小麦TaPRF7基因的克隆及表达  

Cloning and Expression of TaPRF7 Gene in Wheat (Triticum aestivum)

作　　者：段文静[1,2] 白建芳[2] 王鹏[2,3] 王玉昆[2] 苑少华[2] 孙辉[2] 苑国良[2] 张立平[2,3] 赵昌平[2,3] 

机构地区：[1]首都师范大学生命科学学院,北京100048 [2]北京市农林科学院杂交小麦工程技术研究中心/杂交小麦分子遗传北京市重点实验室,北京100097 [3]北京农学院植物科学技术学院,北京102206

出　　处：《农业生物技术学报》2017年第7期1033-1044,共12页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金项目(No.31371699);国家科技支撑计划项目(No.2013BAD04B01和No.2014BAD01B09);北京市自然科学基金项目(No.5131001);北京市科技计划项目(No.Z141100002314018)

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�Profilin （PRF） is a low molecular weight actin binding protein which is linked to the signal transduction cascade via the phosphoinositide pathways in eukaryotes, regulating both actin polymerization and depolymerization. To date, PRF has been identified in many higher plants, such as Arabidopsis thaliana, Zea mays, Akebia trifoliata, Brassica campestris. In this study, a cDNA clone（designated TaPRF7 （GenBank No. KY940299） in accordance with the accession sequence of wheat （Triticum aestivum） PRF in GenBank） encoding PRF gene was isolated from stamen of wheat photoperiod-thermo sensitive genic male sterile （PTGMS） line BS366 using RT-PCR technique. The cDNA sequence contains a 396 bp ORF, coding for 131 amino acids, with an estimated molecular mass of 14.2 kD and a pI of 4.81. Homology comparison revealed that the protein contains conservative PROF domain and actin, PIP2 and poly-L-proline （PLP） binding sites. The amino acid sequence among TaPRF7 and other different species （T. aestivum, Z. mays, Oryza sativa, Glycine max, Hordeum vulgate, Sorghum bicolor, A. thaliana, Nicotiana tabacum, Solarmum tuberosum） compared by Blast, revealing that there was high homology （70%-97%） with profiling protein of other plants and the similarity to TaPRFl（ACE82291.1） protein was the highest with 97%. Phylogenetic analysis showed that TaPRF7 has close relationship with ZmPRF2, ZmPRF4, ZmPRF5, HvPRF1, SbPRF and OsPRF. Subcellular localization analysis indicated that TaPRF7 was targeted to the nucleus and cytoplasm by detecting the fluorescent signals of eukaryotic expression vector in Arabidopsis protoplasts. Expressions of TaPRF7 had been observed in various tissues （root, stem, leaf, stamen, pistal and glume） of wheat, but was highest in stamen, followed by root, suggesting that TaPRF7 was reproductive expression and mainly expressed in reproductive organs. To investigate the involvement of TaPRF7 in signal transduction, TaPRF7 gene expression of wheat seedling in the abiotic stresses was

关 键 词：小麦 小麦前纤维蛋白7(TaPRF7) 非生物胁迫 亚细胞定位 雄性不育 表达分析 

分 类 号：S512.1[农业科学—作物学]