澄黄滨珊瑚ferritin基因部分cDNA序列的获得及分析
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摘要
造礁石珊瑚为珊瑚礁生态系统中的框架生物,研究其重要功能基因对于理解造礁石珊瑚对环境变化的响应有重要指示意义.本研究提取了澄黄滨珊瑚(Porites lutea)的总RNA,通过RT-RCR得到cDNA,并以cDNA为模板设计引物进行test PCR,获得大小为254bp的ferritin基因cDNA序列.分析表明,该部分序列编码了84个氨基酸,其中包含一个亚铁氧化酶活性功能区,而该活性功能区又有一个铁离子通道,其与多孔鹿角珊瑚(Acropora millepora)的ferritin基因相似度较高,达85%,覆盖度为100%,推测该基因序列在种内和种间都较保守,已向GenBank提交序列,并获得登录号为JQ305796.该序列可用于实时定量PCR技术(qRT-PCR)及cDNA末端快速扩增技术(RACE)等后续分子生物学研究.
Coral reef, the main component of the coral reef ecosystem, is extremely sensitive to environmental changes. Research on the functional genes of coral is very valuable. We extracted total RNA from the coral Porites lutea and reversely transcribed it into cDNA. The cDNA was used as a template to amplify part of the ferritin gene by PCR. We obtained a clean 254-bp band, and sequenced. The sequence encodes 83 amino acids of the ferritin protein, containing a ferroxidase di-iron center and an iron ion channel. Similarity searching by BLAST identified 100% coverage with ferritin in the coral Acropora millepora and 85% similarity, suggesting that this gene is highly conserved across species. The sequence can be accessed from the GenBank database with the accession number JQ305796. This sequence is a useful gene for future molecular biological studies such as qRT-PCR and RACE.
Coral reef, the main component of the coral reef ecosystem, is extremely sensitive to environmental changes. Research on the functional genes of coral is very valuable. We extracted total RNA from the coral Porites lutea and reversely transcribed it into cDNA. The cDNA was used as a template to amplify part of the ferritin gene by PCR. We obtained a clean 254-bp band, and sequenced. The sequence encodes 83 amino acids of the ferritin protein, containing a ferroxidase di-iron center and an iron ion channel. Similarity searching by BLAST identified 100% coverage with ferritin in the coral Acropora millepora and 85% similarity, suggesting that this gene is highly conserved across species. The sequence can be accessed from the GenBank database with the accession number JQ305796. This sequence is a useful gene for future molecular biological studies such as qRT-PCR and RACE.
引文
1邹仁林.中国动物志——造礁石珊瑚.北京:科学出版杜,2001.2–7
2Leggat W,Hoegh-Guldberg O,Dove S,et al.Analysis of an EST library from the dinoflagellate(Symbiodinium sp.)symbiont of reef-buildingcorals.J Phycol,2007,43:1010–1021
3Downs C A,Mueller E,Phillips S,et al.A molecular biomarker system for assessing the health of coral(Montastraea faveolata)during heatstress.Mar Biotechnol,2000,2:533–544
4Shinzato C,Shoguchi E,Kawashima T,et al.Using the Acropora digitifera genome to understand coral responses to environmental change.Nature,2011,476:320–323
5张金海,崔成毅,周星飞.单个铁蛋白分子的弹性模量研究.科学通报,2009,54:345–348
6Harrison P M,Arosio P.Ferritins:Molecular properties,iron storage function and cellular regulation.Bba-Bioenergetics,1996,1275:161–203
7Worwood M,Brook J D,Cragg S J,et al.Assignment of human ferritin genes to chromosomes-11and chromosome-19q13.3-19qter.HumGenet,1985,69:371–374
8Youssoufian H,Chance P,Tuckmuller C M,et al.Association of a new chromosomal deletion[DEL(1)(Q32Q42)]with diaphragmatic hernia:Assignment of a human ferritin gene.Hum Genet,1988,78:267–270
9Gasparini P,Calvano S,Memeo E,et al.Assignment of Ferritin L gene(FTL)to human chromosome band19q13.3by in situ hybridization.Ann Genet-Paris,1997,40:227–228
10Theil E C.Ferritin-structure,gene-regulation,and cellular function in animals,plants,and microoganisms.Annu Rev Biochem,1987,56:289–315
11Lawson D M,Treffry A,Artymiuk P J,et al.Identification of the ferroxidase centre in ferritin.Febs Lett,1989,254:207–210
12Schwarz J A,Brokstein P B,Voolstra C,et al.Coral life history and symbiosis:Functional genomic resources for two reef building Caribbeancorals,Acropora palmata and Montastraea faveolata.Bmc Genomics,2008,9:97
13De Zoysa M,Lee J.Two ferritin subunits from disk abalone(Haliotis discus discus):Cloning,characterization and expression analysis.FishShellfish Immun,2007,23:624–635
14Andrews S C,Harrison P M,Yewdall S J,et al.Structure,function,and evolution of ferritins.J Inorg Biochem,1992,47:161–174
15Orino K,Miura T,Muto S,et al.Sequence analysis of canine and equine ferritin H and L subunit cDNAs.DNA Sequence,2005,16:58–64
16Csaszar N B,Seneca F O,van Oppen M J H.Variation in antioxidant gene expression in the scleractinian coral Acropora millepora under la-boratory thermal stress.Mar Ecol-Prog Ser,2009,392:93–102
17Torti F M,Torti S V.Regulation of ferritin genes and protein.Blood,2002,99:3505–3516
18练健生,黄晖,黄良民,等.三亚珊瑚礁及其生物多样性.北京:海洋出版社,2010.18–24
19李淑,余克服,施祺,等.南海北部珊瑚共生虫黄藻密度的种间与空间差异及其对珊瑚礁白化的影响.科学通报,2007,52:2655–2662
20Clausen C D,Roth A A.Effect of temperature and temperature adaption on calcification rate in the hermatypic coral Pocillopora damicornis.Mar Biol,1975,33:93–100
21Cheney D P.The influence of temperature,does level and morphology on the uptake of3H-thmidine by reef corals.In:Proceedings of thesecond intemational symposium on coral reefs.Austrialia:the Great Barrier Reef Committee,1974,32:69–76
22Chomezynski P,Sacchi N.Single-step method of RNA isolation by acid guanidinium thiocyanate phenol chloroform extraction.Anal Bio-chem,1987,162:156–159
23杨婧,牛艳,张可炜.等.一种适用于RT-PCR的微藻总RNA提取方法.海洋科学,2010,34:6–10
24Desalvo M K,Voolstra C R,Sunagawa S,et al.Differential gene expression during thermal stress and bleaching in the Caribbean coral Mon-tastraea faveolata.Mol Ecol,2008,17:3952–3971
25Hofmann G E,Gaines S D.New tools to meet new challenges:Emerging technologies for managing marine ecosystems for resilience.Bio-science,2008,58:43–52
26Downs C A,Fauth J E,Robinson C E.Cellular diagnostics and coral health:Declining coral health in the Florida Keys.Mar Pollut Bull,2005,51:558–569
27Bustin S A,Benes V,Nolan T,et al.Quantitative real-time RT-PCR-a perspective.J Mol Endocrinol,2005,34:597–601
28Tricarico C,Pinzani P,Bianchi S,et al.Quantitative real-time reverse transcription polymerase chain reaction:Normalization to rRNA orsingle housekeeping genes is inappropriate for human tissue biopsies.Anal Biochem,2002,309:293–300
29Dheda K,Huggett J F,Chang J S,et al.The implications of using an inappropriate reference gene for real-time reverse transcription PCR datanormalization.Anal Biochem,2005,344:141–143
30Smith-Keune C,Dove S.Gene expression of a green fluorescent protein homolog as a host-specific biomarker of heat stress within areef-building coral.Mar Biotechnol,2008,10:166–180
31Fukuda I,Imagawa S,Iwao K,et al.Isolation of actin-encoding cDNAs from symbiotic corals.DNA Res,2002,9:217–223
32Venn A A,Quinn J,Jones R,et al.P-glycoprotein(multi-xenobiotic resistance)and heat shock protein gene expression in the reef coral Mon-tastraea franksi in response to environmental toxicants.Aquat Toxicol,2009,93:188–195
2Leggat W,Hoegh-Guldberg O,Dove S,et al.Analysis of an EST library from the dinoflagellate(Symbiodinium sp.)symbiont of reef-buildingcorals.J Phycol,2007,43:1010–1021
3Downs C A,Mueller E,Phillips S,et al.A molecular biomarker system for assessing the health of coral(Montastraea faveolata)during heatstress.Mar Biotechnol,2000,2:533–544
4Shinzato C,Shoguchi E,Kawashima T,et al.Using the Acropora digitifera genome to understand coral responses to environmental change.Nature,2011,476:320–323
5张金海,崔成毅,周星飞.单个铁蛋白分子的弹性模量研究.科学通报,2009,54:345–348
6Harrison P M,Arosio P.Ferritins:Molecular properties,iron storage function and cellular regulation.Bba-Bioenergetics,1996,1275:161–203
7Worwood M,Brook J D,Cragg S J,et al.Assignment of human ferritin genes to chromosomes-11and chromosome-19q13.3-19qter.HumGenet,1985,69:371–374
8Youssoufian H,Chance P,Tuckmuller C M,et al.Association of a new chromosomal deletion[DEL(1)(Q32Q42)]with diaphragmatic hernia:Assignment of a human ferritin gene.Hum Genet,1988,78:267–270
9Gasparini P,Calvano S,Memeo E,et al.Assignment of Ferritin L gene(FTL)to human chromosome band19q13.3by in situ hybridization.Ann Genet-Paris,1997,40:227–228
10Theil E C.Ferritin-structure,gene-regulation,and cellular function in animals,plants,and microoganisms.Annu Rev Biochem,1987,56:289–315
11Lawson D M,Treffry A,Artymiuk P J,et al.Identification of the ferroxidase centre in ferritin.Febs Lett,1989,254:207–210
12Schwarz J A,Brokstein P B,Voolstra C,et al.Coral life history and symbiosis:Functional genomic resources for two reef building Caribbeancorals,Acropora palmata and Montastraea faveolata.Bmc Genomics,2008,9:97
13De Zoysa M,Lee J.Two ferritin subunits from disk abalone(Haliotis discus discus):Cloning,characterization and expression analysis.FishShellfish Immun,2007,23:624–635
14Andrews S C,Harrison P M,Yewdall S J,et al.Structure,function,and evolution of ferritins.J Inorg Biochem,1992,47:161–174
15Orino K,Miura T,Muto S,et al.Sequence analysis of canine and equine ferritin H and L subunit cDNAs.DNA Sequence,2005,16:58–64
16Csaszar N B,Seneca F O,van Oppen M J H.Variation in antioxidant gene expression in the scleractinian coral Acropora millepora under la-boratory thermal stress.Mar Ecol-Prog Ser,2009,392:93–102
17Torti F M,Torti S V.Regulation of ferritin genes and protein.Blood,2002,99:3505–3516
18练健生,黄晖,黄良民,等.三亚珊瑚礁及其生物多样性.北京:海洋出版社,2010.18–24
19李淑,余克服,施祺,等.南海北部珊瑚共生虫黄藻密度的种间与空间差异及其对珊瑚礁白化的影响.科学通报,2007,52:2655–2662
20Clausen C D,Roth A A.Effect of temperature and temperature adaption on calcification rate in the hermatypic coral Pocillopora damicornis.Mar Biol,1975,33:93–100
21Cheney D P.The influence of temperature,does level and morphology on the uptake of3H-thmidine by reef corals.In:Proceedings of thesecond intemational symposium on coral reefs.Austrialia:the Great Barrier Reef Committee,1974,32:69–76
22Chomezynski P,Sacchi N.Single-step method of RNA isolation by acid guanidinium thiocyanate phenol chloroform extraction.Anal Bio-chem,1987,162:156–159
23杨婧,牛艳,张可炜.等.一种适用于RT-PCR的微藻总RNA提取方法.海洋科学,2010,34:6–10
24Desalvo M K,Voolstra C R,Sunagawa S,et al.Differential gene expression during thermal stress and bleaching in the Caribbean coral Mon-tastraea faveolata.Mol Ecol,2008,17:3952–3971
25Hofmann G E,Gaines S D.New tools to meet new challenges:Emerging technologies for managing marine ecosystems for resilience.Bio-science,2008,58:43–52
26Downs C A,Fauth J E,Robinson C E.Cellular diagnostics and coral health:Declining coral health in the Florida Keys.Mar Pollut Bull,2005,51:558–569
27Bustin S A,Benes V,Nolan T,et al.Quantitative real-time RT-PCR-a perspective.J Mol Endocrinol,2005,34:597–601
28Tricarico C,Pinzani P,Bianchi S,et al.Quantitative real-time reverse transcription polymerase chain reaction:Normalization to rRNA orsingle housekeeping genes is inappropriate for human tissue biopsies.Anal Biochem,2002,309:293–300
29Dheda K,Huggett J F,Chang J S,et al.The implications of using an inappropriate reference gene for real-time reverse transcription PCR datanormalization.Anal Biochem,2005,344:141–143
30Smith-Keune C,Dove S.Gene expression of a green fluorescent protein homolog as a host-specific biomarker of heat stress within areef-building coral.Mar Biotechnol,2008,10:166–180
31Fukuda I,Imagawa S,Iwao K,et al.Isolation of actin-encoding cDNAs from symbiotic corals.DNA Res,2002,9:217–223
32Venn A A,Quinn J,Jones R,et al.P-glycoprotein(multi-xenobiotic resistance)and heat shock protein gene expression in the reef coral Mon-tastraea franksi in response to environmental toxicants.Aquat Toxicol,2009,93:188–195