【作者】 周欣；

【导师】 钟建江；

【作者基本信息】 华东理工大学 ， 生物化工， 2011， 博士

【摘要】 前人研究表明优化氮源浓度可以提高植物次级代谢物的产量,比如,改变硝酸根浓度可影响基因转录从而促进次级代谢物合成,但是,有关铵离子浓度对植物次级代谢物合成中基因转录和信号转导的影响及其调控机制,尚未见报道。红豆杉(Taxus spp.)细胞培养是目前工业化生产抗癌物质紫杉醇的一个重要来源,然而紫杉烷类(包括紫杉醇)代谢物的生产率仍不高,其次级代谢调控机制尚有进一步深入研究的必要和空间,这关系到植物细胞培养技术的产业化前景以及生化工程学科基础研究水准的提升。本学位论文以中国红豆杉(Taxus chinensis)悬浮细胞为对象,研究了铵离子浓度对其中云南紫杉烷Tc生物合成及其相关基因表达的影响,并探索了改变铵离子浓度诱导Tc合成中的信号分子响应及细胞内信号分子与次级代谢物积累之间的定量关系。这些结果不仅为解决植物次级代谢产物的低产问题提供有用信息,也有助于加深对低氮调控植物次级代谢规律的认识。首先,本论文研究了培养基中初始铵离子浓度对红豆杉细胞中紫杉烷Tc合成的影响。实验结果表明,当培养基初始铵离子浓度在0到20 mM范围内时,2 mM初始铵离子浓度条件下红豆杉细胞中Tc的最高含量可比对照提高50%,达到8.07 mg/gDW。为进一步探索培养基铵离子浓度对Tc合成的诱导机理,我们对2 mM铵离子浓度下紫杉烷合成相关基因的转录水平进行了检测。荧光定量PCR结果显示,2 mM初始铵使基因转录水平得到显著上调,其中紫杉烷合成关键基因牦牛儿基牦牛儿基焦磷酸合酶基因(GGPPs)和紫杉二烯合酶基因(TS)的转录水平最高可达到对照的47倍和37倍,其他紫杉烷合成相关基因(T5αH, TDAT, T10βH和TaHH)的转录水平也得到不同程度(3-9倍)的提高。以上结果表明改变初始铵离子浓度可以通过提高紫杉烷合成相关基因的转录来诱导Tc的生物合成。由于2 mM初始铵离子浓度不利于红豆杉细胞的生长,为进一步提高Tc产量,我们采用了将细胞在2 mM和20 mM铵培养基中转换的两阶段培养策略。其中,先在2mM铵培养基中培养24小时后转入20 mM铵培养基的Tc产量可以达到154.8 mg/L。这一培养策略使得紫杉烷合成相关基因转录在整个培养周期内一直处于较高水平,说明次级代谢物合成相关基因的高表达与其产量提高有密切关系。在确定改变培养基初始铵离子浓度对Tc积累具有促进作用的基础上,我们探索了其诱导Tc合成的信号转导机制。以2 mM初始铵离子浓度作为典型条件,考察了红豆杉细胞防卫信号H202、水杨酸(SA)和苯丙氨酸氨裂解酶(PAL)的响应情况。结果表明2 mM初始铵刺激了H202和SA信号的合成,并诱导了苯丙氨酸氨裂解酶酶活的增强。使用H202产生的抑制剂二亚苯基碘(DPI)和SA合成途径抑制剂多效唑(Paclobutrazol)都能显著抑制2 mM初始铵诱导的GGPPs和TS等紫杉烷合成相关基因转录水平的上调以及紫杉烷Tc的合成。同时,DPI处理能明显降低2 mM初始铵诱导的细胞内SA水平的增加,而添加Paclobutrazol对H202产生却没有抑制作用。这些结果表明H202和SA信号介导了2mM初始铵诱导紫杉烷的生物合成,而且在信号转导过程中,H202位于SA信号的上游,SA信号通过影响紫杉烷合成相关基因的转录,从而影响紫杉烷的生物合成。至今有关植物细胞内SA作为信号分子参与低氮处理调控植物次级代谢物的合成还未见报道,以上信息为深入研究低氮诱导次级代谢物合成的调控机制提供了一定的基础。为进一步探索植物细胞内信号分子水平与次级代谢物积累之间的关系,我们根据以上获得的SA参与调控Tc合成的信息,通过外源添加不同浓度的SA调节细胞内SA水平,并定量考察了Tc积累的响应。结果表明,随着外源SA浓度的升高,内源SA水平也随之升高。外源添加100μM SA可使细胞内SA含量达到97.1μg/gDW,大约是对照的20倍。同时,Tc合成随内源SA水平升高而增强,外源添加100μM SA时,紫杉烷的最高含量达到10.3 mg/gDW,大约是对照的2倍。在外源添加SA的同时,加入SA合成抑制剂多效唑或氨氧基乙酸(AOA)可部分抑制内源SA水平的升高,而且抑制剂处理也部分降低了紫杉烷合成的升高,暗示胞内SA信号与Tc合成可能存在定量关系。为了揭示两者可能的定量关系,在外源添加不同浓度SA及SA生物合成途径的抑制剂情况下,对胞内SA与紫杉烷两者含量的最大值进行回归分析,结果表明,胞内SA与紫杉烷两者含量的最大值之间存在线性关系(r2=0.8426,p<0.05)。分析胞内SA含量的最大值与紫杉烷合成关键基因GGPPs和TS转录水平的最大值之间的关系,GGPPs转录的最大值与SA含量存在良好的线性关系(r2=0.9574,p<0.05)而TS转录的最大值与SA含量的线性趋势较弱(r2=0.8091)。这些信息对基于信号转导提高植物细胞中次级代谢物合成的策略设计具有借鉴作用。总之,本论文获得的关于初始铵离子浓度对红豆杉细胞紫杉烷合成的影响,特别是其对紫杉烷合成途径基因转录的诱导以及对红豆杉细胞信号传导途径影响的信息,对于深入研究低氮诱导植物细胞次级代谢物合成的作用机制,以便于今后有目的地调节次级代谢物合成奠定了一定的基础。本论文提出的通过调节细胞内源信号分子水平来提高次级代谢物产量的策略,对其它细胞大规模培养高效生产有用代谢物也具有一定的借鉴作用。更多还原

【Abstract】 Optimization of nitrogen concentration in medium was previously reported to efficiently improve secondary metabolite biosynthesis in plant cell cultures, as changing nitrate concentration could enhance bioactive compounds production through influencing gene expression. But the influence of ammonium on gene transcription and signal transduction and its regulation had not been known. At present, Taxus spp. cell culture is one of important sources for pharmaceutically active taxol production in industry. However, the productivity of taxoids (including taxol) is still low. Further investigation on the regulation mechanism of secondary metabolite biosynthesis will be helpful for industrial application of plant cell culture and basic researches in the field of biotechnology. By taking Taxus chinensis suspension cells for example, this Ph.D. dissertation was aimed to focus on the effects of medium initial ammonium concentration on bioactive taxuyunnanine C (Tc) accumulation and on the expression of related gene. The signals mediating induction role of changing ammonium concentration on Tc biosynthesis and quantitative relation between intracellular signal level and secondary metabolite accumulation were also studied. This work provided us important information to improve the low yield of bioactive metabolite in plant cell culture, and also is useful for understanding the induction mechanism of low nitrogen on secondary metabolites.At first, effect of medium initial ammonium concentration on Tc biosynthesis in Taxus chinensis cells was investigated When medium initial ammonium was in the range of 0-20 mM, the highest Tc content of 8.07 mg/gDW was achieved at 2 mM initial ammonium, which was about 50% higher than that of control. To provide an insight into the unknown inducible mechanism of initial ammonium, the expression of important genes in taxoid biosynthetic pathway were examined. The quantitative Real Time PCR results showed that the transcription of all taxoid biosynthetic genes were up-regulated when T. chinensis cells was cultivated in medium of 2 mM initial ammonium. Among them, transcription of geranylgeranyl diphosphate synthase (GGPPs) and taxa-4(5),11(12)-diene synthase (TS), key genes in taxoid biosynthesis, achieved maximum at 47-fold and 37-fold compared to control, respectively. Other four taxoid biosynthetic related genes(T5aH, TDAT, T10βH和TaH) were also induced by 3-9 folds. The above results indicated the stimulation of taxoid synthesis by low initial ammonium may be resulted from induced transcription of taxoid biosynthetic genes.However, the cell growth was very poor in the 2 mM initial ammonium medium compared to control (at 20 mM of initial ammonium). To further improve the Tc production, strategy of two stage culture between 2 mM and 20 mM ammonium medium was used. Among them, the maximal Tc production of 154.8 mg/L was obtained in the cells which was cultivated in 2 mM ammonium medium for the first 24 h and then transferred to 20 mM ammonium medium. This two stage culture strategy made the Tc biosynthetic gene transcription maintained at a high level during cultivation period, suggesting that high expression level of biosynthetic related gene may be closely related to higher secondary metabolite production.Based on the stimulation effects of changing initial ammonium concentration on Tc biosynthesis, the unknown signal transduction pathway in low ammonium induction of secondary metabolism was investigated. Choosing the 2 mM initial ammonium as a typical condition, defense signals of H2O2, salicylic acid (SA) and phenylalanine ammonia-lyase (PAL) were detected were examined in cell cultures of T. chinensis. The oxidative burst (induced H2O2 production) was confirmed, and the induction of PAL activity and intracellular SA synthesis were found. Application of diphenylene iodonium (DPI), the H2O2 production inhibitor, and paclobutrazol, the SA biosynthesis inhibitor, inhibited the 2 mM initial ammonium-induced up-regulation of taxoid biosynthetic genes such as GGPPs and TS, as well as induction of Tc accumulation. DPI treatment effectively depressed the 2 mM initial ammonium-stimulated SA accumulation, while paclobutrazol addition didn’t affect induced H2O2 production. The above results suggested that both H2O2 and SA signals were involved in 2 mM initial ammonium-induced Tc biosynthesis, and H2O2 was upstream of SA in signal transduction pathway, SA may influence Tc synthesis through mediating the induction of Tc biosynthetic genes expression. To the best of our knowledge, intracellular SA could be a mediatory signal under low nitrogen is proposed for the first time. The obtained information about signal transduction cascade from defense signal response to activated transcription of taxoid biosynthetic genes and enhanced Tc production is helpful for further investigation on low nitrogen induction of secondary metabolism.To further study the relation between intracellular signal level and secondary metabolite accumulation in plant cells, endogenous SA level was regulated by applying different concentrations of exogenous SA based on the mediatory role of intracellular SA in Tc biosynthesis, and the quantitative response of Tc accumulation was also investigated. The results showed that intracellular SA increased with increase of exogenous applied SA concentration. The maximal endogenous SA of 97.1μg/gDW was observed at 1 h after addition of 100μM exogenous SA, approximately 20 folds higher than intracellular SA before exogenous SA elicitation. Tc content increased with increase of internal SA, which achieved maximum at 10.3 mg/gDW,2-fold over control upon 100μM exogenous SA. Addition of SA biosynthesis inhibitor, paclobutrazol or (BOC-aminooxy) acetic acid (AOA), with exogenous SA, led to a partial inhibitory effect on endogenous SA accumulation, as well as Tc induction, suggesting Tc biosynthesis was dependent on internal SA level. In order to identify potential quantitative relationships between intracellular SA and Tc biosynthesis, regression analyses were performed for maximum values of intracellular SA and Tc accumulation under different concentrations of exogenous SA and its biosynthesis inhibitor treatments. There was a linear correlation between maximal intracellular SA level and maximal induced Tc biosynthesis (r2=0.8426, p<0.05). As for gene expression, maximal induction of GGPPs transcription showed a good dose-dependent relation with the highest intracellular SA level (r2=0.9574, p<0.05), but this trend was a bit weakly observed in case of TS expression with maximal intracellular SA level (r2=0.8091). The signal-product quantitative relationship observed here could be useful for strategy development based on signal transduction for enhancement secondary metabolite production in plant cell cultures.Collectively, the information of initial ammonium concentration affected Tc biosynthesis in Taxus chinensis cells, especially information on low initial ammonium induced gene transcription and signal transduction pathway, will be useful for the exploitation of the inducible mechanisms of low nitrogen-regulated secondary metabolite biosynthesis and for the objective manipulation of bioactive products production. The strategy based on intracellular signal engineering proposed here will also be helpful for useful metabolites production in other plant cell cultures.更多还原