小麦TaPRF7基因的克隆及表达

发布时间：2018-10-17 19:21

【摘要】：前纤维蛋白(profilin,PRF)是一种植物中广泛存在的微丝结合蛋白,对微丝动态重排进行双向调控,在植物的各项生命活动中起非常重要的作用。为研究其在小麦(Triticum aestivum)中的功能,本研究以小麦光温敏不育系BS366雄蕊的cDNA为模板,通过电子克隆和RT-PCR方法克隆获得1个PRF类基因,并根据Gen Bank登录顺序命名为TaPRF7(Gen Bank No.KY940299)。利用生物信息学软件对TaPRF7及其蛋白进行分析,结果表明,TaPRF7编码区含有396个核苷酸,编码131个氨基酸;其蛋白分子量约为14.2 KD,p I约为4.81,属于稳定蛋白;含有保守域PROF,有肌动蛋白(actin)、磷脂酰肌醇4,5-二磷酸(phosphatidyl inositol 4,5-diphosphate,PIP2)、多聚脯氨酸结合位点。TaPRF7基因与玉米(Zea mays)ZmPRF2、ZmPRF4、ZmPRF5,大麦(Hordeum vulgare)Hv PRF1,高粱(Sorghum bicolor)Sb PRF,水稻(Oryza sativa)Os PRF亲缘关系较近,同源性也比较高。构建TaPRF7-16318h GFP融合蛋白表达载体,观察其在拟南芥(Arabidopsis thaliana)原生质体亚细胞定位,显示定位于细胞核和细胞质中;运用qRT-PCR分析小麦BS366不同组织及脱落酸(abscisic acid,ABA)、生长素(indoleaceticacid,IAA)、茉莉酸甲酯(methyl jasmonate,MeJA)、盐(NaCl)、模拟干旱(PEG 6000)、水杨酸(salicylic acid,SA)、赤霉素(gibberellin,GA)、低温(10℃)处理下TaPRF7表达特性。分析表明,TaPRF7在雄蕊中表达量最高,属于生殖型表达。在ABA、PEG、GA、NaCl、IAA和低温6种处理下,TaPRF7表达量都呈现出先升后降的趋势,而在Me JA和SA处理下,TaPRF7的表达被显著抑制。利用qRT-PCR分析该基因在不育系BS366和恢复系京411不育和可育环境下花药发育3个时期的表达情况,结果表明,相比BS366,该基因在京411中3个时期表达量都很低,且在BS366不育环境下表达量明显高于可育环境,随着花药发育时期的推进,在不育环境下该基因表达量呈上升趋势。综上结果推测TaPRF7可能参与了花药开裂和低温诱导不育等小麦分子信号转导通路,为进一步研究TaPRF7基因在光温敏小麦不育的分子机制提供了一定的理论依据。
[Abstract]:Prefibrin (profilin,PRF) is a widely existed microfilament binding protein in plants. It regulates the dynamic rearrangement of microfilaments in both directions and plays an important role in the life activities of plants. In order to study its function in wheat (Triticum aestivum), a PRF gene was obtained by electronic cloning and RT-PCR cloning using cDNA of wheat photo-thermosensitive sterile line BS366 stamen as template, and named TaPRF7 (Gen Bank No.KY940299 according to Gen Bank login sequence. TaPRF7 and its protein were analyzed by bioinformatics software. The results showed that the coding region of TaPRF7 contained 396 nucleotides encoding 131 amino acids, and its molecular weight was about 14.2 KD,p I about 4.81, which was a stable protein. The conserved domain PROF, contained actin (actin), phosphatidylinositol (actin), 5-diphosphatePIP2), and the polyproline binding site. The TaPRF7 gene was closely related to (Zea mays) ZmPRF2,ZmPRF4,ZmPRF5, barley (Hordeum vulgare) Hv PRF1, sorghum (Sorghum bicolor) Sb PRF, rice (Oryza sativa) Os PRF and had high homology. The expression vector of TaPRF7-16318h GFP fusion protein was constructed, and its localization in (Arabidopsis thaliana) protoplast subcellular of Arabidopsis thaliana was observed, which showed that it was located in the nucleus and cytoplasm of Arabidopsis thaliana. QRT-PCR was used to analyze the TaPRF7 expression characteristics of wheat BS366 treated with abscisic acid (abscisic acid,ABA), auxin (indoleaceticacid,IAA), methyl jasmonate (methyl jasmonate,MeJA), salt (NaCl), simulated drought (PEG 6000), salicylic acid (salicylic acid,SA), gibberellin (gibberellin,GA) and low temperature (10 鈩，

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