【作者】 马光；

【导师】 李玉花；

【作者基本信息】 东北林业大学 ， 发育生物学， 2008， 博士

【摘要】 依赖于蓝光的花青素合成现象是植物中普遍存在的一种现象,近期研究表明这种现象是由植物蓝光受体—隐花色素（Cryptochrome,CRY）所介导的。本研究对克隆花青素合成依光型芜菁品种“津田”芜菁中的花青素合成关键基因F3H（flavanone 3-hydroxylase）的启动子区序列以及蓝光受体CRY1基因进行了克隆和功能分析,为揭示植物隐花色素对花青素合成的调节机制开展了必要的前期工作。1 F3H启动子区序列的克隆、分析与瞬时表达根据已经公布的Brassica rapa subsp.pekinensis中含有F3H基因的KBrS001M03克隆序列,采用常规PCR的方法,克隆得到“津田”芜菁F3H基因启动子区序列。在线比对和生物信息学分析表明此序列是位于“津田”芜菁F3H基因上游的启动子区序列,具有启动子特征。利用plantcare工具分析统计此序列中的各个功能元件,发现其中除具有一般真核生物启动子结构所普遍具有的保守CAAT box和TATA box等序列外,还有许多其他的保守序列。采用研究启动子的pKGWFS7载体,利用Gateway技术将此序列替换到Gus基因的上游,驱动Gus基因在“津田”芜菁无菌苗的离体胚轴和子叶中瞬时表达。检测结果显示,胚轴和子叶中均有Gus基因表达。而且无论是在光下还是在黑暗中,克隆到的F3H启动子区序列均可驱动Gus基因正常表达。可见所克隆的序列具有启动子功能。2芜菁CRY1基因的克隆与功能初步分析1)CRY1基因的克隆及其功能分析采用RT-PCR的方法克隆芜菁的CRY1基因,对克隆得到序列进行和系统进化树分析,初步确定其为CRY1基因。研究了此基因在不同波长光下的表达情况,结果表明芜菁CRY1基因的表达量基本一致,为组成型表达。这说明CRY1与其他因子发生作用来传递光信号影响花青素的合成。利用酵母双杂交系统研究芜菁CRY1与COP1的相互作用的结果表明两者之间具有相互作用,表明其具有与拟南芥中蓝光受体传递光信号的类似机制。2) CRY1-dsRNAi表达载体的构建与遗传转化为更深入的研究芜菁CRY1的生理功能,采用Gateway克隆系统,根据克隆到的“津田”芜菁CRY1基因序列设计RNA干涉引物,成功构建了芜菁CRY1双链RNA干扰表达载体:CRY1-dsRNAi。采用农杆菌介导的方法转化芜菁,对影响芜菁离体再生和农杆菌介导的遗传转化的各个因素进行了研究。通过对外植体类型、激素种类、AgNO₃浓度、无菌苗苗龄、预培养时间等因素的优化,获得了再生频率达90%左右的离体再生体系,可以满足遗传转化的要求。研究发现,采用TDZ代替前人常采用的BA来与NAA配合更适于“津田”芜菁的离体再生,适合再生的激素组合为TDZ 7.0 mg/L+NAA 1.0 mg/L。5.0 mg/L AgNO₃对再生来讲是必须的。外植体以苗龄为5d的带柄子叶为好。采用含1.0 mg/L的2,4-D的MS培养基预培养2d可以一定程度上提高再生的频率。在已经建立的芜菁离体再生体系的基础上,研究了不同抗生素种类、农杆菌菌液不同浓度、不同侵染时间、不同共培养时间等因素对遗传转化的影响。得到了较好的转化程序。结果表明采用OD₆₀₀为0.6的农杆菌菌液侵染20 min后共培养3 d,用300 mg/L的羧苄青霉素脱菌,在含300 mg/L羧苄青霉素和20 mg/L潮霉素的培养基中筛选为好。对转化植株后代进行PCR和Southern-blot杂交分析,结果表明干涉片段已经整合到芜菁基因组中。更多还原

【Abstract】 Biosynthesis of anthocyanidinmany induced by blue light is common in plant. Cryptochrome（CRY）,a receptor of blue light,plays a key role in the biosynthesis.In this study, CRY1 gene and promoter sequence of flavanone 3-hydroxylase（F3H） gene in turnip ’Tsuda’ （Brassica rapa L.ssp.rapifera） were cloned,and their function was studied.The works in this study were part of investigation on the mechanism that CRY regulate the biosynthesis of anthocyanidinmany.1 Cloning and transient expression of F3H promoter in turnip ’Tsuda’With primers designed according to the sequence of Brassica rapa subsp,pekinensis clone KBrS001M03 which including F3H gene,and total DNA of turnip ’Tsuda’ as templat,the promoter of F3H was cloned by PCR.The results of blast and analysis of bioinformation showed the cloned sequence was upstream sequence of F3H and has characters of promoter. The statistical analysis of elements in the sequence was carried out with software Plantcare. The elements such as CAAT box and TATA box in eukaryote promoters were found.The cloned sequence was replaced into Gateway vector pKGWFS7 at the position upstream of Gus to induce the expression of Gus in excised hypocotyl and cotyledon of turnip ’Tsuda’ seedling.The results showed that Gus was expressed both in hypocotyl and cotyledon. Moreover,either in light or in dark,the cloned sequence of F3H promoter can induce expression Gus stably.So,it is concluded that the cloned sequence had function of promoter.2Cloning of CRY1 gene and its function1)Sequence and function of CRY1CRY1 gene of turnip ’Tsuda’ was cloned by RT-PCR.Homology analysis of cloned sequence suggested the sequence was homologous with CRY1 of Brassica napus and Arabidopsis thaliana.The expression of this gene in light with different wavelength was studied.The results suggested the expression was steady and not influenced by wavelength. The interaction of CRY1 and COP1 in turnip ’Tsuda’ was confirmed by two hybrid system.2)Construction of vector CRY1-dsRNAi and its genetic transformationTo investigate the function of CRY1 by RNAi,the factors influence the regeneration and genetic transformation of turnip ’Tsuda’ were studied.With cotyledonary petiole and hypocotyl explants,the regeneration frequencies of turnip ’Tsuda’ cukivars were examined.To achieve a high-frequency regeneration system,the hormone combination of thidiazuron（TDZ） and naphthaleneacetic acid（NAA） was compared with combination of benzyladenine（BA） and NAA on shoot regeneration.The results show that cotyledonary petioles were the best explant and that Murashige and Skoog（MS） medium containing 7.0mg/L TDZ and 1.0 mg/L NAA was suitable recipe for getting high-frequency shoot regeneration.Based the recipe,the effects of AgNO₃ concentration,seedling age,pre-culture time of 2,4-Dichlorophenoxyacetic acid （2,4-D） were investigated to optimize the shoot regeneration system.The results suggseted that petiolate cotyledon with seedling age of 5 d cultured in MS medium containing TDZ 7.0 mg/L+NAA1.0mg/L+AgNO₃ 5.0 mg/L followed by pre-culture with 2,4-D 1.0 mg/L for 2 d can be induced with highest-frequency regeneration.The highest shoot formation rate was about 90%.The rooting percentage of shoots was 100%on MS supplemented with 0.1 mg/L indole-3-butyric acid（IBA）.The 95%rooted shoots survived in a greenhouseThe pivotal factors,which influenced the transformation frequency,were compared,and the parameters of transformation system were optimized.It was showed that selective medium containing 300 mg/L carbenicillin,which inhibited growth of Agrobacterium,led to production of more shoots resistant to hygromycin;the time required for infecting and co-culture with Agrobacterium was 20min and three days respectively;and the efficiency of screening with 20 mg/L hygromycin was highest.Molecular analysis of seedlings resisting to hygromycin showed transformants were positive.With Gateway clonging system,expression vector,CRY1-dsRNAi was constructed with primers designed acording to the sequence of CRY1.The vector was transformed to turnip ’Tsuda’ mediamed by agrobacterium successfully.更多还原