枳SPL9和SPL13全长cDNA克隆、亚细胞定位和表达分析  

Cloning,Subcellular Localization and Expression Analysis of SPL9 and SPL13 Genes from Poncirus trifoliata

作　　者：宋长年[1] 钱剑林[2] 房经贵[1] 王化坤[2] 邱学林[2] 章镇[1] 张晓莹[1] 

机构地区：[1]南京农业大学园艺学院,南京210095 [2]江苏省太湖常绿果树技术推广中心,江苏苏州215107

出　　处：《中国农业科学》2010年第10期2105-2114,共10页Scientia Agricultura Sinica

基　　金：教育部科学技术研究重点项目(109084)

摘　　要：【目的】从枳[Poncirus trifoliata(L.)Raf.]中克隆SBP类[SQUAMOSA(SQUA)promoter-binding-like]转录因子基因SPL9和SPL13全长,构建SPL9和SPL13亚细胞定位表达载体验证其是否具有核定位功能,利用荧光定量PCR研究其在枳不同组织的表达特性,初步确定SPL9和SPL13在枳生长发育过程中的作用。【方法】利用生物信息学结合RACE技术以枳花器官的cDNA为模板,克隆出SPL9和SPL13基因全长,分别命名Pt-SPL9和Pt-SPL13,大小分别是1519bp和1824bp,在GenBank的登录号分别是FJ502237和FJ502238;构建Pt-SPL9和Pt-SPL13亚细胞定位载体35S-GW-FJ502237/FJ502238-GFP,基因枪转化洋葱表皮细胞,暗培养24h后激光共聚焦显微镜下观察;利用SYBR Green I实时定量RT-PCR方法检测Pt-SPL9和Pt-SPL13在根、茎、叶、花序、花和果等不同组织中的表达。【结果】生物信息学分析表明,Pt-SPL9和Pt-SPL13的cDNA序列中都有microRNA156的识别位点,Pt-SPL9与金鱼草、拟南芥和玉米SPL9的同源性分别为48.9%、42.5%和41.7%;Pt-SPL13与拟南芥SPL13、水稻的SPL16和玉米的TGA1同源性分别为40.8%、38.1%和35.8%。Pt-SPL9和Pt-SPL13与其它植物的SBP一样有着高度保守的序列,即SBP结构域和一个双向核定位信号KRXXXRRRK。亚细胞定位结果表明,Pt-SPL9和Pt-SPL13均定位于细胞核中。SYBR Green I实时定量RT-PCR结果表明,Pt-SPL9和Pt-SPL13在各个器官均有表达,但表达量不同,Pt-SPL9在茎中的表达量最高,在花和叶中的表达量次之,在根、花芽和幼果中的表达量最低;Pt-SPL13在幼果中的表达量最高,在茎和花芽中的表达量相当,其次为叶,在花和根中的表达量很低。【结论】转录因子Pt-SPL9和Pt-SPL13均具有核定位功能,Pt-SPL9和Pt-SPL13对枳的茎和果实的发育可能有着重要作用。【Objective】 This study aimed to clone two full length cDNA of SPL9 and SPL13 SBPs （SQUAMOSA promoter binding proteins） transcription factors from Poncirus trifoliata （L.） Raf. and construct expression vectors of SPL9 and SPL13 for subcellular location analysis. Real time PCR was used to determine the tissue expression patterns of SPL9 and SPL13 for analysis of the role of SPL9 and SPL13 during growth and development in adult P. trifoliata. 【Method】 Bioinformatics analysis and RACE technology showed that the complete cDNAs cloned,designated as Pt-spl9 and Pt-spl13,was 1 519 bp and 1 824 bp in length,respectively. The sequences were deposited in GenBank database with accession no. of FJ502237 and FJ502238. Recombinant plasmid 35S-GW-FJ502237/FJ502238-GFP was introduced into onion epidermal cells by the particle bombardment method with a PDS1000/He. Transformed cells were incubated for 24 h at 22℃ in the dark and green fluorescence was monitored under a laser scanning confocal microscope. The SYBR Green I Real-time qRT-PCR was employed to analyze the expression of Pt-SPL9 and Pt-SPL13 in young leaf,stem,root,bud flower,flower,fruit and other organs. 【Result】 Bioinformatics analysis showed that the cDNA of Pt-SPL9 and Pt-SPL13 had the recognition sites of microRNA156. The deduced amino acid sequence of Pt-SPL9 and Pt-SPL13 were 388 and 379 residues,which were 48.9 %,42.5 %,41.7 % identical,respectively,with SPL9 of Antirrhinum majus,Arabidopsis thaliana and Zea mays; and 40.8%,38.1%,35.8% identical respectively with SPL13 of Arabidopsis thaliana,SPL16 of Oryza sativa,and TGA1 of Zea mays,respectively. Pt-SPL9 and Pt-SPL13 and other plant SBPs have the same amino acid sequences that are highly conserved as the designed SBP domain and two-way nuclear localization signal. Subcellular localization results showed that the Pt-SPL9 and Pt-SPL13 were localized in the nucleus. SYBR Green I real-time quantitative RT-PCR results showed that the Pt-SPL9 and Pt-SPL13 were expressed ubiquitously in various or

关 键 词：枳 SPL9和SPL13 亚细胞定位 荧光定量RT-PCR 

分 类 号：S666[农业科学—果树学]