宋順 金志強(qiáng) 徐碧玉 黃東梅 胡偉 李凱 許奕
摘 要 從香蕉中克隆了1個(gè)乙烯響應(yīng)因子(ERF)MaERF-1。序列分析表明,該基因存在1個(gè)完整的開放閱讀框(ORF)729 bp,編碼243個(gè)氨基酸。多序列比對和進(jìn)化樹分析表明,MaERF-1所編碼的蛋白與其他植物中ERF編碼的蛋白具有較高的一致性。其中與馬來西亞野生香蕉同源性最高達(dá)98%,與油棕、菠蘿、海棗、葡萄、荷花、煙草的MaERF編碼的氨基酸序列的同源性分別為65%、60%、59%、54%、53%、51%。MaERF-1編碼的蛋白質(zhì)分子量為26 139.03 u,理論等電點(diǎn)pI為7.81,其親水性氨基酸均勻分布在整個(gè)肽鏈中,多于疏水性氨基酸。通過PCR和酶切反應(yīng)鑒定成功構(gòu)建該基因的表達(dá)載體。
關(guān)鍵詞 乙烯響應(yīng)因子;ERF;香蕉;生物信息學(xué);表達(dá)載體
中圖分類號 S668.1 文獻(xiàn)標(biāo)識碼 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
植物會(huì)經(jīng)常遭受到許多環(huán)境的非生物脅迫變化,如高鹽、干旱或者極端的溫度?,F(xiàn)代農(nóng)業(yè)的模式會(huì)導(dǎo)致鹽堿地的面積增加。預(yù)測到2050年,多余50%的土地均會(huì)遭受到鹽堿化[1]。干旱的發(fā)生在許多地方都很普遍并且會(huì)不斷擴(kuò)大[2]。香蕉是世界水果貿(mào)易量及消費(fèi)量最大宗的鮮果,被聯(lián)合國糧農(nóng)組織(FAO)定位為發(fā)展中國家僅次于水稻、小麥、玉米之后的第四大糧食作物。然而香蕉前期植株較小,根系淺生,易受旱,其葉面積大,蒸騰量大,早在其發(fā)生萎蔫之前,就已表現(xiàn)出缺水現(xiàn)狀。尤其是在高溫季節(jié),香蕉受到干旱后,葉片失水,相對含水量下降,水分脅迫導(dǎo)致香蕉葉片的細(xì)胞質(zhì)膜透性增大,極大地傷害香蕉正常的生理代謝活動(dòng),造成香蕉減產(chǎn),品質(zhì)下降,嚴(yán)重影響植株的生長發(fā)育[3]。所以,提高香蕉抗旱能力對于香蕉生產(chǎn)至關(guān)重要。通過研究相關(guān)的功能基因來提高香蕉抵御干旱及非生物脅迫的能力是一個(gè)重要的途徑。
在植物中,轉(zhuǎn)錄因子在響應(yīng)非生物脅迫中起著很重要的作用。ERF是一個(gè)轉(zhuǎn)錄因子家族,其在植物中能夠響應(yīng)許多非生物脅迫[4-8]。ERF是一個(gè)大家族,在水稻、擬南芥和黃豆基因組中分別有157、147和148個(gè)成員[9-11]。ERF可以激活或抑制,在其啟動(dòng)子具有GCC盒(AGCC GCC)基因的轉(zhuǎn)錄[12]。一些ERF能夠結(jié)合CRT/DRE(A/GCCGAC)結(jié)構(gòu)從而調(diào)控基因響應(yīng)生物或者非生物脅迫[13-16]。ERF參與許多生物過程,包括一些代謝過程,花和種子的發(fā)育,根系的發(fā)育[17-20]。例如,小麥TaERF1通過結(jié)合GCC盒和CRT/DRE元件,能夠激活一些與非生物脅迫相關(guān)的基因,如低溫脅迫、干旱脅迫和高鹽脅迫[21]。Mishra從罌粟中分離出了PsAP2基因,其能夠與DRE和GCC元件結(jié)合,將該基因轉(zhuǎn)化煙草,轉(zhuǎn)基因植株能夠增強(qiáng)對脅迫以及非生物脅迫的耐受力[22]。Gao等[23]從番茄中克隆了TERF1基因,并將其轉(zhuǎn)化水稻。在轉(zhuǎn)基因水稻植株中,其脯氨酸增加,減少了水分的丟失,提高對高鹽的干旱的耐受性。同時(shí),TERF1誘導(dǎo)了LIP5、 Wcor4131、OsPrx和OsABA2的表達(dá)。但是,也有一些ERF基因會(huì)降低對脅迫的耐受性,如在擬南芥中過表達(dá)AtERF4降低了對ABA的敏感度和對Nacl的耐受力[24-25]。在OsERF922轉(zhuǎn)基因植株中顯示了,該植株減低了對高鹽脅迫的耐受性,其Na+/K+比高于野生型[26]。在StERF3的轉(zhuǎn)基因煙草中,其減低了對高鹽脅迫的耐受性,然而,在StERF3 RNAi抑制的植株中能夠提高對高鹽脅迫的耐受性,同時(shí)在該植株中,防御相關(guān)基因PR1、NPR1和 WRKY1也被誘導(dǎo)表達(dá)[27]。在土豆中沉默ERF3的表達(dá)能夠增強(qiáng)其對晚疫病的抵御能力[28]。同樣,在水稻中用RNAi沉默ERF922的表達(dá)能夠增強(qiáng)其對青枯病的抵御能力[29]。
本研究通過克隆香蕉水通道蛋白基因MaERF-1,利用生物信息學(xué)對該基因的功能進(jìn)行進(jìn)一步的分析,推測其生物學(xué)功能,同時(shí)成功構(gòu)建了該基因的植物表達(dá)載體,為進(jìn)一步研究ERF基因的表達(dá)調(diào)控以及功能分析奠定了基礎(chǔ)。
1 材料與方法
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、莖、葉、花、果,用無菌水清洗后立即放置于液氮中速凍,于 -70 ℃冰箱中保存?zhèn)溆谩?shí)驗(yàn)所用香蕉均采自中國熱帶農(nóng)業(yè)科學(xué)院熱帶生物技術(shù)研究所。
1.1.2 載體與菌株 克隆載體pMD18-T載體購自TaKaRa公司產(chǎn)品,大腸桿菌感受態(tài)DH5α購自天根公司。植物表達(dá)載體Pcambia 1304為實(shí)驗(yàn)室保存。
1.1.3 酶與化學(xué)試劑 DNA Marker、限制性內(nèi)切酶、LA Taq酶、LA PCR in vitro Cloning Kit購自TaKaRa公司。RevertAidTM First Strand cDNA Synthesis Kit購自Fermentas公司。質(zhì)粒提取試劑盒、DNA回收試劑盒和RNA提取試劑盒購自O(shè)mega Bio-Tek公司,其他化學(xué)藥品為分析純。
1.2 方法
1.2.1 總RNA提取及cDNA獲得 取巴西蕉根、莖、葉、花、果組織,采用改良的CTAB法 / 植物RNA試劑盒(Omega)結(jié)合法提取和純化總RNA。以RevertAidTM First Strand cDNA Synthesis Kit 將RNA反轉(zhuǎn)錄成cDNA并置于-80 ℃冰箱中保存?zhèn)溆谩?/p>
1.2.2 目的基因的獲得 從香蕉A基因組測序數(shù)據(jù)庫中得到一個(gè)ERF家族基因MaERF-1,根據(jù)其序列設(shè)計(jì)一對引物F1,R1(F1:5′-CATGCCATGG
CGATGGATTTTGAGGATTCCTCCTCCA-3′,R1:5′-GGACTAGTTCGGGTGGACGAACACCACATGG-3′,斜體堿基為酶切位點(diǎn)序列),擴(kuò)增MaERF-1全長序列。PCR擴(kuò)增程序?yàn)椋?5 ℃預(yù)變性3 min;94 ℃變性40 s,55 ℃退火40 s,72 ℃延伸50 s,共 35個(gè)循環(huán)。按照分子克隆實(shí)驗(yàn)指南進(jìn)行PCR 擴(kuò)增產(chǎn)物回收、連接、轉(zhuǎn)化和鑒定。對已鑒定的陽性克隆進(jìn)行測序分析。
1.2.3 生物信息學(xué)分析 將基因MaERF-1的cDNA序列和開放閱讀框(open reading frame,ORF)編碼的氨基酸序列在NCBI數(shù)據(jù)庫中的BLASTx進(jìn)行同源性搜索和比對;利用Clustal X1.81和MEGA 3.1 軟件分析MaERF-1蛋白與其他植物的ERF蛋白的進(jìn)化關(guān)系,構(gòu)建分子進(jìn)化樹;利用expasy數(shù) 據(jù) 庫(http://web.expasy.org/compute_pi/)預(yù)測蛋白質(zhì)pI和分子質(zhì)量;用Protscale(http://www.expasy.ch/tools/protscale.html/)工具對MaERF-1編碼的蛋白質(zhì)產(chǎn)物疏水性和親水性進(jìn)行分析。
1.2.4 MaERF-1表達(dá)載體的構(gòu)建 選用Pcambia 1304植物表達(dá)載體構(gòu)建MaERF-1表達(dá)基因融合載體,以NcoI和SpeI酶切pMD18-T/MaERF-1重組質(zhì)粒和Pcambia 1304空載體,分別回收啟動(dòng)子片段和線性載體,連接,轉(zhuǎn)化DH5α后進(jìn)行菌液PCR及酶切檢測,重組質(zhì)粒命名為MaERF-1::1304。
2 結(jié)果與分析
2.1 MaERF-1基因的克隆與鑒定
以巴西蕉各器官組織混合cDNA為模版,F(xiàn)1和R1為引物,從香蕉栽培品種巴西蕉中克隆到一個(gè)ERF家族基因MaERF-1,其ORF為729 bp,PCR結(jié)果如圖1所示。
2.2 生物信息學(xué)分析
2.2.1 MaERF-1基因蛋白質(zhì)序列差異的比對 利用DNAman將MaERF-1 cDNA推導(dǎo)的氨基酸序列與NCBI中已登錄的其他高等植物的ERF氨基酸序列進(jìn)行同源關(guān)系的比較,結(jié)果(圖2)顯示,各種植物ERF編碼的氨基酸序列存在較高的同源性,多數(shù)都達(dá)到55%以上。BLASTX分析表明,MaERF-1編碼的氨基酸序列與馬來西亞野生蕉MaERF-1-like(XP_009379780.1)、菠蘿AcERF-2(OAY63143.160)、海棗PcERF-2(XP_008807522.1)、荷花NnERF-2(XP_010243826.1)、葡萄VvERF-2(XP_002267961.1)、煙草NtERF-2(XP_016448076.1)、油棕EgERF-2(XP_010940400.1)編碼的氨基酸序列具有較高的一致性,分別為98%、60%、59%、53%、54%、51%、65%。
2.2.2 MaERF-1基因進(jìn)化樹分析 利用Clustal X1.81和MEGA 3.1軟件,將MaERF-1 cDNA推導(dǎo)的氨基酸序列與NCBI中已登錄的其他植物的SIP氨基酸序列進(jìn)行系統(tǒng)進(jìn)化樹的比對分析。結(jié)果表明本研究得到的巴西蕉MaERF-1基因所編碼的氨基酸序列與馬來西亞野生蕉MaERF-1-like具有較近的親緣關(guān)系(圖3)。
2.2.3 MaERF-1編碼蛋白質(zhì)理化性質(zhì)分析 利用Expasy分析MaERF-1編碼蛋白質(zhì)的理化性質(zhì),如圖4顯示,該蛋白具有243個(gè)氨基酸,分子量為26 139.03 u,理論等電點(diǎn)pI為7.81。
2.2.4 MaERF-1編碼蛋白質(zhì)產(chǎn)物的親水性、疏水性分析 對MaERF-1編碼蛋白質(zhì)產(chǎn)物親水性、疏水性分析結(jié)果表明(圖5),MaERF-1多肽鏈中精氨酸Arg具有最低的分值-2.800,異亮氨酸Ile具有最高分值1.600。依據(jù)氨基酸分值越低親水性越強(qiáng)和分值越高疏水性越強(qiáng)的規(guī)律,可見,精氨酸Arg親水性最強(qiáng),而異亮氨酸Ile疏水性最強(qiáng),而就整體來看,親水性氨基酸均勻分布在整個(gè)肽鏈中,多于疏水性氨基酸。
2.3 MaERF-1表達(dá)載體的構(gòu)建
在上游引物F1和下游引物R1分別加入NcoI和SpeI內(nèi)切酶,將pMD18-T/MaERF-1和Pcambia 1304載體同時(shí)用NcoI和SpeI內(nèi)切酶酶切后,回收目的片段后連接轉(zhuǎn)化,PCR以及雙酶切鑒定如圖6,結(jié)果顯示,PCR產(chǎn)物和酶切條帶均為729 bp。
3 討論
香蕉是一年生草本植物,喜高溫多濕,缺水時(shí)葉片變薄、果指變短,生長延緩,導(dǎo)致減產(chǎn),因此對香蕉抗逆品種的選育是十分必要的。近年來,通過挖掘香蕉本身所具有的抗逆基因資源,研究該基因的功能來進(jìn)一步提高香蕉的抗逆性就成為一個(gè)重要的手段。
本實(shí)驗(yàn)克隆到1個(gè)香蕉乙烯響應(yīng)因子MaERF-1,通過在NCBI上BLAST分析各高等植物中ERF氨基酸序列的同源關(guān)系,結(jié)果顯示MaERF-1編碼的氨基酸與各種植物中ERF編碼的氨基酸序列存在較高的同源性,達(dá)到55%以上,其中與馬來西亞野生蕉同源性最高為98%。對各種植物ERF氨基酸序列比對和系統(tǒng)進(jìn)化樹分析發(fā)現(xiàn),不同植物的ERF序列相似性較高,以ERF氨基酸序列構(gòu)建的進(jìn)化樹能準(zhǔn)確反映不同植物間的親緣關(guān)系,也可作為評價(jià)香蕉等植物種植資源的重要依據(jù)。通過對ERF編碼蛋白質(zhì)的親水性和疏水性進(jìn)行分析,表明其親水性氨基酸多于疏水性氨基酸。將MaERF-1成功構(gòu)建到植物表達(dá)載體中,后續(xù)實(shí)驗(yàn)可以通過將表達(dá)載體轉(zhuǎn)化到如擬南芥、煙草等模式植物中,對該基因進(jìn)行進(jìn)一步的功能驗(yàn)證和分析。
當(dāng)植物遭受到外界的非生物脅迫時(shí),植物自身有復(fù)雜的系統(tǒng)去保護(hù)其去抵御不同的脅迫。ERF在植物中是屬于(AP2/ERF)轉(zhuǎn)錄因子家族中的亞族,對ERF的功能研究主要集中在對其在抗逆性方面的研究[30-32],如抗旱、抗鹽、抗寒等。例如,水稻ERF基因OsBIERF1、OsBIERF3 和OsBIERF4不僅能被稻瘟病菌誘導(dǎo)表達(dá),還能受到高鹽、冷害、干旱、傷害脅迫而上調(diào)表達(dá)[33]。在煙草中過表達(dá)ERF11能夠提高植物抵御青枯病的能力。在擬南芥中,AtERF71能夠顯著被高鹽、甘露醇和ABA誘導(dǎo)表達(dá),在高鹽脅迫下,植株中的活性氧(ROS)累積顯著減少[34]。轉(zhuǎn)化GmERF7的煙草能夠提高對高鹽脅迫的耐受性,該轉(zhuǎn)基因植株中葉綠素和糖含量顯著增加[35]。因此,本實(shí)驗(yàn)通過對該基因的功能進(jìn)行生物信息學(xué)分析,并且將其連接到植物表達(dá)載體中,該實(shí)驗(yàn)結(jié)果為進(jìn)一步研究ERF基因的表達(dá)調(diào)控以及后續(xù)轉(zhuǎn)基因分析基因功能奠定了基礎(chǔ)。
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