【作者】 蔡诚；

【导师】 项艳；

【作者基本信息】 安徽农业大学 ， 园林植物与观赏园艺， 2008， 硕士

【摘要】 木质素是地球上数量仅次于纤维素的有机物,林木中木质素占木材干重的15%～36%,木质素生物合成及调节在植物生长和发育中发挥重要作用,另一方面,木质素是制浆造纸业的不利因素,脱除纤维素中的木质素,需要消耗大量能源和使用有毒化学物质,提高了造纸成本并污染环境。近十几年来,随着人们对木质素生物合成研究的深入,木质素生物合成途径许多酶的编码基因被不断分离克隆出来,调控木质素生物合成途径中重要酶基因的表达,通过抑制酶的活性,从而阻断木质素生物合成途径,降低木质素的合成量或单体组成,已经在烟草和杨树等植株上取得了成功。因此,利用生物技术手段降低木质素含量或改变其组成,提高杨树的制浆性能,创造出符合造纸原料要求的林木品种,对于降低制浆造纸的经济成本和保护环境等都具有重要意义。CCR是木质素生物合成途径的关键酶之一,在木质素生物合成途径中处于终端位置。RNAi(RNA interference)是一种重要的基因沉默方式,在植物基因功能和遗传改良研究中广泛应用。为优化和建立杨树遗传转化体系,探讨RNAi技术在杨树木质素改良中的应用效果,建立杨树RNAi的分子育种新方法,本文以南林95叶片为外植体,对农杆菌介导的遗传转化体系进行较系统地研究,构建了肉桂酰辅酶A还原酶基因CCR干涉表达载体pBI121+2F,开展了农杆菌介导的杨树遗传转化,并对转化植株进行了筛选、PCR检测和木质素含量检测,获得了如下主要结果:1、采用改良的CTAB法提取南林95的基因组DNA,利用PCR技术克隆得到CCR基因的第4个外显子部分序列,并将其正向和反向克隆到pUCCRNAi载体上,通过酶切、纯化、连接、转化等一系列基因工程操作方法构建了含CaMV35S组成型启动子和卡那霉素筛选基因的RNAi表达载体pBI121+2F,并将其成功的导入农杆菌LBA4404中。2、以南林95为试材,对其叶片再生体系进行了优化,建立了高效的叶盘法农杆菌遗传转化体系,并获得了经抗Kan筛选的RNAi片段的转基因植株。(1)以MS为基本培养基附加不同浓度的6-BA和NAA,优化了南林95的组织培养扩繁体系,外植体培养基(MS+6-BA 1.0 mg/L+NAA 0.2 mg/L+蔗糖20g/L+琼脂8 g/L)诱导芽增殖,生根培养基为[1/2MS+NAA 0.01 mg/L+IBA 0.2 mg/L+蔗糖15 g/L+琼脂8 g/L]。(2)用导入pBI121质粒的农杆菌液转化南林95叶片,通过正交试验设计,建立了最佳遗传转化体系,即外植体预培养7 d,在含AS 200μmol/L的菌液浓度为0.6的农杆菌液中侵染20 min,共培养3 d,其中适宜的Kan分化筛选浓度为50 mg/L,生根筛选浓度为30 mg/L。(3)利用筛选出来的最佳遗传转化体系转化目的片段,获得了具有RNAi片段的转基因植株。3、对获得的转基因植株进行PCR检测,获得了RNAi片段的转基因植株。对培养3个月的转基因植株进行Klason木质素分析,与对照植株相比,木质素含量降低20%～30%,且转基因植株的形态和生长发育未见异常。结果表明,利用RNAi技术抑制CCR基因表达,能有效降低植株的木质素含量。综上所述,通过构建CCR基因RNAi表达载体,建立杨树优良无性系叶片遗传转化体系,培育出速生、优质的杨树新品种,为进一步深入了解木质素生物合成提供了研究材料,也为其他树种的木质素基因工程改良奠定理论基础。更多还原

【Abstract】 Lignin is,second to cellulose,the most abundant organic compound in the terrestrial biosphere.In different tree species,lignin content varies between 15 and 36%of the dry weight of wood.Lignin is a major constituent of cell walls of fibers and tracheary elements and provides these cells rigidity for structural support and impermeability for water transport.For the production of high-quality paper,lignin is considered as a negative factor because it must be extracted from the cellulose fraction by energy-requiring and polluting methods.For this reason,there is considerable interest in modifying lignin by genetic engineering to improve its extractability from wood.CCR:Cinnamoyl-CoA reductase is a key enzyme,which decided the last step of lignin biosynthesis.RNAi,as an important gene silencing methods,has a significant application to the research of gene function and genetic melioration.In order to select and establish the regeneration and transformation system of poplar,to investigate effects of RNAi in poplar lignin improvement and to found a new molecular breeding method in poplar,we systematically explored the characteristics and parameters in inheritable transformation system by Agrobacterium,and constructed the expression vector of pBI121+2F then Agrobactirium mediated transformation.The integration of targeted gene was proved by PCR analysis,finally,the lignin content was tested.The main results are summarized as follow:1、The total Poplar DNA were extracted following a modification of CTAB method with some adjustments,Fourth extron（340bp） fragment from genome of CCR gene have been amplified by PCR amplification.The positive oriention and negative oriention of the fragment is ligated into pUCCRNAi,expression vector pBI121+2F have constructed by gene engeneering measure which containing kanamycin selecting gene,such as digestion、purification、ligation and transformation.Then transformed it into Agrobactirium tumefaciens LBA4404.2、In vitro micropropagation and a efficient leaf disc regeneration system have been optimized and established for nanlin95,and transgenic plants were screened using Kan in root induced-media.（1） Take MS as basic mediation with different concentration of 6-BA & NAA,the system of nanlin95 tissue culture has been optimized.The optimum culture medium for differentiation was（MS+6-BA 1.0 mg/L+NAA 0.2 mg/L+Sucrose 20 g/L+Agar 8 g/L）,and[1/2MS+NAA 0.01 mg/L+IBA 0.2 mg/L+Sucrose 15 g/L+Agar 8 g/L]was optimum for root induced-media.（2） Through orthogonal design,an efficient transformation and screening system of nanlin95 mediated by Agrobateriurn tumefaciens with pBI121 was developed.The system was 7 days preculturation,20 min Agrobacterium tumefaciens(OD₆₀₀=0.6) infection with 200μmol/L acetosyringone,and 2 days’ co-infection,the critical kanamycin sensitive concentrations for inducing shoots and roots of the poplar were 50 mg/L and 30 mg/L respectly.（3） With the optimized system of Agrobacterium-mediated transformation,the aimed fragment was transformed and generated transgenic plants.3、Transgenic plants of nanlin95 cultivar confirmed by PCR were transferred into soil and grown for further characterization.Developing xylems were collected from three-month-old transgenic plants of nanlin95.The lignin content of transformed plants reduced by 20%～30%without abnormal morphogenesis and growth compared to that in the controls.Transgenic plants with RNAi fragment of CCR for poplar cultivars will not only give novel insight into regulatory aspects of the pathway and on the structure and biological roles of lignin,but also provide a picture of genetic engineering for modified lignin composition and content.更多还原