摘要
根据毛果杨AP3同源基因(PTD)设计一对PCR引物,以毛白杨基因组DNA为模板,通过PCR技术分离克隆了毛白杨AP3同源基因PtAP3.分析表明该基因全长1 813 bp(包括引入的酶切位点),包括7个外显子和6个内含子,编码238个氨基酸.在GenBank中进行blast检索,发现其氨基酸序列与大丁草、矮牵牛、百合、玫瑰、苹果、毛果杨AP3同源基因的氨基酸序列具有52%～82%的同源性.PtAP3蛋白具有非常保守的MADS-box序列,推测其为转录因子.Southern杂交分析发现,该基因在毛白杨雄株中为双拷贝,而在雌株中则为单拷贝.为进行功能分析,构建了正反义表达载体,通过农杆菌介导转化获得了一些转基因烟草植株.
A pair of primers were designed according to published Populus trichocarpa gene （ PTD）, and PtAP3 , an AP3 homologous gene from P. tomentosa was isolated by PCR using genomic DNA of male clone of Populus tomentosa （L50） as template. The result indicated that the sequence was 1 813 bp （ BamH I and Sac I were introduced at the 5＇ and 3＇ end） including 7 exons and 6 introns, coding 238 amino acids. It was found to have 52% - 82% homology to proteins from Lilium regale （AF503913）, Petunia hybrida （ AF230704 ） , Gerbera hybrida （AJ009724）, Rosa rugosa （AB055966）, Malus domestica （AJ251116） and P. trichocarpa （AF057708） by blast analysis in GenBank. There was a highly conserved MADS-box motif in the protein of PtAP3 , so it was putatived to be a transcription factor. The result of Seuthem blot analysis indicated that there were double copies of PtAP3 or two members which had a high homology to each other in P. tomentosa （L50, male） genomic DNA, and there was single copy PtAP3 in P. tomentosa （5082, female） genomic DNA. Sense and anti-sense expression vectors of PtAP3 were constructed by PCR and restriction enzymes digestion identification, and transformed into tobacco （Nicotiana tabacum） by Agrobacterium GV3101 and LBA4404. Some transgenic tobacco plantlets were obtained by PCR identification. The results above mentioned have provided important data to understand the molecular mechanism of male flower development of P. tomentosa, and also contributed to study on controlling flowering of P. tomentosa by gene engineering.
出处
《林业科学》
EI
CAS
CSCD
北大核心
2005年第6期35-42,共8页
Scientia Silvae Sinicae
基金
国家自然科学基金(30371175)
中国博士后基金(2002032041)资助。
关键词
毛白杨
AP3同源基因
克隆
转化
Populus tomentosa
APETALA3 homologous gene
cloning
transformation
