火龙果线粒体细胞色素b基因克隆及其应用潜力分析
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摘要
火龙果(Hylocereus undulatus Britt.)种质资源评价是开展火龙果育种的基础,也可为火龙果的其它方面研究提供指导。线粒体细胞色素b基因(Mt-cyt b)是物种鉴定、系统进化及遗传多样性分析等方面研究的潜在遗传分子标记。本研究通过同源克隆和RACE技术,从红肉火龙果获得了长度为3 093 bp的Pmt-cyt b基因片段,包括1 628 bp的5'UTR、1 182 bp的CDS及283 bp的3'UTR,预测编码393个氨基酸。系统进化分析表明火龙果与白花蝇子草、白玉草、甜菜和奎藜的进化关系最近。对来自中国不同地方的21份火龙果种质资源的进行Pmt-cyt b序列比对分析发现:不同火龙果Pmt-cyt b基因序列完全一样,说明Pmt-cyt b可能不适用于火龙果的品种鉴定及遗传多样性研究。我们的研究补充了火龙果Mt-cyt b种质鉴定的认识,在一定程度上为火龙果种质资源的评价工作和研究仙人掌科其它植物Mt-cyt b基因提供序列参考。
Evaluation of dragon fruit(Hylocereus undulatus Britt.) germplasm resources is an important aspect of dragon fruit breeding research, which can also provide guidance for studies on other aspects of dragon fruit. The mitochondrial cytochrome b gene(Mt-cyt b) is a potential genetic marker for species identification, phylogenetic evolution and genetic diversity analysis. The Pmt-cyt b gene fragment with the length of 3 093 bp was obtained from Hylocereus polyrhizus by homologous cloning and RACE technology in this study, including 1 628 bp 5'UTR,1 182 bp CDS and 283 bp 3'UTR, which encoded 393 amino acids. The phylogenetic analysis showed that dragon fruit had the closest evolutionary relationship with Chenopodium quinoa, Silene latifolia, Beta vulgaris and Silene vulgaris. The sequence alignment of Pmt-cyt b was carried out on 21 germplasm resources of dragon fruit from different regions of China. The analysis showed that different dragon fruit Pmt-cyt b gene sequences were exactly same, which indicated that Pmt-cyt b gene might not be suitable for variety identification and genetic diversity research of dragon fruit. Our study supplemented the understanding of dragon fruit Mt-cyt b in germplasm identification, which could provide sequence reference for the evaluation of dragon fruit germplasm resources and studies on Mt-cyt b gene in other plants of Cactaceae to some extent.
Evaluation of dragon fruit(Hylocereus undulatus Britt.) germplasm resources is an important aspect of dragon fruit breeding research, which can also provide guidance for studies on other aspects of dragon fruit. The mitochondrial cytochrome b gene(Mt-cyt b) is a potential genetic marker for species identification, phylogenetic evolution and genetic diversity analysis. The Pmt-cyt b gene fragment with the length of 3 093 bp was obtained from Hylocereus polyrhizus by homologous cloning and RACE technology in this study, including 1 628 bp 5'UTR,1 182 bp CDS and 283 bp 3'UTR, which encoded 393 amino acids. The phylogenetic analysis showed that dragon fruit had the closest evolutionary relationship with Chenopodium quinoa, Silene latifolia, Beta vulgaris and Silene vulgaris. The sequence alignment of Pmt-cyt b was carried out on 21 germplasm resources of dragon fruit from different regions of China. The analysis showed that different dragon fruit Pmt-cyt b gene sequences were exactly same, which indicated that Pmt-cyt b gene might not be suitable for variety identification and genetic diversity research of dragon fruit. Our study supplemented the understanding of dragon fruit Mt-cyt b in germplasm identification, which could provide sequence reference for the evaluation of dragon fruit germplasm resources and studies on Mt-cyt b gene in other plants of Cactaceae to some extent.
引文
Arnold A.E.,Andersen E.M.,Taylor M.J.,and Steidl R.J.,2017,Using cytochrome b to identify nests and museum specimens of cryptic songbirds,Conservation Genet.Resour.,9(3):451-458
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Fan J.X.,Deng R.J.,Wang Y.Q.,Liu H.,Peng Z.J.,and Liu T.,2017b,Embryogenic callus induction via anther culture of purple pitaya(Hylocereus costaricensis cv.Zihonglong)and their genetic stability,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),15(1):258-266(范建新,邓仁菊,王永清,刘红,彭志军,刘涛,2017b,火龙果花药培养诱导胚性愈伤组织及遗传稳定性,分子植物育种,15(1):258-266)
Fu X.Y.,2011,Genetic diversity of mitochondrial cytochromes b in black carp from the Yangtze and the pearl river,Thesis for M.S.,Jinan University,Supervisor:Li G.S.,pp.30-46(付晓艳,2011,长江和珠江水系青鱼线粒体细胞色素b基因遗传多样性分析,硕士学位论文,暨南大学,导师:李贵生,pp.30-46)
Hao G.Y.,Yang G.Y.,Gu X.B.,Yang A.G.,Yang Y.D.,and Wang S.X.,2012,Analysis of genetic variation in different populations of Cysticercus tenuicallis based on the mitochondrial cytochrome b gene,Xumu Shouyi Xuebao(Acta Veterinaria et Zootechnica Sinica),43(1):119-125(郝桂英,杨光友,古小彬,阳爱国,杨应东,王淑贤,2012,基于线粒体细胞色素b基因的细颈囊尾蚴种群遗传多样性研究,畜牧兽医学报,43(1):119-125)
Hu Q.,Zheng Y.C.,Jin S.Y.,Wang Y.Y.,Chen C.,Zhou J.,and Liang Z.,2007,Identification of Yak meat by amplification of cytochrome b gene with universal primers,Shipin Kexue(Food Science),28(11):319-321(胡强,郑玉才,金素钰,王延云,陈琛,周静,梁梓,2007,用通用引物扩增细胞色素b基因进行牦牛肉的鉴定,食品科学,28(11):319-321)
Junqueira K.P.,Faleiro F.G.,Junqueira N.T.V.,Bellon G.,Lima C.A.D.,and Souza L.S.D.,2010,Genetic diversity of native pitaya from Brazilian Savannas with basis on RAPD markers,Revista Brasileira De Fruticultura,32(3):819-824
Li F.,2008,The cloning and analysis of salt-tolerant gene from mangrove,Thesis for M.S.,Xiamen University,Supervisor:Zhou H.T.,pp.50-68(李芳,2008,红树植物耐盐基因的克隆与分析,硕士学位论文,厦门大学,导师:周涵韬,pp.50-68)
Li Y.Q.,Zhao H.K.,Liu X.D.,Zhang C.B.,and Dong Y.S.,2011,Cloning and phylogenetic analysis of mitochondrial cytochrome b gene from soybean(Glycine max),Dadou Kexue(Soybean Science),30(5):738-742(李玉秋,赵洪锟,刘晓东,张春宝,董英山,2011,大豆线粒体细胞色素b基因的克隆及系统分析,大豆科学,30(5):738-742)
Mahmoudi A.,Darvish J.,Aliabadian M.,Moghaddam F.Y.,and Krystufek B.,2017,New insight into the cradle of the grey voles(subgenus Microtus)inferred from mitochondrial cytochrome b sequences,Mammalia,81(6):583-593
Qi X.Z.,Yin S.W.,Lou T.T.,Liao J.Q.,Chen G.H.,and Zhang B.,2007,Cloning and sequence analyses of cytochrome b gene of Anguilla marmorata from Hainan,Hainan Daxue Xuebao(Natural Sciences Journal of Hainan University),25(4):397-401(齐兴柱,尹绍武,娄甜甜,廖经球,陈国华,张本,2007,海南产花鳗鲡细胞色素b基因的克隆及序列分析,海南大学学报(自然科学版),25(4):397-401)
Qin J.C.,2011,The mitochondrial genome sequences of Isonychia ignota and Siphluriscus chinensis,Thesis for M.S.,Nanjing Normal University,Supervisor:Zhou C.F.,pp.40-54(秦加长,2011,莫名等蜉Isonychia ignota和中国拟短丝蜉Siphluriscus chinensis线粒体基因全序列的测定,硕士学位论文,南京师范大学,导师:周长发,pp.40-54)
Da Silva A.C.C.,Sabiao R.R.,Chiamolera F.M.,Segantini D.M.,and Martins A.B.G.,2017,Morphological traits as tool to verify genetic variability of interspecific dragon fruit hybrids,Rev.Bras.Frutic.,39(1):e168
Thanakiatkrai P.,and Kitpipit T.,2017,Meat species identification by two direct-triplex real-time PCR assays using low resolution melting,Food Chemistry,233:144-150
Tian X.M.,Li H.L.,He Y.,Hong Q.M.,Hu W.B.,and Li Q.,2015,Research progress of pitaya,Beifang Yuanyi(Northern Horticulture),(18):188-193(田新民,李洪立,何云,洪青梅,胡文斌,李琼,2015,火龙果研究现状,北方园艺,(18):188-193)
Tu J.F.,Wang L.,Xing X.M.,Li Y.Q.,and Yang F.H.,2010,Cloning and sequence analysis of mitochondrial cytochrome b gene(Cytb)in mink(Mustela vison)and its evolutionary relationships with the Mustela,Nongye Shengwu Jishu Xuebao(Journal of Agricultural Biotechnology),18(5):975-980(涂剑锋,王雷,邢秀梅,李一清,杨福合,2010,水貂细胞色素b基因(Cytb)克隆、序列分析及与鼬属动物的进化关系,农业生物技术学报,18(5):975-980)
Wang Y.L.,Hu W.R.,Liu S.Z.,Pan Y.Z.,Yi G.J.,and Sun Q.M.,2018,Effects of boric acid,sugar and pH on in vitro pollen germination and pollen tube length of pitaya,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),16(1):240-247(王玉林,胡位荣,刘顺枝,潘玉珠,易干军,孙清明,2018,硼酸、蔗糖和pH值对火龙果花粉离体萌发和花粉管伸长的影响,分子植物育种,16(1):240-247)
Wares J.P.,2014,Mitochondrial cytochrome b sequence data are not an improvement for species identification in Scleractinian corals,Peer J.,2:e564
Wu L.,2016,The evaluation and breeding research of pitaya germplasm resources,Thesis for M.S.,Guangxi University,Supervisor:Huang G.X.,pp.50-62(吴琳,2016,火龙果种质资源评价及繁育研究,硕士学位论文,广西大学,导师:黄桂香,pp.50-62)
Yang C.J.,Ge J.,Chen S.J.,Liu Y.J.,Wu S.Q.,Yang G.Z.,Gu Z.L.,2015,Analysis of molecular cloning,gene polymorphism and evolution of Mc1r in colored rex rabbits,Jinyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),34(8):1695-1699(杨翠军,葛剑,陈赛娟,刘亚娟,吴淑琴,杨国忠,谷子林,2015,彩色獭兔Mc1r基因克隆、多态性研究及分子进化分析,基因组学与应用生物学,34(8):1695-1699)
Yang L.Y.,Li H.,Zhao X.Q.,and Liu G.Q.,2012,Comparison of phylogenetic relationships constructed by using protein-coding sequences,intergenic sequences and amino acid sequences,Shengwu Wuli Xuebao(Acta Biophysica Sinica),28(2):157-168(杨林源,李宏,赵小庆,刘国庆,2012,基于编码序列、基因间序列和氨基酸序列构建的系统发生关系比较,生物物理学报,28(2):157-168)
Yang Q.,Bai X.F.,Gao Y.,Yi L.J.,Cong J.Y.,Wang R.G.,and Li G.J.,2009,cDNA cloning of CBF/DREB1 transcription factor of Ammopiptanthus mongolicus and its sequence analysis,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),28(6):1043-1048(杨杞,白肖飞,高阳,伊莉佳,丛靖宇,王瑞刚,李国婧,2009,沙冬青CBF/DREB1转录因子cDNA的克隆及序列分析,基因组学与应用生物学,28(6):1043-1048)
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Zhang B.X.,Fan F.H.,Qiao G.,Song S.,Wen X.P.,and Liu T.,2013,DNA fingerprint construction and genetic relationship analysis of dragon fruit germplasm in Guizhou by IS-SR markers,Guoshu Xuebao(Journal of Fruit Science),30(4):573-577(张冰雪,范付华,乔光,宋莎,文晓鹏,刘涛,2013,贵州地方火龙果芽变种质DNA指纹图谱及遗传多样性的ISSR分析,果树学报,30(4):573-577)
Zhang B.,2007,Identification,classification,and phylogenetic relationships of pathogenic fungi based on mitochondrial cytochrome b gene and translation elongation factor gene analysis,Thesis for Ph.D.,Jilin University,Supervisor:Wang L.,pp.50-61(张波,2007,线粒体细胞色素b和翻译延伸因子基因分析用于病原真菌的分类、鉴定和系统发生,博士学位论文,吉林大学,导师:王丽,pp.50-61)
Zhang J.P.,Liu Z.,Zhang B.,Yin X.Y.,Wang L.,Shi H.N.,and Kang Y.J.,2015,Genetic diversity and taxonomic status of Gymnocypris chilianensis based on the mitochondrial DNAcytochrome b gene,Genetics and Molecular Research,14(3):9253-9260
Zhong L.Q.,Liu P.P.,Pan J.L.,Wang M.H.,Chen Y.P.,Qin Q.,Bian W.J.,and Chen X.H.,2013,Genetic variation analysis of yellow catfish(Pelteobagrus fulvidraco)from five lakes in the middle and lower reaches of the Yangtze River based on mitochondrial DNA cyt b,Hupo Kexue(Journal of Lake Sciences),25(2):302-308(钟立强,刘朋朋,潘建林,王明华,陈友明,秦钦,边文冀,陈校辉,2013,长江中下游5个湖泊黄颡鱼(Pelteobagrus fulvidraco)种群线粒体细胞色素b基因的遗传变异分析,湖泊科学,25(2):302-308)
Fan J.X.,Deng R.J.,Wang Y.Q.,Liu R.,Han S.Q.,and Zhu A.L.,2017a,Morphological characteristics of pitaya microspore at different developmental stages,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),15(2):710-715(范建新,邓仁菊,王永清,刘荣,韩树全,朱安乐,2017a,火龙果小孢子不同发育时期的形态特征,分子植物育种,15(2):710-715)
Fan J.X.,Deng R.J.,Wang Y.Q.,Liu H.,Peng Z.J.,and Liu T.,2017b,Embryogenic callus induction via anther culture of purple pitaya(Hylocereus costaricensis cv.Zihonglong)and their genetic stability,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),15(1):258-266(范建新,邓仁菊,王永清,刘红,彭志军,刘涛,2017b,火龙果花药培养诱导胚性愈伤组织及遗传稳定性,分子植物育种,15(1):258-266)
Fu X.Y.,2011,Genetic diversity of mitochondrial cytochromes b in black carp from the Yangtze and the pearl river,Thesis for M.S.,Jinan University,Supervisor:Li G.S.,pp.30-46(付晓艳,2011,长江和珠江水系青鱼线粒体细胞色素b基因遗传多样性分析,硕士学位论文,暨南大学,导师:李贵生,pp.30-46)
Hao G.Y.,Yang G.Y.,Gu X.B.,Yang A.G.,Yang Y.D.,and Wang S.X.,2012,Analysis of genetic variation in different populations of Cysticercus tenuicallis based on the mitochondrial cytochrome b gene,Xumu Shouyi Xuebao(Acta Veterinaria et Zootechnica Sinica),43(1):119-125(郝桂英,杨光友,古小彬,阳爱国,杨应东,王淑贤,2012,基于线粒体细胞色素b基因的细颈囊尾蚴种群遗传多样性研究,畜牧兽医学报,43(1):119-125)
Hu Q.,Zheng Y.C.,Jin S.Y.,Wang Y.Y.,Chen C.,Zhou J.,and Liang Z.,2007,Identification of Yak meat by amplification of cytochrome b gene with universal primers,Shipin Kexue(Food Science),28(11):319-321(胡强,郑玉才,金素钰,王延云,陈琛,周静,梁梓,2007,用通用引物扩增细胞色素b基因进行牦牛肉的鉴定,食品科学,28(11):319-321)
Junqueira K.P.,Faleiro F.G.,Junqueira N.T.V.,Bellon G.,Lima C.A.D.,and Souza L.S.D.,2010,Genetic diversity of native pitaya from Brazilian Savannas with basis on RAPD markers,Revista Brasileira De Fruticultura,32(3):819-824
Li F.,2008,The cloning and analysis of salt-tolerant gene from mangrove,Thesis for M.S.,Xiamen University,Supervisor:Zhou H.T.,pp.50-68(李芳,2008,红树植物耐盐基因的克隆与分析,硕士学位论文,厦门大学,导师:周涵韬,pp.50-68)
Li Y.Q.,Zhao H.K.,Liu X.D.,Zhang C.B.,and Dong Y.S.,2011,Cloning and phylogenetic analysis of mitochondrial cytochrome b gene from soybean(Glycine max),Dadou Kexue(Soybean Science),30(5):738-742(李玉秋,赵洪锟,刘晓东,张春宝,董英山,2011,大豆线粒体细胞色素b基因的克隆及系统分析,大豆科学,30(5):738-742)
Mahmoudi A.,Darvish J.,Aliabadian M.,Moghaddam F.Y.,and Krystufek B.,2017,New insight into the cradle of the grey voles(subgenus Microtus)inferred from mitochondrial cytochrome b sequences,Mammalia,81(6):583-593
Qi X.Z.,Yin S.W.,Lou T.T.,Liao J.Q.,Chen G.H.,and Zhang B.,2007,Cloning and sequence analyses of cytochrome b gene of Anguilla marmorata from Hainan,Hainan Daxue Xuebao(Natural Sciences Journal of Hainan University),25(4):397-401(齐兴柱,尹绍武,娄甜甜,廖经球,陈国华,张本,2007,海南产花鳗鲡细胞色素b基因的克隆及序列分析,海南大学学报(自然科学版),25(4):397-401)
Qin J.C.,2011,The mitochondrial genome sequences of Isonychia ignota and Siphluriscus chinensis,Thesis for M.S.,Nanjing Normal University,Supervisor:Zhou C.F.,pp.40-54(秦加长,2011,莫名等蜉Isonychia ignota和中国拟短丝蜉Siphluriscus chinensis线粒体基因全序列的测定,硕士学位论文,南京师范大学,导师:周长发,pp.40-54)
Da Silva A.C.C.,Sabiao R.R.,Chiamolera F.M.,Segantini D.M.,and Martins A.B.G.,2017,Morphological traits as tool to verify genetic variability of interspecific dragon fruit hybrids,Rev.Bras.Frutic.,39(1):e168
Thanakiatkrai P.,and Kitpipit T.,2017,Meat species identification by two direct-triplex real-time PCR assays using low resolution melting,Food Chemistry,233:144-150
Tian X.M.,Li H.L.,He Y.,Hong Q.M.,Hu W.B.,and Li Q.,2015,Research progress of pitaya,Beifang Yuanyi(Northern Horticulture),(18):188-193(田新民,李洪立,何云,洪青梅,胡文斌,李琼,2015,火龙果研究现状,北方园艺,(18):188-193)
Tu J.F.,Wang L.,Xing X.M.,Li Y.Q.,and Yang F.H.,2010,Cloning and sequence analysis of mitochondrial cytochrome b gene(Cytb)in mink(Mustela vison)and its evolutionary relationships with the Mustela,Nongye Shengwu Jishu Xuebao(Journal of Agricultural Biotechnology),18(5):975-980(涂剑锋,王雷,邢秀梅,李一清,杨福合,2010,水貂细胞色素b基因(Cytb)克隆、序列分析及与鼬属动物的进化关系,农业生物技术学报,18(5):975-980)
Wang Y.L.,Hu W.R.,Liu S.Z.,Pan Y.Z.,Yi G.J.,and Sun Q.M.,2018,Effects of boric acid,sugar and pH on in vitro pollen germination and pollen tube length of pitaya,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),16(1):240-247(王玉林,胡位荣,刘顺枝,潘玉珠,易干军,孙清明,2018,硼酸、蔗糖和pH值对火龙果花粉离体萌发和花粉管伸长的影响,分子植物育种,16(1):240-247)
Wares J.P.,2014,Mitochondrial cytochrome b sequence data are not an improvement for species identification in Scleractinian corals,Peer J.,2:e564
Wu L.,2016,The evaluation and breeding research of pitaya germplasm resources,Thesis for M.S.,Guangxi University,Supervisor:Huang G.X.,pp.50-62(吴琳,2016,火龙果种质资源评价及繁育研究,硕士学位论文,广西大学,导师:黄桂香,pp.50-62)
Yang C.J.,Ge J.,Chen S.J.,Liu Y.J.,Wu S.Q.,Yang G.Z.,Gu Z.L.,2015,Analysis of molecular cloning,gene polymorphism and evolution of Mc1r in colored rex rabbits,Jinyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),34(8):1695-1699(杨翠军,葛剑,陈赛娟,刘亚娟,吴淑琴,杨国忠,谷子林,2015,彩色獭兔Mc1r基因克隆、多态性研究及分子进化分析,基因组学与应用生物学,34(8):1695-1699)
Yang L.Y.,Li H.,Zhao X.Q.,and Liu G.Q.,2012,Comparison of phylogenetic relationships constructed by using protein-coding sequences,intergenic sequences and amino acid sequences,Shengwu Wuli Xuebao(Acta Biophysica Sinica),28(2):157-168(杨林源,李宏,赵小庆,刘国庆,2012,基于编码序列、基因间序列和氨基酸序列构建的系统发生关系比较,生物物理学报,28(2):157-168)
Yang Q.,Bai X.F.,Gao Y.,Yi L.J.,Cong J.Y.,Wang R.G.,and Li G.J.,2009,cDNA cloning of CBF/DREB1 transcription factor of Ammopiptanthus mongolicus and its sequence analysis,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),28(6):1043-1048(杨杞,白肖飞,高阳,伊莉佳,丛靖宇,王瑞刚,李国婧,2009,沙冬青CBF/DREB1转录因子cDNA的克隆及序列分析,基因组学与应用生物学,28(6):1043-1048)
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