【作者】 李丽琴；

【导师】 余龙江；

【作者基本信息】 华中科技大学 ， 生物制药工程， 2009， 博士

【摘要】 植物材料在离体培养过程中常发生遗传变异。这些变异包括外部形态、育性、生长势、抗性和次生代谢等各个方面。植物细胞在离体培养过程中发生的遗传变异往往导致细胞在大规模培养过程中生产次生代谢产物的含量不稳定可控。具体表现为：第一，在不同的培养时期和培养环境下，细胞表观形态、生长速率和合成次生代谢产物的能力发生变化；第二，在相同的传代次数和培养条件下，不同小细胞团的形态颜色发生变化、生长速率不一致、细胞有丝分裂周期不同步、合成次生代谢产物能力发生变化。这些现象导致科学家们筛选得到的高产优质的种质细胞资源无法获得长期有效的应用，影响植物细胞大规模培养生产次生代谢产物的工业化进程。因此，研究植物细胞在离体培养过程中发生遗传变异的影响因素，探讨变异发生的遗传基础，揭示次生代谢产物在细胞离体培养中发生变化的分子调控机理是解决植物细胞工业化应用的前提和基础。本文以离体培养的红豆杉细胞为研究对象，从细胞学、紫杉醇合成关键酶基因的表达谱、基因组DNA遗传和表观遗传学等层面系统研究红豆杉细胞在离体培养过程中遗传变异规律及其分子机理和影响因素，揭示红豆杉细胞合成紫杉醇的分子调控机理，为红豆杉细胞大规模培养生产紫杉醇奠定基础，取得了以下结果：建立了红豆杉细胞遗传变异分析方法和技术体系，如实时荧光定量PCR技术检测紫杉醇合成关键酶基因表达量、AFLP和MSAP分子标记技术检测红豆杉细胞遗传和表观遗传变异、HPLC技术检测红豆杉细胞DNA甲基化水平。离体培养过程中红豆杉细胞遗传变异的分子机理：随着离体培养时间延长，细胞褐化减轻、生长率增加、细胞出现多核和不对称分裂现象、染色体出现四倍体和非整倍体变异、紫杉醇和紫杉烷类含量下降、紫杉醇合成关键酶基因的表达量下降；AFLP分析表明基因组DNA序列的稳定性较好，未出现显著变异，HPLC和MSAP分析显示DNA甲基化水平和模式在红豆杉细胞长期离体培养过程中发生改变，基因组DNA的甲基化修饰水平逐渐升高，且紫杉醇生物合成与DNA甲基化水平密切相关。环境因子影响红豆杉细胞紫杉醇含量的分子机理：改变固液培养方式、光照条件、添加条件培养液等措施仅能在短期内改变紫杉醇合成能力。调整培养基激素浓度能长期影响红豆杉细胞中紫杉醇的合成。在高激素浓度培养基上长期离体培养的红豆杉细胞紫杉醇含量下降、细胞小不均一，形态不规则，出现巨型细胞、空泡细胞和小细胞、紫杉醇合成关键酶基因的表达量下降。基因组DNA序列比较稳定，但是出现了DNA甲基化水平和模式的显著变化。在低激素浓度培养基上长期离体培养的红豆杉细胞的遗传变异现象与之相反。说明植物激素作用可能是影响红豆杉细胞长期离体培养过程中发生遗传变异的因素之一，且与高激素条件下引起的细胞中DNA甲基化水平变异密切相关。低温缓慢生长法培养红豆杉细胞可以减少红豆杉细胞在长期离体培养过程中的传代次数，但在恢复培养过程中仍发生了遗传变异：红豆杉细胞分别在低温保存30 d、60 d、180 d和1 a后恢复培养30 d、60 d、180 d过程中，随着紫杉醇和紫杉烷含量的改变，紫杉醇合成关键酶基因的表达谱、基因组DNA序列稳定性、DNA甲基化水平和模式均发生了相应的改变，其中，DNA甲基化水平的变异与紫杉醇含量的改变密切相关。上述研究结果均证明紫杉醇含量与DNA甲基化水平始终密切相关。外源添加5-Aza-CdR，能抑制红豆杉DNA甲基转移酶(CMT)的活性，降低红豆杉DNA甲基化水平，提高紫杉醇合成关键酶基因的表达，从而提高细胞合成紫杉醇的能力。在相同的传代次数和培养条件下，同步化处理能有效控制部分小细胞团引起的紫杉醇合成能力发生变化的现象。研究表明未经低温同步化的红豆杉细胞中的关键酶基因DXR、HMGR、GGPPS和DBAT的表达量在MJ诱导24 h后均迅速下降，但在低温同步化的细胞中DBAT基因表达量增加，4个关键基因的表达量下降减缓，诱导60h后仍维持较高水平。说明DBAT基因表达量增加以及一系列其他紫杉醇合成关键酶基因在转录水平的高效持续表达是同步化稳定提高紫杉醇产量的主要分子机理之一。红豆杉细胞在离体培养过程中，紫杉醇生物合成能力受到合成紫杉醇关键酶基因转录水平的调控。DNA甲基化／去甲基化模式可能是启动关键酶基因转录的“开关”，从而表现为DNA甲基化水平与与紫杉醇合成能力的密切相关。因此，DNA甲基化引起的紫杉醇合成关键酶基因表达量的改变可能是红豆杉细胞离体培养过程中调控紫杉醇合成的重要分子机理之一。更多还原

【Abstract】 In vitro culture of plant materials often led to genetic variation,described assomaclonal variation,including morphology,fertility,growth potentiality,resistibility andsecondary metabolism aspects.Genetic variation can result in unstable and uncontrollablesecondary metabolite production in large-scale plant cell culture:firstly,in differentgrowing periods and culture environments,cell morphology,growth rate and ability ofsecondary metabolite production have changed;secondly,in the same passage times andculture environments,different small calli demonstrated as different morphology,color,growth rate,asynchronization of cell mitosis cycle and ability of secondary metaboliteproduction.For these reasons,the high-yield germplasm cell can not be applied insecondary metabolite production by large-scale plant cell culture efficiently.Thus,study onimpacting factors and molecular mechanism of genetic variation during the process of invitro plant cell culture is the foundation of plant cell industrial application.In this study,wetake Taxus media cv.Hicksii as research object and try to discover molecular regulationmechanism of taxol biosynthesis in Taxus media cv.Hicksii cell line at chromatosome,genetranscript level,genetic level and epigenetic level.Detailed study as followed:Genetic variation of Taxus during long-term in vitro taxus cell culture:Gradual loss ofpaclitaxel yield in taxus cell line during 5 years in vitro cell culture was observed,withdecreasing cell browning,increasing growth rate and chromatosome variation.Transcriptlevel of four genes encoding key enzymes in the paclitaxel biosynthesis pathway wereheavily lower and little DNA sequence polymorphism were found by AFLP assayed.While,both HPLC and MSAP methods displayed variation of DNA methylation pattern ofdifferent samples.Effect of environment on genetic stability of Taxus media cv.Hicksii cell duringlong-term in vitro taxus cell culture:compared with environment factors,as culture method,light condition,condition medium,plant auxin play an important role in taxol contentvariation.Auxin content impacted not only taxol production,but also cell phenotype,transcript level of four genes encoding key enzymes in the paclitaxel biosynthesis pathway,DNA methylation level and pattern.Taxus cell line genetic stability during process of low temperature preservation:taxus cell line was cultured in low temperature for 30 d,60 d,180 d and 1 a,and recovered tonormal temperature for 30 d,60 d,180 d,separately.Along with taxol and 10-DAB contentvariation,cell phenotype,transcript level of four genes encoding key enzymes in thepaclitaxel biosynthesis pathway,DNA methylation level and pattern happened variation.Repeated low temperature preservation could maintain taxus cell line genetic stability inlong-term.Correlation analysis between taxol content of Taxus media cv.Hicksii and cellbiomass,key enzyme expression,DNA methylation suggested that DNA methylationshowed a significant correlation with taxol content of Taxus media cv.Hicksii.Taxus cellline treated with 5-Aza-CdR could inhibit CMT activity and lower DNA methylationlevel.De-methylation progress maybe stimulate the expression of serial genes related withtaxol biosynthesis.Therefore,DNA methylation maybe regulate taxol production in Taxuscell line.In addition,molecular regulation mechanism of cold synchronization treatment wasstudied.The expression levels of DXR,HMGR,GGPPS and DBAT decreased rapidly after24 h induced by MJ in cells untreated with cold.On the contrary,expression level of DBATwas improved and expression level of those genes decreased slowly in cells with coldtreatment,and the genes expression maintained higher levels after 60 h induced by MJ.Incells with cold synchronization treatment and MJ induction,the high and steady expressionof taxol biosynthetic pathway key genes maybe one reason for the steady improvement oftaxol content.更多还原