河八王分蘖基因NpD53克隆及其生物信息学分析  

Cloning and bioinformatics analysis of tillering-related gene NpD53 from Narenga porphyrocoma(Hance)Bor

作　　者：刘昔辉[1,2] 张荣华 桂意云[1] 张小秋[1] 韦金菊 周会[1] 区惠平[1] 刘新龙 LIU Xi-hui;ZHANG Rong-hua;GUI Yi-yun;ZHANG Xiao-qiu;WEI Jin-ju;ZHOU Hui;OU Hui-ping;LIU Xin-long(Guangxi Key Laboratory of Sugarcane Genetic Improvement/Key Laboratory of Sugarcane Biotechnology and Genetic Improvement(Guangxi),Ministry of Agriculture/Sugarcane Research Center,Chinese Academy of Agricultural Sciences/Sugarcane Research Institute,Guangxi Academy of Agricultural Sciences,Nanni;Yunnan Key Laboratory of Sugarcane Genetic Improvement,Kaiyuan,Yunnan 661600,China)

机构地区：[1]广西甘蔗遗传改良重点实验室/农业部广西甘蔗生物技术与遗传改良重点实验室/中国农业科学院甘蔗研究中心/广西农业科学院甘蔗研究所,南宁530007 [2]云南省甘蔗遗传改良重点实验室,云南开远661600

出　　处：《南方农业学报》2017年第9期1554-1559,共6页Journal of Southern Agriculture

基　　金：国家自然科学基金项目(31101195);云南甘蔗遗传改良重点实验室项目(2015DG015-03);广西"八桂学者"和特聘专家专项项目([2013]3号);广西农业科学院科技发展基金项目(桂农科2016JM07)

摘　　要：【目的】克隆河八王[Narenga porphyrocoma(Hance)Bor]分蘖基因NpD53,并分析其序列特征,为甘蔗野生种分蘖的分子机理研究提供理论参考。【方法】以河八王为试验材料,提取其叶片总RNA,反转录获得cDNA,通过RACE(cDNA末端快增)技术扩增NpD53基因,并采用生物信息学方法对其核苷酸序列及编码蛋白的氨基酸序列进行分析。【结果】NpD53基因(Gen Bank登录号MF593461)的中间序列长度760 bp、5'端序列长度1497 bp、3'端序列长度1233 bp,拼接后其cDNA序列全长为2597 bp,包含1个2037 bp的开放阅读框(ORF),编码678个氨基酸,蛋白分子量75.02 k D,等电点6.59,亲水性平均数-0.506,表明其为亲水性蛋白,位于细胞核内(可信度94.1%)。NpD53基因编码蛋白(NpD53)存在P-loop_NTPase和Clp B_D2-small超家族核心序列,含有4个非特异性位点,与其他物种的D53蛋白均具有相同的保守结构域Clp B_D2-small;其二级结构仅有4种卷曲类型,以无规则卷曲最多,占42.77%,其次是α-螺旋,占35.55%,延伸链和β-转角分别占15.34%和6.34%。NpD53蛋白的氨基酸序列同源性比对及系统进化树分析结果显示,河八王与禾本科物种(高粱和山羊草)的亲缘关系较近,与海枣、油棕、芭蕉等物种的亲缘关系较远。【结论】河八王分蘖基因NpD53编码蛋白与其他物种的D53蛋白具有相同的保守结构域,且具有2个I类Clp ATP酶的非特异性位点,推测NpD53为一种分子伴侣,与河八王自身的耐热性相关。【Objective】This research was aimed to clone tillering-related gene NpD53from Narenga porphyrocoma(Hance)Bor,and analyze the sequence characteristics of NpD53and provide theoretical basis for the study on molecular mechanism of wild sugarcane tillering.【Method】Total RNAwas isolated from the leaves of N.porphyrocoma and the cDNA was obtained by reverse transcription.NpD53was amplified by RACE(rapid amplification of cDNA end)and its nucleotide sequence and amino acid sequence of encoding protein were analyzed by bioinformatics methods.【Result】The cloned Gene NpD53(GeneBank accession No.MF593461)contained midregion sequence of760bp,5'terminal sequence of1497bp,3'terminal sequence of1233bp.Combining the three parts,its cDNA sequence full-length was2597bp.It containedan open reading frame(ORF)of2037bp,encoded678amino acids with molecular mass of75.02KD,pI of6.59,and average hydrophilicity of-0.506,which meant it was a hydrophilic protein.The protein was situated in cell nucleus(94.1%confidence).The encoding protein(NpD53)of NpD53had two super family core sequences,namely P-loop_NTPase and ClpB_D2-small,with four non-specific sites,and shared the same ClpB_D2-small conserved domain with D53protein of other species.Its secondary structure had only four coil types,among which random coils were in the majority,accounting for42.77%,followed by alpha helix,accounting for35.55%,while extended strand and beta turn accounted for15.34%and6.34%respectively.According to the homology comparison and phylogenetic tree analysis for amino acid sequences of NpD53protein,N.porphyrocoma had a close phylogenetic relationship with gramineous plants(Sorghum bicolor and Aegilops tauschii),and had a distant phylogenetic relationship with Phoenix dactylifera,Elaeis guineensis and Musa acuminate.【Conclusion】The encoding protein of tillering-related gene NpD53from N.porphyrocoma shares the same conserved domain with D53protein of other species,and has two non-specific sites of class-I Clp ATPase.It is predicted that NpD53may be a

关 键 词：河八王 NpD53基因 克隆 序列分析 RACE 

分 类 号：S566.1[农业科学—作物学]