甘菊BADH基因启动子的克隆与瞬时表达分析  

Cloning and Transient Expression Assay of BADH Gene Promoters from Dendronthema lavandulifolium

作　　者：刘振林[1,2] 曹华雯[1] 夏新莉[3] 尹伟伦[3] 戴思兰[1,4] 

机构地区：[1]北京林业大学园林学院,北京100083 [2]河北科技师范学院园艺园林系,河北昌黎066600 [3]北京林业大学生物科学与技术学院,北京100083 [4]国家花卉工程技术研究中心,北京100083

出　　处：《园艺学报》2008年第12期1787-1794,共8页Acta Horticulturae Sinica

基　　金：国家自然科学基金项目(30471419);北京市自然科学基金项目(502208);国家‘863’计划项目(2006AA100109;2007AA021403)

摘　　要：为了给菊花[Dendronthema×grandiflora (Ramat.) Kitam.]的转基因育种提供诱导型启动子,借鉴5′RACE策略,利用锚定PCR步移的方法克隆了甘菊[Dendranthema lavandulifolium(Fisch.ex Trautv.)Makino]甜菜碱醛脱氢酶(betaine aldehyde dehydrogenase,BADH)基因的4个启动子序列,分别命名为DBP11、DBP12、DBP21和DBP22(GenBank登录号:DQ497620～DQ497623),序列长度分别为1230、1249、1273和574bp。4个启动子序列对应区域的同源性在89%以上。其中DBP12和DBP21分別是甘菊BADH基因DlBADH1和DlBADH2(GenBank登录号:DQ011151和DQ011152)的启动子序列,DBP11和DBP22为甘菊BADH基因家族中其它成员的启动子序列。序列分析表明,上述启动子序列中均含有多处与水分胁迫和脱落酸诱导相关的顺式作用元件。在表达载体pCAMBIA1305.2的基础上,用所克隆的4个甘菊BADH基因启动子序列分别置换驱动其报告基因表达的35S启动子,建立了新的植物表达载体。用其转化农杆菌,并用叶盘法侵染甘菊,瞬时表达的结果表明这些启动子序列均具备驱动报告基因表达的功能。In order to provide inducible promoter for chrysanthemum [ Dendronthema × grandiflora （Ramat. ） Kitam. ] transgenic breeding, referring to the strategy of 5′RACE, four promoter sequences of betaine aldehyde dehydrogenase （BADH） gene from Dendranthema lavandulifolium （ Fisch. ex Trautv. ） Makino were cloned by anchored PCR walking, which were named DBPll, DBP12, DBP21 and DBP22 （GenBank accession No. DQ497620 -DQ497623）. The four sequences are 1 230 bp, 1 249 bp, 1 273 bp and 574 bp respectively. The homology of the corresponding regions between every two sequences is above 89%. DBP12 and DBP21 are the promoters of DIBADH1 and DIBADH2 （GenBank accession No. DQOl1151 and DQ011152） , DBP11 and DBP22 are the promoters of other members in BADH gene family from Dendranthema lavandulifolium. Many cis-acting elements related to water stress and ABA inducement were found in all the sequences. New expression vectors were constructed by replacing the 35S-CaMV promoter with the above promoter sequences to drive the reporter gene GUSplus of the expression vector nCAMBIA1305 .2 The mew vectors were transferred into Agrobacterium to infect leaf disks of Dendranthema lavandulifolium. The result of transient expression indicated that all the sequences had the function to drive reporter gene.

关 键 词：甘菊 甜菜碱醛脱氢酶基因 启动子 克隆 瞬时表达 

分 类 号：S682.11[农业科学—观赏园艺]