矮牵牛花同源异型基因fbp2的克隆及其对烟草花形态的影响  

Cloning of the Homeotic Gene fbp2 from Petunia hybrida and Its Effects in Tobacco Flower

作　　者：于静娟[1] 国凤利[1] 赵德刚[1] 傅永福[1] 韩玉珍[1] 敖光明[1] 孟繁静[1] 

机构地区：[1]中国农业大学生物学院,北京100094

出　　处：《Acta Botanica Sinica》1999年第1期45-50,共6页Acta Botanica Sinica（植物学报：英文版）

摘　　要：以矮牵牛（Ｐｅｔｕｎｉａ　ｈｙｂｒｉｄａ　Ｌ．）栽培品种为材料，取开放前的花蕾分离ｍＲＮＡ，反转录合成ｃＤＮＡ，以ｃＤＮＡ为模板，通过ＰＣＲ扩增，对获得的目的片段进行序列分析。结果表明，分离的目的片段含有６８６个核苷酸（含有起始密码和终止密码）。核苷酸序列与文献报道相比，同源率为９９．６％，只有３个碱基发生改变，５’端的ＭＡＤＳ盒区域完全相同。将得到的矮牵牛花同源异型基因ｆｂｐ２的ｃＤＮＡ（ｙｆｂｐ２）与ＣａＭＶ３５５启动子和ＮＯＳ３’终止子融合，构建了表达载体ｐＢＢＰ２。表达载体通过农杆菌（Ａｇｒｏｂａｃｔｅｒｉｕｍ　ｔｕｍｅｆａｃｉｅｎｓ）ＬＢＡ４４０４（ｐＡＬ４４０４）介导转化烟草（ＮｉｃｏｔｉａｎａｔａｂａｃｕｍＬ．）叶片，在含有１００ｍｇ／Ｌ卡那霉素的抗性培养基上再生成株。对抗性株进行总ＤＮＡＳｏｕｔｈｅｒｎ杂交和总ＲＮＡ的点杂交，证明目的基因已导入烟草细胞中，整合到烟草基因组上，并且在烟草细胞中转录。同源异型基因ｆｂｐ２导入烟草后导致烟草花型改变，在雄蕊上产生了花瓣。Messenger RNAs were isolated from the petunia (Petunia hybrida L. ) flower buds and cDNAs were synthesized from reverse transcription. The petunia homeotic gene fbp2 was amplified by PCR usingcDNAs as templates. Sequence analysis indicated that the isolated fragment was composed of 686 bp and theregion flanked by two primers showing 99. 6% homology to the sequence reported by Angenent et al. theebases changed, and MADS domain was conserved. Using petunia flower homeotic gene fbp2 cDNA (yfbp2) astarget gene, the expression vector pBBP2 containing CaMV 355 promoter /yfbp2/nos terminator was constructed and was transformed into Agrobacterium tumefacicns strain LBA4404(pAL44404) by direct transformationmethod. Incubating the leaf explants of tobacco (Nicotiana tabacum L. var. samsum) with LBA4404(pal4404) and selecting in the meditun containing 100 mg/L kanamycin, the regenerated resistant plantswere obtained. Polymerase chain reaction and Southern blotting demonstrated that the target gene was integrat- ed into the genome of tobacco. The flowers of the transgenic tobacco were aberrant resulted in expression offbp2, with petal formed on the stamen.

关 键 词：矮牵牛 同源异型基因 CDNA 烟草 表达 花 

分 类 号：S681.610.1[农业科学—观赏园艺] S572.03[农业科学—园艺学]