国家杰出青年基金(30825030),国家自然科学基金(90717110),国家高技术研究发展计划(2007AA10Z122)和新疆建设兵团博士基金(2008JC06)资助项目
This work was supported by grants from The National Science Fund of China for Distinguished Young Scholars (30825030), The National Natural Science Foundation of China (90717110), Hi-Tech Research and Development Program of China (2007AA10Z122) and Doctor Fund of Xinjiang Construction Corps (2008JC06)
克隆番茄(Solanum lycopersicum) ARF2基因,并分析其分子特性和亚细胞定位,为研究其功能提供基础.通过生物信息学方法分析SlARF2基因编码蛋白的理化性质和分子特性.采用RT-PCR技术从番茄果实cDNA中扩增SlARF2基因全长,并构建与黄色荧光蛋白(YFP)融合的pBA-ARF2-YFP表达载体,进而再通过农杆菌介导的遗传转化方法,将重组质粒转化到野生型番茄中,将得到的T1代转基因种子萌发,然后取根尖通过荧光显微镜观察了融合蛋白在活细胞内分布的特点.生物信息学分析结果表明,SlARF2是富含Ser、Leu、Gly和Pro以及具有ARF家族典型结构域的可溶性蛋白,其氨基酸序列与葡萄、木薯和拟南芥的同源性分别为70.08 %、66.94 %和60.87 %.经酶切和测序分析证实pBA-ARF2-YFP融合表达载体构建成功,此外,PCR分析表明融合蛋白在转基因植株中得到表达.经荧光显微镜观察,ARF2定位在细胞核中.表明转录因子SlARF2定位在细胞核中,对番茄果实发育和成熟起重要作用.
Auxin response factors (ARFs) are important transcription factors involved in auxin signal transduction pathway. In order to elucidate the function of tomato ARF2, we isolated the SlARF2 gene and analyzed its molecular features, in addition, we observed the subcellular localization of ARF2 in transgenic tomato plants. Physicochemical properties and molecular features of ARF2 were predicted by bioinformatic approaches including physical and chemical properties analysis, hydrophobicity analysis, domain analysis, phylogenetic tree analysis and subcellular localization analysis. Moreover, the full-length of SlARF2 gene was amplified by RT-PCR, and a binary vector consisting of ARF2 fused with the yellow fluorescent protein (YFP) coding sequence was further constructed. Using the method of Agrobacterium-mediated transformation, the recombinant vector was transformed into wild-type tomato, and the transgenic tomato plants were confirmed by PCR. The roots of transgenic tomato seedlings were observed under a fluorescence microscope. Bioinformatic analysis showed that the SlARF2 is an unstable soluble protein, which is rich in Ser, Gly, Pro and Leu residues, and it has typical structure and functional domain of ARF family. Tomato ARF2 shares 70.08 %, 66.94 %, and 60.87 % similarity in amino acid sequences with grapes, cassava and Arabidopsis, respectively. Analyses of restriction enzyme digestion and sequencing confirmed the expression vector pBA-ARF2-YFP was successfully constructed. PCR results showed the fusion protein was expressed in transgenic tomato plants. The subcellular localization of SlARF2 was found mostly in the nucleus. SlARF2 has the key characteristics of transcription factor, which is localized in the nucleus, and it might play a pivotal role in tomato fruit development and ripening. This might provide references for studying the function of SlARF2 gene.
冯媛媛,侯佩,李颖楠,刘永胜.番茄ARF2蛋白的生物信息学分析与亚细胞定位[J].生物化学与生物物理进展,2012,39(1):51-58
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