新疆雪莲全长cDNA文库构建及序列分析
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摘要
从新疆野生雪莲中提取总RNA，运用“SMART cDNA文库构建试剂盒”进行LD-PCR合成双链cDNA；产物经蛋白酶K消化，SfiI酶切，Chroma Spin-400分级分离，回收400bp以上片段。并与λTripLEx2载体连接，体外包装，产生未扩增文库，文库滴度为4.03×107 pfu/ml，重组率为93％。经扩增后滴度为1.75×109pfu/ml。随机挑选120个阳性菌落,碱裂解提取质粒DNA,经测序得到80个有效EST序列。GenBank网上进行BLAST同源性分析，有52个序列有同源,其中10个为未知功能蛋白；42个为已知功能蛋白。
The full-length cDNA library of Saussurea involucrata Kar.et was constructed by using SMART(switching mechanism at 5＇end of RNA transcript) technology .The total RNA was extracted and was generated full-length cDNA, The PCR product were digested by proteinase K and SfiI ，and fractionated with the Chroma spin -400 Column.The cDNAs of more than 400bp were collected and ligated intoλTripLEx2 vector. The λ-phage packaging reaction was used for the final construction of the cDNA library. The titer of unamplified cDNA library consisted of 4.03×107 independent clones with a recombinants rate of 93%.While the amplified library 1.75×109 pfu/ml. 120 positive clones were randomly selected for plasmid DNA extraction and sequencing. 80 EST sequences were obtained. After BLAST identity analysis, 42 genes of known function and 10 genes of unknown function were obtained.
The full-length cDNA library of Saussurea involucrata Kar.et was constructed by using SMART(switching mechanism at 5＇end of RNA transcript) technology .The total RNA was extracted and was generated full-length cDNA, The PCR product were digested by proteinase K and SfiI ，and fractionated with the Chroma spin -400 Column.The cDNAs of more than 400bp were collected and ligated intoλTripLEx2 vector. The λ-phage packaging reaction was used for the final construction of the cDNA library. The titer of unamplified cDNA library consisted of 4.03×107 independent clones with a recombinants rate of 93%.While the amplified library 1.75×109 pfu/ml. 120 positive clones were randomly selected for plasmid DNA extraction and sequencing. 80 EST sequences were obtained. After BLAST identity analysis, 42 genes of known function and 10 genes of unknown function were obtained.
引文
1.高健，许晓风，乌慧玲等.2004，特异种质烟草全长cDNA文库的构建及质量分析.安徽农业大学学报，31（1）：22-25.
2.韩新燕 许梓荣,中国兽医科技,2003,33(11).28-32.
3.廖乾生,林福呈,李德葆.2003,植物防御素及其研究进展.浙江大学学报,29(1):113-118.
4.卢圣栋.1999.现代分子生物学实验技术(第2版).北京:中国协和医科大学出版社,1-813.
5.罗静初，江涛等. 1998，分子生物信息镜像系统和数据库.高技术通讯， 10（10）：61-63，53.
6.彭秀玲，袁汉英等1998，基因工程实验技术（第2版）.长沙:湖南科学技术出版社,1-350.
7.邱咏梅，夏庆友.2002，EST规模测序在家蚕功能基因组中的应用.蚕学通讯，22（4）：16-19.
8.萨其拉,李文彬,孙勇如.2001,植物病程相关蛋白的基因表达调控.高技术通讯,1:100-103.
9.唐莲 ，刘振中， 蒋任飞，环境保护科学，2003,29(6).-33-36.
10.夏云，雷二庆等. 1997，Internet 实用技术与生物医学应用.北京：军事医学出版社，341-354.
11.徐丽霞、彭新君，湖南中医药导报，2002,8(4).-152-153
12.杨官品,沈怀舜等.2002,条斑紫菜丝状孢子体表达序列标签分析.高技术通讯2:93-97.
13.周晓馥，肖乃仲等.2002，植物功能基因组学的研究策略，中国生物工程杂志，22（6）：13-17.
14.Adams. MD, Kelley JM, Gocayne JD, et al. 1991, Complementary DNA sequencing : Expressed Sequence Tag s and Human Genome Project. Science, 252:11651-11656.
15.Anderson I and Brass A.1998,Searching DNA databases for similarities to DNA sequences:when is a match significant? Bioinformatics ,14(4):349-356
16.Arumuganathan, E. And Earle, 1991，E D Nuclear DNA content of some important plant species. Plant Mol. Biol Rep, 9:208-218.
17.benson DA，Lipman DJ，Ostell J，Wheeler DL.2003,GeneBank.Nucl Acids Res31(1):23-27.
18.Bevan M, Bancroft I, Bent E, Love K, Goodman H, Dean C, Bergkamp R, et al. 1998,Analysis of 1.9Mb of contiguous sequence from chromosome 4 of Arabidopsis thaliana.Nature, 391(6666):485-488.
19.Boguski MS, Lowe TMJ, Tolstoshev CM. 1993，dbEST-database for‘expressed sequence tags’.
NatGenel, 4:332-333.
20.Bouchez M.L et al. 1998, Plant Physiol. 86:607-614.
21.Cooke R，Raynal M，et al.1996,Further progress towards a catalogue of all Arabidopsis genes: analysis of a set of 5000 non-redundant ESTs.Plant J9(1):101-124.
22.Costanzo F, et al. 1983,Cloning of several cDNA segments coding rot human liver proteins. EMBO J, 2:57-60.
23.Crock J，Wildung M，Croteau R. 1997,Isolation and bacterial expression of a sesquiterpene synthase cDNA clone frome peppermint(Mentha x piperita,L.)that produces the aphid alarm pheromone(E)-beta-farnesene[J].Proc Natl Acad Sci. 94:12833-12838.
24.David J. Graves. 1999， Powerful tools for genetic analysis come of age. TIBTECH,17:127-134.
25.David J.Lockhart ＆ Elizabeth A. Winzeler. Genomics. 2000 ，gene expression and DNA arrays. Nature,405:827-836.
26.Devas K. Beales J et al.1999,Genome Res 9:825-829.
27.Epple P,Aple K, Bohlmann H.1997,ESTs reveal a multigene family for plant defensions in Arabidopsis thaliana. FEBS Lett 400(2):168-172.
28.Ewing RM, Kahla AB,Poirot O, et al. 1999, Large-scale statistical analysis of rice ESTs reveal correlated patterns of gene expression. Genome Research, 9:950-959.
29.Gale M.Hara C et al.1998,J Plant physiol 152:158-166.
30.Gelbart WM. 1998,Database in genetic research.Science, 282(5389):659-661.
31.Goff SA, Ricke D, Lan TH, et al. 2002, A draft sequence of the rice genome ( Oryza sativa L.ssp.japonica). Science, 296:92-100.
32.Hatey F, Tosser-Klopp G, Clouscard-martinato C, et al. 1998, Expressed sequence tags for genes: a review. Genet Sel Evol, 30(5):521-541.
33.Jeffrey L Bennetzen. 1999， Plant genomics takes root, branches out. TIG, 15:86-87.
34.Lawrence CW. 1991， Classical mutagenesis techniques Methods Enzymol,194:273-280.
35.Leipe D D .Genome and DNA sequence database. Curr Opin GenDevel,1996,6(6):686-691.
36.Lin XY, Kaul S,et al.1999, sequence and analysis of chromosome 2 of the plant Arabidopsis
thaliana.Nature 402(6763):761-768.
37.Luo J C，Jiang T，Li B，et al.Mirror system of molecular bioinformation and database. Advanced Technology Communciation,1998,10(10):61-63,53
38.Michalek W, Weschke W,Pleissner KP,et al.2002,EST analysis in barley defines a unigene set comprising 4000 genes. Theor Appl Genet 104(1):97-103.
39.Meinke, D.W.et al. 1991，Arabidopsis thaliana: A model plant spectes for genome analysis. Science 282,662-682.
40.Mekhedov S, Martinnez de llarduya O, Ohlrogge J. 2000, Towards a functional catalog of the plant genome : a survey of genes for lipid biosynthesis. Plant Physiol, 122:389-401.
41.Newman T，Bruijn FJD，Green P，Keegstra K，et al.1994,Genes galore: A summary of methods for accessing results from large-scale partial sequencing of anonymous Arabidopsis cDNA clones. Plant Physiol.106(4):1241-1255.
42.Ohlrogge J, Benning C. 2000, Unraveling plant metabolism by EST analysis. Current Opinion in Plant Biology, 3:224-228.
43.Okubo K, Hori N, Matoba R, et al. 1992,Large scale cDNA sequencing for analysis of quantitative and qualitative aspects of gene expression. Nature genetics, 2:173-179.
44.Pear JR, Kawagoe Y,Schreckengost WE, et al. 1996,Higher plants cotains homologs of the bacterial celA genes encoding the catalytic subunit of cellulose synthase[J].Proc Natl Acad Sci USA, 93:12637-12642.
45.Rounsley S, Xiaoying Lin, Karen A Ketchum. 1998,Large-scale sequencing of plant genomes. Current Opinion in Plant Biology, 1:136-141.
46.Schmide R.2000,Proc Natl Acad Sci USA 97:3789-3791.
47.Sequence Tags from Developing Seeds.2001, The Metabolic Pathway from Carbohydrates to Seed Oil. Plant Physiology, 124:1582-1594.
48.Shen B,Carneiro N,et al.1994,Partial sequencing and mapping of clones from two maize cDNA library. Plant Mol Biol 26(4):1085-1101.
49.Skinner W,Keon J,Hargreaves J.2001,Gene information for fungal plant pathogens from expressed sequences.Curr Opin Microbiol 4(4):381-386.
50.Steve Rounsley, Xiaoying Lin and Karen A Ketchum. 1998，Large-scale sequencing of plant
genomes. Current Opinion in Plant Biology,1:136-141.
51.Venter JC, Adams MD, Eugene WM, et al. 2001,The sequence of the Human Genome. Science, 291:1304-1352.
52.White JA White JA, Todd J, Newman T, et al. 2000,A new set of Arbidopsis Expressed Sasaki T, Benjamin Burr. International Rice Genome Sequencing Project:the effort to completely sequence the rice genome. Current Opinion in Plant Biology, 3:138-141.
53.Wu J, Yamamoto S, Machara T, et al. 2000,A comprehensive map of rice expressed sequence tags (ESTs):it’s characteristics and application to genome analysis. 6th International congress of Plant Molecular Biology, Supplement to reporter, 18:91-92.
54.Yamamoto K, Sasaki T. 1997, Large-scale EST sequencing in rice. Plant Mol Biol,35:135-144.Summarizes the progress to date in the rice EST sequencing program in Japan.
55.Yuan Q, Quackenbush J, Sultana R, Pertea M, Salzberg SL, Buell CR. 2001, Rice bioinformatics.Analysis of rice sequence data and leveraging the data to other plant specties. Plant Physiol , 125(3):1166-1174.
56.Van de Loo FJ, Turner S, Broun P, Somerville C. 1995, Expressed sequence tags from developing castor seeds. Plant Physiol, 108:1441-1150.
2.韩新燕 许梓荣,中国兽医科技,2003,33(11).28-32.
3.廖乾生,林福呈,李德葆.2003,植物防御素及其研究进展.浙江大学学报,29(1):113-118.
4.卢圣栋.1999.现代分子生物学实验技术(第2版).北京:中国协和医科大学出版社,1-813.
5.罗静初，江涛等. 1998，分子生物信息镜像系统和数据库.高技术通讯， 10（10）：61-63，53.
6.彭秀玲，袁汉英等1998，基因工程实验技术（第2版）.长沙:湖南科学技术出版社,1-350.
7.邱咏梅，夏庆友.2002，EST规模测序在家蚕功能基因组中的应用.蚕学通讯，22（4）：16-19.
8.萨其拉,李文彬,孙勇如.2001,植物病程相关蛋白的基因表达调控.高技术通讯,1:100-103.
9.唐莲 ，刘振中， 蒋任飞，环境保护科学，2003,29(6).-33-36.
10.夏云，雷二庆等. 1997，Internet 实用技术与生物医学应用.北京：军事医学出版社，341-354.
11.徐丽霞、彭新君，湖南中医药导报，2002,8(4).-152-153
12.杨官品,沈怀舜等.2002,条斑紫菜丝状孢子体表达序列标签分析.高技术通讯2:93-97.
13.周晓馥，肖乃仲等.2002，植物功能基因组学的研究策略，中国生物工程杂志，22（6）：13-17.
14.Adams. MD, Kelley JM, Gocayne JD, et al. 1991, Complementary DNA sequencing : Expressed Sequence Tag s and Human Genome Project. Science, 252:11651-11656.
15.Anderson I and Brass A.1998,Searching DNA databases for similarities to DNA sequences:when is a match significant? Bioinformatics ,14(4):349-356
16.Arumuganathan, E. And Earle, 1991，E D Nuclear DNA content of some important plant species. Plant Mol. Biol Rep, 9:208-218.
17.benson DA，Lipman DJ，Ostell J，Wheeler DL.2003,GeneBank.Nucl Acids Res31(1):23-27.
18.Bevan M, Bancroft I, Bent E, Love K, Goodman H, Dean C, Bergkamp R, et al. 1998,Analysis of 1.9Mb of contiguous sequence from chromosome 4 of Arabidopsis thaliana.Nature, 391(6666):485-488.
19.Boguski MS, Lowe TMJ, Tolstoshev CM. 1993，dbEST-database for‘expressed sequence tags’.
NatGenel, 4:332-333.
20.Bouchez M.L et al. 1998, Plant Physiol. 86:607-614.
21.Cooke R，Raynal M，et al.1996,Further progress towards a catalogue of all Arabidopsis genes: analysis of a set of 5000 non-redundant ESTs.Plant J9(1):101-124.
22.Costanzo F, et al. 1983,Cloning of several cDNA segments coding rot human liver proteins. EMBO J, 2:57-60.
23.Crock J，Wildung M，Croteau R. 1997,Isolation and bacterial expression of a sesquiterpene synthase cDNA clone frome peppermint(Mentha x piperita,L.)that produces the aphid alarm pheromone(E)-beta-farnesene[J].Proc Natl Acad Sci. 94:12833-12838.
24.David J. Graves. 1999， Powerful tools for genetic analysis come of age. TIBTECH,17:127-134.
25.David J.Lockhart ＆ Elizabeth A. Winzeler. Genomics. 2000 ，gene expression and DNA arrays. Nature,405:827-836.
26.Devas K. Beales J et al.1999,Genome Res 9:825-829.
27.Epple P,Aple K, Bohlmann H.1997,ESTs reveal a multigene family for plant defensions in Arabidopsis thaliana. FEBS Lett 400(2):168-172.
28.Ewing RM, Kahla AB,Poirot O, et al. 1999, Large-scale statistical analysis of rice ESTs reveal correlated patterns of gene expression. Genome Research, 9:950-959.
29.Gale M.Hara C et al.1998,J Plant physiol 152:158-166.
30.Gelbart WM. 1998,Database in genetic research.Science, 282(5389):659-661.
31.Goff SA, Ricke D, Lan TH, et al. 2002, A draft sequence of the rice genome ( Oryza sativa L.ssp.japonica). Science, 296:92-100.
32.Hatey F, Tosser-Klopp G, Clouscard-martinato C, et al. 1998, Expressed sequence tags for genes: a review. Genet Sel Evol, 30(5):521-541.
33.Jeffrey L Bennetzen. 1999， Plant genomics takes root, branches out. TIG, 15:86-87.
34.Lawrence CW. 1991， Classical mutagenesis techniques Methods Enzymol,194:273-280.
35.Leipe D D .Genome and DNA sequence database. Curr Opin GenDevel,1996,6(6):686-691.
36.Lin XY, Kaul S,et al.1999, sequence and analysis of chromosome 2 of the plant Arabidopsis
thaliana.Nature 402(6763):761-768.
37.Luo J C，Jiang T，Li B，et al.Mirror system of molecular bioinformation and database. Advanced Technology Communciation,1998,10(10):61-63,53
38.Michalek W, Weschke W,Pleissner KP,et al.2002,EST analysis in barley defines a unigene set comprising 4000 genes. Theor Appl Genet 104(1):97-103.
39.Meinke, D.W.et al. 1991，Arabidopsis thaliana: A model plant spectes for genome analysis. Science 282,662-682.
40.Mekhedov S, Martinnez de llarduya O, Ohlrogge J. 2000, Towards a functional catalog of the plant genome : a survey of genes for lipid biosynthesis. Plant Physiol, 122:389-401.
41.Newman T，Bruijn FJD，Green P，Keegstra K，et al.1994,Genes galore: A summary of methods for accessing results from large-scale partial sequencing of anonymous Arabidopsis cDNA clones. Plant Physiol.106(4):1241-1255.
42.Ohlrogge J, Benning C. 2000, Unraveling plant metabolism by EST analysis. Current Opinion in Plant Biology, 3:224-228.
43.Okubo K, Hori N, Matoba R, et al. 1992,Large scale cDNA sequencing for analysis of quantitative and qualitative aspects of gene expression. Nature genetics, 2:173-179.
44.Pear JR, Kawagoe Y,Schreckengost WE, et al. 1996,Higher plants cotains homologs of the bacterial celA genes encoding the catalytic subunit of cellulose synthase[J].Proc Natl Acad Sci USA, 93:12637-12642.
45.Rounsley S, Xiaoying Lin, Karen A Ketchum. 1998,Large-scale sequencing of plant genomes. Current Opinion in Plant Biology, 1:136-141.
46.Schmide R.2000,Proc Natl Acad Sci USA 97:3789-3791.
47.Sequence Tags from Developing Seeds.2001, The Metabolic Pathway from Carbohydrates to Seed Oil. Plant Physiology, 124:1582-1594.
48.Shen B,Carneiro N,et al.1994,Partial sequencing and mapping of clones from two maize cDNA library. Plant Mol Biol 26(4):1085-1101.
49.Skinner W,Keon J,Hargreaves J.2001,Gene information for fungal plant pathogens from expressed sequences.Curr Opin Microbiol 4(4):381-386.
50.Steve Rounsley, Xiaoying Lin and Karen A Ketchum. 1998，Large-scale sequencing of plant
genomes. Current Opinion in Plant Biology,1:136-141.
51.Venter JC, Adams MD, Eugene WM, et al. 2001,The sequence of the Human Genome. Science, 291:1304-1352.
52.White JA White JA, Todd J, Newman T, et al. 2000,A new set of Arbidopsis Expressed Sasaki T, Benjamin Burr. International Rice Genome Sequencing Project:the effort to completely sequence the rice genome. Current Opinion in Plant Biology, 3:138-141.
53.Wu J, Yamamoto S, Machara T, et al. 2000,A comprehensive map of rice expressed sequence tags (ESTs):it’s characteristics and application to genome analysis. 6th International congress of Plant Molecular Biology, Supplement to reporter, 18:91-92.
54.Yamamoto K, Sasaki T. 1997, Large-scale EST sequencing in rice. Plant Mol Biol,35:135-144.Summarizes the progress to date in the rice EST sequencing program in Japan.
55.Yuan Q, Quackenbush J, Sultana R, Pertea M, Salzberg SL, Buell CR. 2001, Rice bioinformatics.Analysis of rice sequence data and leveraging the data to other plant specties. Plant Physiol , 125(3):1166-1174.
56.Van de Loo FJ, Turner S, Broun P, Somerville C. 1995, Expressed sequence tags from developing castor seeds. Plant Physiol, 108:1441-1150.