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香蕉MaERF—1基因的克隆、序列分析及表达载体构建

2017-05-30宋顺金志强徐碧玉黄东梅胡伟李凯许奕

热带作物学报 2017年1期
关键词:生物信息学香蕉

宋顺 金志强 徐碧玉 黄东梅 胡伟 李凯 许奕

摘 要 从香蕉中克隆了1个乙烯响应因子(ERF)MaERF-1。序列分析表明,该基因存在1个完整的开放阅读框(ORF)729 bp,编码243个氨基酸。多序列比对和进化树分析表明,MaERF-1所编码的蛋白与其他植物中ERF编码的蛋白具有较高的一致性。其中与马来西亚野生香蕉同源性最高达98%,与油棕、菠萝、海枣、葡萄、荷花、烟草的MaERF编码的氨基酸序列的同源性分别为65%、60%、59%、54%、53%、51%。MaERF-1編码的蛋白质分子量为26 139.03 u,理论等电点pI为7.81,其亲水性氨基酸均匀分布在整个肽链中,多于疏水性氨基酸。通过PCR和酶切反应鉴定成功构建该基因的表达载体。

关键词 乙烯响应因子;ERF;香蕉;生物信息学;表达载体

中图分类号 S668.1 文献标识码 A

Gene Cloning, Sequence Analysis and Expression Vector

Construction of MaERF-1 from

Musa acuminate L. AAA

SONG Shun1, JIN Zhiqiang1, XU Biyu2, HUANG Dongmei1,

HU Wei2, LI Kai2, XU Yi1 *

1 Haikou Experimental Station, CATAS / Hainan Key Laboratory of Banana Genetic Improvement , Haikou, Hainan 570102, China

2 Institute of Tropical Bioscience and Biotechnology, CATAS / Key Laboratory of Biology and Genetic Resources of Tropical Crops,

Ministry of Agriculture, Haikou, Hainan 571101, China

Abstract We have isolated an ethylene response factor from banana, and designated as MaERF-1. The sequence analysis indicated that the complete ORF of MaERF-1 was 729 bp, and it encoded 243 amino acids. Alignment of amino acid sequences and phylogenetic analysis indicated that the protein encoded by MaERF-1 was in high similarity with ERF-encoding protein in other known plants and highly homologous with amino acid sequences in Musa acuminate by 98%, The similarity with Elaeis guineensis, Ananas comosus, Phoenix dactylifera, Nelumbo nucifera,Nicotiana tabacum was 65%, 60%, 59%, 54%, 53%, 51%, respectively. The protein molecular weight of MaERF-1 was 26 139.03 u, and the theoretical isoelectric point pI was 7.81. The hydrophilic amino acids were uniformly distributed throughout the peptide chain and more than hydrophobic amino acids. The expression vector of the gene was successfully constructed and identified by PCR and restriction enzyme digestion reactions. This experiment could be the basis for further study of MaERF-1 gene expression regulation and functional analysis.

Key words ethylene response factor; ERF; banana; bioinformatics; expression vector

doi 10.3969/j.issn.1000-2561.2017.01.014

植物会经常遭受到许多环境的非生物胁迫变化,如高盐、干旱或者极端的温度。现代农业的模式会导致盐碱地的面积增加。预测到2050年,多余50%的土地均会遭受到盐碱化[1]。干旱的发生在许多地方都很普遍并且会不断扩大[2]。香蕉是世界水果贸易量及消费量最大宗的鲜果,被联合国粮农组织(FAO)定位为发展中国家仅次于水稻、小麦、玉米之后的第四大粮食作物。然而香蕉前期植株较小,根系浅生,易受旱,其叶面积大,蒸腾量大,早在其发生萎蔫之前,就已表现出缺水现状。尤其是在高温季节,香蕉受到干旱后,叶片失水,相对含水量下降,水分胁迫导致香蕉叶片的细胞质膜透性增大,极大地伤害香蕉正常的生理代谢活动,造成香蕉减产,品质下降,严重影响植株的生长发育[3]。所以,提高香蕉抗旱能力对于香蕉生产至关重要。通过研究相关的功能基因来提高香蕉抵御干旱及非生物胁迫的能力是一个重要的途径。

在植物中,转录因子在响应非生物胁迫中起着很重要的作用。ERF是一个转录因子家族,其在植物中能够响应许多非生物胁迫[4-8]。ERF是一个大家族,在水稻、拟南芥和黄豆基因组中分别有157、147和148个成员[9-11]。ERF可以激活或抑制,在其启动子具有GCC盒(AGCC GCC)基因的转录[12]。一些ERF能够结合CRT/DRE(A/GCCGAC)结构从而调控基因响应生物或者非生物胁迫[13-16]。ERF参与许多生物过程,包括一些代谢过程,花和种子的发育,根系的发育[17-20]。例如,小麦TaERF1通过结合GCC盒和CRT/DRE元件,能够激活一些与非生物胁迫相关的基因,如低温胁迫、干旱胁迫和高盐胁迫[21]。Mishra从罂粟中分离出了PsAP2基因,其能够与DRE和GCC元件结合,将该基因转化烟草,转基因植株能够增强对胁迫以及非生物胁迫的耐受力[22]。Gao等[23]从番茄中克隆了TERF1基因,并将其转化水稻。在转基因水稻植株中,其脯氨酸增加,减少了水分的丢失,提高对高盐的干旱的耐受性。同时,TERF1诱导了LIP5、 Wcor4131、OsPrx和OsABA2的表达。但是,也有一些ERF基因会降低对胁迫的耐受性,如在拟南芥中过表达AtERF4降低了对ABA的敏感度和对Nacl的耐受力[24-25]。在OsERF922转基因植株中显示了,该植株减低了对高盐胁迫的耐受性,其Na+/K+比高于野生型[26]。在StERF3的转基因烟草中,其减低了对高盐胁迫的耐受性,然而,在StERF3 RNAi抑制的植株中能够提高对高盐胁迫的耐受性,同时在该植株中,防御相关基因PR1、NPR1和 WRKY1也被诱导表达[27]。在土豆中沉默ERF3的表达能够增强其对晚疫病的抵御能力[28]。同样,在水稻中用RNAi沉默ERF922的表达能够增强其对青枯病的抵御能力[29]。

本研究通过克隆香蕉水通道蛋白基因MaERF-1,利用生物信息学对该基因的功能进行进一步的分析,推测其生物学功能,同时成功构建了该基因的植物表达载体,为进一步研究ERF基因的表达调控以及功能分析奠定了基础。

1 材料与方法

1.1 材料

1.1.1 植物材料 2016年1月初采集巴西香蕉(Musa acuminata L. AAA group‘Brazilian)的根、茎、叶、花、果,用无菌水清洗后立即放置于液氮中速冻,于 -70 ℃冰箱中保存备用。实验所用香蕉均采自中国热带农业科学院热带生物技术研究所。

1.1.2 载体与菌株 克隆载体pMD18-T载体购自TaKaRa公司产品,大肠杆菌感受态DH5α购自天根公司。植物表达载体Pcambia 1304为实验室保存。

1.1.3 酶与化学试剂 DNA Marker、限制性内切酶、LA Taq酶、LA PCR in vitro Cloning Kit购自TaKaRa公司。RevertAidTM First Strand cDNA Synthesis Kit购自Fermentas公司。质粒提取试剂盒、DNA回收试剂盒和RNA提取试剂盒购自Omega Bio-Tek公司,其他化学药品为分析纯。

1.2 方法

1.2.1 总RNA提取及cDNA获得 取巴西蕉根、茎、叶、花、果组织,采用改良的CTAB法 / 植物RNA试剂盒(Omega)结合法提取和纯化总RNA。以RevertAidTM First Strand cDNA Synthesis Kit 将RNA反转录成cDNA并置于-80 ℃冰箱中保存备用。

1.2.2 目的基因的获得 从香蕉A基因组测序数据库中得到一个ERF家族基因MaERF-1,根据其序列设计一对引物F1,R1(F1:5′-CATGCCATGG

CGATGGATTTTGAGGATTCCTCCTCCA-3′,R1:5′-GGACTAGTTCGGGTGGACGAACACCACATGG-3′,斜体碱基为酶切位点序列),扩增MaERF-1全长序列。PCR扩增程序为:95 ℃预变性3 min;94 ℃变性40 s,55 ℃退火40 s,72 ℃延伸50 s,共 35个循环。按照分子克隆實验指南进行PCR 扩增产物回收、连接、转化和鉴定。对已鉴定的阳性克隆进行测序分析。

1.2.3 生物信息学分析 将基因MaERF-1的cDNA序列和开放阅读框(open reading frame,ORF)编码的氨基酸序列在NCBI数据库中的BLASTx进行同源性搜索和比对;利用Clustal X1.81和MEGA 3.1 软件分析MaERF-1蛋白与其他植物的ERF蛋白的进化关系,构建分子进化树;利用expasy数 据 库(http://web.expasy.org/compute_pi/)预测蛋白质pI和分子质量;用Protscale(http://www.expasy.ch/tools/protscale.html/)工具对MaERF-1编码的蛋白质产物疏水性和亲水性进行分析。

1.2.4 MaERF-1表达载体的构建 选用Pcambia 1304植物表达载体构建MaERF-1表达基因融合载体,以NcoI和SpeI酶切pMD18-T/MaERF-1重组质粒和Pcambia 1304空载体,分别回收启动子片段和线性载体,连接,转化DH5α后进行菌液PCR及酶切检测,重组质粒命名为MaERF-1::1304。

2 结果与分析

2.1 MaERF-1基因的克隆与鉴定

以巴西蕉各器官组织混合cDNA为模版,F1和R1为引物,从香蕉栽培品种巴西蕉中克隆到一个ERF家族基因MaERF-1,其ORF为729 bp,PCR结果如图1所示。

2.2 生物信息学分析

2.2.1 MaERF-1基因蛋白质序列差异的比对 利用DNAman将MaERF-1 cDNA推导的氨基酸序列与NCBI中已登录的其他高等植物的ERF氨基酸序列进行同源关系的比较,结果(图2)显示,各种植物ERF编码的氨基酸序列存在较高的同源性,多数都达到55%以上。BLASTX分析表明,MaERF-1编码的氨基酸序列与马来西亚野生蕉MaERF-1-like(XP_009379780.1)、菠萝AcERF-2(OAY63143.160)、海枣PcERF-2(XP_008807522.1)、荷花NnERF-2(XP_010243826.1)、葡萄VvERF-2(XP_002267961.1)、烟草NtERF-2(XP_016448076.1)、油棕EgERF-2(XP_010940400.1)编码的氨基酸序列具有较高的一致性,分别为98%、60%、59%、53%、54%、51%、65%。

2.2.2 MaERF-1基因进化树分析 利用Clustal X1.81和MEGA 3.1软件,将MaERF-1 cDNA推导的氨基酸序列与NCBI中已登录的其他植物的SIP氨基酸序列进行系统进化树的比对分析。结果表明本研究得到的巴西蕉MaERF-1基因所编码的氨基酸序列与马来西亚野生蕉MaERF-1-like具有较近的亲缘关系(图3)。

2.2.3 MaERF-1编码蛋白质理化性质分析 利用Expasy分析MaERF-1编码蛋白质的理化性质,如图4显示,该蛋白具有243个氨基酸,分子量为26 139.03 u,理论等电点pI为7.81。

2.2.4 MaERF-1编码蛋白质产物的亲水性、疏水性分析 对MaERF-1编码蛋白质产物亲水性、疏水性分析结果表明(图5),MaERF-1多肽链中精氨酸Arg具有最低的分值-2.800,异亮氨酸Ile具有最高分值1.600。依据氨基酸分值越低亲水性越强和分值越高疏水性越强的规律,可见,精氨酸Arg亲水性最强,而异亮氨酸Ile疏水性最强,而就整体来看,亲水性氨基酸均匀分布在整个肽链中,多于疏水性氨基酸。

2.3 MaERF-1表达载体的构建

在上游引物F1和下游引物R1分别加入NcoI和SpeI内切酶,将pMD18-T/MaERF-1和Pcambia 1304载体同时用NcoI和SpeI内切酶酶切后,回收目的片段后连接转化,PCR以及双酶切鉴定如图6,结果显示,PCR产物和酶切条带均为729 bp。

3 讨论

香蕉是一年生草本植物,喜高温多湿,缺水时叶片变薄、果指变短,生长延缓,导致减产,因此对香蕉抗逆品种的选育是十分必要的。近年来,通过挖掘香蕉本身所具有的抗逆基因资源,研究该基因的功能来进一步提高香蕉的抗逆性就成为一个重要的手段。

本实验克隆到1个香蕉乙烯响应因子MaERF-1,通过在NCBI上BLAST分析各高等植物中ERF氨基酸序列的同源关系,结果显示MaERF-1编码的氨基酸与各种植物中ERF编码的氨基酸序列存在较高的同源性,达到55%以上,其中与马来西亚野生蕉同源性最高为98%。对各种植物ERF氨基酸序列比对和系统进化树分析发现,不同植物的ERF序列相似性较高,以ERF氨基酸序列构建的进化树能准确反映不同植物间的亲缘关系,也可作为评价香蕉等植物种植资源的重要依据。通过对ERF编码蛋白质的亲水性和疏水性进行分析,表明其亲水性氨基酸多于疏水性氨基酸。将MaERF-1成功构建到植物表达载体中,后续实验可以通过将表达载体转化到如拟南芥、烟草等模式植物中,对该基因进行进一步的功能验证和分析。

当植物遭受到外界的非生物胁迫时,植物自身有复杂的系统去保护其去抵御不同的胁迫。ERF在植物中是属于(AP2/ERF)转录因子家族中的亚族,对ERF的功能研究主要集中在对其在抗逆性方面的研究[30-32],如抗旱、抗盐、抗寒等。例如,水稻ERF基因OsBIERF1、OsBIERF3 和OsBIERF4不仅能被稻瘟病菌诱导表达,还能受到高盐、冷害、干旱、伤害胁迫而上调表达[33]。在烟草中过表达ERF11能够提高植物抵御青枯病的能力。在拟南芥中,AtERF71能够显著被高盐、甘露醇和ABA诱导表达,在高盐胁迫下,植株中的活性氧(ROS)累积显著减少[34]。转化GmERF7的烟草能够提高对高盐胁迫的耐受性,该转基因植株中叶绿素和糖含量显著增加[35]。因此,本实验通过对该基因的功能进行生物信息学分析,并且將其连接到植物表达载体中,该实验结果为进一步研究ERF基因的表达调控以及后续转基因分析基因功能奠定了基础。

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