国生物工程杂志, 2003, 23(10): 71-75.

Viewed

Full text

Abstract

Cited

Shared

Discussed