【作者】 曹华雯；

【导师】 戴思兰；

【作者基本信息】 北京林业大学 ， 园林植物与观赏园艺， 2010， 博士

【摘要】 菊花(Chrysanthemum×morifolium Ramat.)是我国传统十大名花和世界四大鲜切花之一,近年来利用植物基因工程技术培育菊花新品种已经成为研究热点,并取得了初步成果。目前在菊花转基因研究中主要使用花椰菜花叶病毒CaMV 35S启动子,该启动子为组成型表达启动子,在转基因植株的各个器官和生长发育的所有时期均表达,容易打破转基因植株的代谢平衡,对其正常生长发育造成影响,有时在转基因植株中还易引起外源基因的沉默。诱导型启动子可以利用外界物理或化学条件的变化启动其表达活性,在非诱导条件下其在转基因植株中表达水平较低,对受体植株的伤害较小,因此利用诱导型启动子进行菊花转基因工程研究,具有重要的理论和实践意义。甜菜碱醛脱氢酶(betaine aldehyde dehydrogenase, BADH)基因是甜菜碱合成途径中的重要基因,植物中研究表明该基因的表达受干旱、盐、ABA和低温等的胁迫诱导。本课题组前期已经从菊科菊属植物甘菊[Chrysanthemum lavandulifolium (Fisch. ex Trautv.) Makino]中克隆得到了4个BADH基因启动子序列DBP11、DBP12、DBP21和DBP22 (GenBank登录号分别为DQ497620-23),并利用瞬时表达分析证明4个序列均具有启动GUS报告基因的功能。本研究在此基础上利用转基因的方法研究4个启动子的胁迫诱导活性,并选取其中表达活性较高的启动子进行缺失试验,研究其上顺式作用元件的功能。本研究主要取得如下成果：1.利用实时荧光定量PCR和雷氏盐紫外分光光度计法分别测定了400mmol／LNaCl胁迫处理0h、0.5h、2h、12h、1d、2d、4d、6d、8d、10d和12d,20%PEG-6000胁迫处理0h、0.5h、2h、12h、1d、2d、6d,甘菊叶片中BADH基因表达量和甜菜碱含量的变化。试验结果表明：在高盐胁迫下甘菊叶片中BADH基因表达量和甜菜碱含量均呈现先上升后下降的趋势。在处理初期(0.5h和2h)BADH基因的表达量与对照相比略有下降,此后随处理时间的增加BADH基因表达持续增大,在胁迫处理6d时BADH基因表达量最大为对照的4.6倍,此后BADH基因表达量逐渐降低。甜菜碱含量在NaCl处理0.5h明显增加,此后其含量出现了小幅的震荡上升,在胁迫处理4d时达到了最大值,之后随胁迫处理时间的增加甜菜碱含量逐渐降低。二者之间的变化并不是同步的,而是存在滞后性,分析认为甘菊叶片中BADH基因表达与甜菜碱积累间存在相互抑制的作用。甘菊BADH基因在20%PEG-6000胁迫处理0.5h表达量明显增大,之后活性降低,在胁迫处理1d时表达量达到最大值。2.将甘菊BADH基因启动子DBP11、DBP12、DBP21和DBP22与GUSplus报告基因融合构建植物表达载体,转化烟草。对转基因植株进行NaCl、干旱、ABA、SA和低温胁迫处理,通过测定GUS活性鉴定启动子的诱导性。结果表明：DBP11、DBP12和DBP21启动子均响应NaCl、干旱、ABA和低温胁迫处理,其中DBP12启动子诱导活性更强,DBP22启动子只响应NaCl和低温胁迫处理,且其基本活性仅为DBP11、DBP12和DBP21的0.1倍。3.选取诱导活性较强的DBP12启动子,根据其上顺式作用元件的分布位置设计不同的引物,利用PCR的方法对DBP12启动子进行5’和3’端缺失,将7个缺失片段与GUSplus报告基因融合构建植物表达载体。用农杆菌介导的叶盘转化法转化甘菊叶片,瞬时表达的结果表明,7个载体构建成功。4.将7个DBP12启动子的缺失片段转化烟草,对转基因植株进行NaCl、干旱、ABA和低温胁迫处理,利用GUS活性变化检测启动子各片段的表达差异。DBP12启动子上同时含有抑制子和增强子；在DBP12启动子上响应NaCl、干旱、ABA和低温胁迫诱导的顺式作用元件为MYB、MYC识别位点和ACACNNG core;同时DBP12启动子上所含有的抑制子也和顺式元件一起共同作用调控BADH基因对胁迫信号的响应；此外BADH基因对于启动子基础活性及响应干旱、NaCl、ABA和低温诱导具有重要作用,5’-UTR对于BADH基因响应低温胁迫诱导的作用是唯一的。5.利用接头染色体步移法,以甘菊盐胁迫条件下表达的6条EST序列为基础,从甘菊Genome Walker库中克隆得到了其中4条片段上游DNA序列,经启动子软件分析结果显示,片段24.1(ADP核糖焦磷酸水解酶基因)上含有与水分和生物胁迫相关的顺式作用元件。利用双酶切的方法将克隆得到的24.1启动子片段替换载体pBI121上的35S启动子,将新构建的植物表达载体转入农杆菌中,并用叶盘法侵染甘菊和菊花叶片,进行瞬时表达研究。试验结果表明,所克隆的24.1的启动子片段具有驱动下游报告基因表达的功能。本研究鉴定了甘菊BADH基因启动子响应干旱、高盐、ABA和低温胁迫诱导的顺式作用元件的种类和分布的位置。为菊花转基因工程中诱导型启动子的应用奠定了基础。更多还原

【Abstract】 Chrysanthemum (Chrysanthemum×morifolium Ramat.) is one of the ten traditional famous flowers in China and the four famous cutting flowers in the world. In recent years, chrysanthemum breeding by genetic transformation has become a hotspot, and some primary achievements have been attained. Studies on transgene expression in chrysanthemum have focused on the CaMV (Cauliflower mosaic virus) 35S promoter, which is a constitutive promoter. It was expression in the various organs and all periods of growth and development in transgenic plants, it would break the metabolic balance and normal growth and development of host plant. Sometimes, it was also cause exogenous gene silence in the transgenic plants. Inducible promoter activity could increase under physical or chemical treatment, and had relatively low expression level at normal conditions, in transgenic plants it caused less ham compared with constitutive promoter. Studies on the use of inducible promoter into chrysanthemum transgenic engineering have important theoretical and practical significance. Betaine aldehyde dehydrogenase (BADH) gene is very important in the anabolism of betaine in many plants, which is induced by drought, salinity, abscisic acid (ABA) and low temperature. In perious work of our group had cloned 4 promoters of BADH gene from Chrysanthemum lavandulifolium, named DBP11, DBP12, DBP21 and DBP22 (GenBank accession number DQ497620-23). The result of transient expression indicated that all the sequences had the function to drive reporter gene. On the base of the previous work, this paper study the expression pattern of the 4 promoters in transgenic plant, and selected the most sensitive promoter to the abiotic stress, to understand the function of cis-acting elements on the promoter. The main results are summarized as follows:1-In this study, we analysis the expression of BADH gene by real-time fluorescence PCR and the content of betaine in leaves from Chrysanthemum lavandulifolium under 400mmol/L NaCl treatment with 0,0.5h,2h,12h, 1d,2d,4d,6d,8d, lOd and 12d, discussed the relationship between BADH gene and betaine. The results showed that, under NaCl treatment both BADH gene expression and the content of betaine had exhibited the tendency that first increased and then decreased. The expression level of BADH gene was down slightly compared with the untreated control at 0.5h and 2h. With the extension of treatment time, the level of BADH gene was continuous increased and reached the maximum at 6d, which were 4.6-higher than untreated control. The activity of BADH gene was decreased gradually after 6d under NaCl treatment. The content of betaine was a sudden increase in NaCl treated with 0.5h, since then the content of betaine fluctuate raise until treatment at 4d reached a maximum. Thereafter the synthesis of betaine was drop drown slowly. The changes between BADH gene and betaine were not at the same time, but had a lag. The BADH gene expression and betaine expression were inhibition by each other. BADH gene expression was significantly increased under 20% PEG-6000 treatment at 0.5h, and then decreased until 1d reached its maximum expression.2.DBP11, DBP12, DBP21 and DBP22 promter were fused with GUSplus reporter gene, to construct of plant expression vectors, the 4 new vectors were then introduced into tobacco. The transgenic plants were treated with NaCl, drought, ABA, SA and low temperature. The results of GUS activities showed that, DBP11, DBP12, DBP21 promoters were responsed to NaCl, drought, ABA and low temperature stresses, and DBP21 promoter was more sensitive than other two promoters. DBP22 promoter was only induced by NaCl and low temperature treatment, and its basic activity was 0.1 times of other three promoters.3. Selected the strongest inducible promoter DBP12, designed different primers according to the cis-acting elements locations on the promoter, using the PCR method to obtain the 5’and 3’end of deletion fragments of DBP12 promoter. New expression vectors were constructed by replacing the CaMV 35S promoter with the 7 deletion fragment sequence respectively to drive the reporter gene GUSplus of the expression vector pCAMBIA1305.2. The new vector was transferred into Agrobacterium to infect leaf disks of Chrysanthemum lavandulifolium. The result of transient expression indicated that the 7 vectors were successfully constructed.4. The 7 vectors were transformed into tobacco, and the transgenic plants were treated with NaCl, drought, ABA and low temperature stresses. The GUS activities illustrated that, there were both inhibitors and enhansers in the sequence of DBP12 promoter. The cis-acting elements, which regulated the BADH gene of Dendranthema lavandulifolium responsed to NaCl, drought, ABA and low temperature stresses were MYB/MYC recognition sites and ACACNNG core. The inhibitors on DBP12 promoter were control the BADH gene responsed to the stress with the cis-acting element at the same time. In addition,5’-UTR had significant function to regulate the BADH gene response to NaCl, drought, ABA and low temperature treatments, and its role of response to low temperature stress was unique.5. Using chromosome walking method isolated 4 DNA fragments in Genome Walker library of Chrysanthemum lavandulifolium, based on 6 EST sequences which were expression under salt stress. The promoter analysis results showed that, the 24.1 (ADP-ribose pyrophosphatase gene) fragment contains cis-acting element that related to water and biotic stress. New expression vector was constructed by replacing the CaMV 35S promoter with the 24.1 fragment sequence to drive the reporter gene GUS of the expression vector pBI121. The new vector was transferred into Agrobacterium to infect leaf disks of Chrysanthemum lavandulifolium and Chrysanthemum. The result of transient expression indicated that the 24.1 sequence had the function to drive reporter gene.This study identified the cis-acting elements that responded to drought, high salt, ABA and low temperature on the promoter sequence of BADH gene from Chrysanthemum lavandulifolium. And the research serves as an important basis for chrysanthemum transgenic engineering.更多还原