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1、植物資源與環(huán)境學(xué)報(bào)2014,23(2):11—18JournalofPlantResourcesandEnvironment喜鹽鳶尾Na+/H+逆轉(zhuǎn)運(yùn)蛋白基因IhNHX1的克隆及序列分析趙沿海,原海燕,顧春筍,黃蘇珍①[江蘇省·中國(guó)科學(xué)院植物研究所(南京中山植物園)江蘇省植物遷地保護(hù)重點(diǎn)實(shí)驗(yàn)室,江蘇南京2100147摘要:采用同源克隆和RACE法克隆獲得喜鹽鳶尾(IrishalophilaPal1.)Na/H逆轉(zhuǎn)運(yùn)蛋白基因IhNHX1的全長(zhǎng)序列,該基因序列的全長(zhǎng)為1946bp,包含1個(gè)長(zhǎng)度為1611bp的開(kāi)
2、放閱讀框(ORF),編碼537個(gè)氨基酸。序列對(duì)比及系統(tǒng)樹(shù)分析結(jié)果表明:IhNHX1基因編碼的氨基酸序列與另外11種植物NHX1基因編碼的氨基酸序列的一致性高達(dá)96.2%,相同序列占61.7%,表明該氨基酸序列保守性較高;在系統(tǒng)樹(shù)上喜鹽鳶尾與其他植物的分支長(zhǎng)均大于1.2,表明它們的親緣關(guān)系均較遠(yuǎn);IhNHX1基因編碼的氨基酸序列含有2個(gè)保守結(jié)構(gòu)域,即氨氯吡嗪結(jié)合位點(diǎn)和CaM結(jié)合結(jié)構(gòu)域,分別是NHX1蛋白的標(biāo)志性結(jié)合位點(diǎn)和重要調(diào)節(jié)區(qū)域。該蛋白質(zhì)的二級(jí)結(jié)構(gòu)和跨膜結(jié)構(gòu)域分析結(jié)果表明:在IhNHX1基因編碼的蛋白質(zhì)的
3、二級(jí)結(jié)構(gòu)中,ot螺旋占48.60%、不規(guī)則卷曲占32.03%、延伸鏈占14.71%、氫鍵轉(zhuǎn)角占4.66%;該蛋白質(zhì)含有10個(gè)跨膜結(jié)構(gòu)域。此外,對(duì)5RACE方法中5端正向引物的優(yōu)化設(shè)計(jì)步驟進(jìn)行了歸納,以提高PCR擴(kuò)增的特異性。關(guān)鍵詞:喜鹽鳶尾;Na/H逆轉(zhuǎn)運(yùn)蛋白基因IhNHX1;基因克??;序列分析;系統(tǒng)樹(shù);跨膜結(jié)構(gòu)域中圖分類(lèi)號(hào):Q943.2;Q785文獻(xiàn)標(biāo)志碼:A文章編號(hào):1674—7895(2014)02-0011—08DOI:10.3969/j.issn.1674—7895.2014.02.02Cloni
4、ngandsequenceanalysisofNa/HantiportergeneIhNHX1ofIrishalophilaZHAOYanhai,YUANHaiyan,GUChunsun,HUANGSuzhen①(JiangsuProvinceKeyLaboratoryforPlantEx—situConservation,InstituteofBotany,JiangsuProvinceandtheChineseAcademyofSciences,Nanjing210014,China),PlantRes
5、our.&Environ.2014,23(2):11-18Abstract:Throughhomology—basedcloningandRACE(rapidamplificationofcDNAends)methods,thewholesequenceofNa/Hantiportergene/hNHXlofIrishalophilaPal1.wascloned.Thewholelengthofthisgeneis1946bp,whichcontainsanopenreadingfragment(ORF)w
6、ithalengthofl6l1bpandencodes537aminoacids.Theresuhsofsequencecomparisonandphylogenetietreeanalysisshowthattheidentityofaminoacidsequencesencodedby/hNHxlgeneandbyNHxlgeneofotherelevenspeciesisupto96.2%.thesamesequenceaccountsfor61.7%,meaningthattheconservat
7、ionofthisaminoacidsequenceishigher.Thebranchlengthofhalophilaandotherspeciesinphylogenetictreeislargerthan1.2.indicatingthattheirgeneticrelationshipisfaraway.Therearetwoconserveddomainsinaminoacidsequenceencodedby/hNHxlgene.namelyammoniachloridepyrazinebin
8、dingsiteandCaMbindingdomain.whichisthetypicalbindingsiteandimportantregulationdomainofNHX1protein,respectively.Theanalysisresultsofthesecondstructureandtransmembranedomainofthisproteinshowthatinthesecondstruc