小麦组蛋白基因的表达与其耐盐性
摘要
盐胁迫作用造成植物气孔关闭,光合降低,能耗增加,养分离子吸收不平衡,严重地损害植物质膜的正常功能。目前已从许多植物中分离了一些盐胁迫诱导的基因及基因上游序列,研究了其盐胁迫的信号传导途径及分子机理。有些耐盐基因已被成功转入植物中,提高了植物的耐盐性。
因H2A,H2B,H3,H4共同构成了真核生物染色体核小体的八聚体,已发现组蛋白具有多种翻译后修饰现象(如甲基化、乙酰化、磷酸化等),表明其在基因转录调控过程中可发挥重要的作用。邱生平等从盐胁迫处理的水稻幼苗组织中分离了一个新的水稻组蛋白H3基因RH3.2A,在高盐和ABA胁迫下的表达,研究发现在水稻根部RH3.2A基因受高盐的强烈诱导,而在叶片RH3.2A基因的表达则不受高盐诱导,此外RH3.2A基因也受外源ABA的诱导,可能参与了依赖于ABA的高盐胁迫应答反应。
为了进一步了解山融三号耐盐小麦在盐胁迫条件下的基因表达的规律,本实验提取了普通小麦盐胁迫下组织的总RNA,经反转录荧光标记制备cDNA探针,并将其与小麦基因芯片进行杂交,扫描采集数据后并进行分析。根据芯片杂交结果,小麦在盐胁迫下其组蛋白基因的表达具有共同下调的现象,但不同组蛋白基因的表达程度差异很大,甚至有个别组蛋白基因发生上调。在济南177中,有22条组蛋白在叶中上调而有6条组蛋白在叶中下调,有9条组蛋白在根中上调而有124条组蛋白在根中下调。在山融三号中有5条组蛋白在叶中上调而有122条组蛋白在叶中下调,有37条组蛋白在根中上调而有20条组蛋白在根中下调。为了证实小麦芯片杂交结果的正确性,我们采用了real-time PCR对芯片中表达特异的(5个组蛋白基因)组蛋白进一步分析,结果证明大多数组蛋白基因的表达趋势与芯片结果基本相符。最后,我们选择了其中Ⅰ-H2B基因转入拟南芥中,目前获得了TO代,为进一步的验证在耐盐中的功能奠定基础。
Salt stress induces stoma-closing, photosynthesis-decreasing,over energy-consuming and unbalance of inorganic ion absorbing and damages the functions of plasma membrane.Now many salt-inducible genes and upstream sequences have been cloned, and there was certainly progress on Molecular mechanism of plant biotic and abiotic stress signaling. Some salt-inducible genes have been transformed into plants, and it was evidenced that exogenous gene can enforce the ability to tolerate salt stress.
The nucleosome consists of an octamer of two copies of the four core histones H2A, H2B, H3 and H4 in eukaryotes;many histone post-translational modifications can serve a distinct function in transcriptonal regulation (e.g.methylation,acetylation,phosp-horylation),and that indicated they could play a key role on the molecular mechanisms of salt-stress tolerance. RH3.2A was cloned through RT-PCR from salt-treated rice seedlings by Qiu Sheng Ping.The mRNA expression analysis of RH3.2A revealed that RH3.2A gene was upregulated by salt stress in rice roots and ABA treatment in seedlings. The potential role of RH3.2A during salt stress was discussed.
For investigating the Shan Rong 3 wheat (salt-tolerant wheat) transcriptional profiling salt stress,RNA samples were collected at different hours mixly influence dye-labeled complimentary DNA were used to hybridize with the whole genome wheat gene chip. Data analysis indicated that most of the histone genes was down-regulated simultaneously but interestingly,there were great changes of different expressed histone genes,even individual histone genes were up-regulated. In Jinan 177,22 histone genes were upregulated by salt stress in leaves and 9 histone genes were downregulated by salt stress in leaves; 9 histone genes were upregulated by salt stress in roots and 124 histone genes were downregulated by salt stress in roots.In Shan rong 3,5 histone genes were upregulated by salt stress in leaves and 122 histone genes were downregulated by salt stress in leaves; 37 histone genes were upregulated by salt stress in roots and 20 histone genes were downregulated by salt stress in roots. This were further verified to be true by Real Time PCR. Generally speaking,Real Time PCR analysis indicated that five cloned histone genes expressed the same as gene chip's data.At last we have chosen one of them named I-H2B to Transform of Arabidopsis.Now,we have TO seeds for investigating its function in plant.
因H2A,H2B,H3,H4共同构成了真核生物染色体核小体的八聚体,已发现组蛋白具有多种翻译后修饰现象(如甲基化、乙酰化、磷酸化等),表明其在基因转录调控过程中可发挥重要的作用。邱生平等从盐胁迫处理的水稻幼苗组织中分离了一个新的水稻组蛋白H3基因RH3.2A,在高盐和ABA胁迫下的表达,研究发现在水稻根部RH3.2A基因受高盐的强烈诱导,而在叶片RH3.2A基因的表达则不受高盐诱导,此外RH3.2A基因也受外源ABA的诱导,可能参与了依赖于ABA的高盐胁迫应答反应。
为了进一步了解山融三号耐盐小麦在盐胁迫条件下的基因表达的规律,本实验提取了普通小麦盐胁迫下组织的总RNA,经反转录荧光标记制备cDNA探针,并将其与小麦基因芯片进行杂交,扫描采集数据后并进行分析。根据芯片杂交结果,小麦在盐胁迫下其组蛋白基因的表达具有共同下调的现象,但不同组蛋白基因的表达程度差异很大,甚至有个别组蛋白基因发生上调。在济南177中,有22条组蛋白在叶中上调而有6条组蛋白在叶中下调,有9条组蛋白在根中上调而有124条组蛋白在根中下调。在山融三号中有5条组蛋白在叶中上调而有122条组蛋白在叶中下调,有37条组蛋白在根中上调而有20条组蛋白在根中下调。为了证实小麦芯片杂交结果的正确性,我们采用了real-time PCR对芯片中表达特异的(5个组蛋白基因)组蛋白进一步分析,结果证明大多数组蛋白基因的表达趋势与芯片结果基本相符。最后,我们选择了其中Ⅰ-H2B基因转入拟南芥中,目前获得了TO代,为进一步的验证在耐盐中的功能奠定基础。
Salt stress induces stoma-closing, photosynthesis-decreasing,over energy-consuming and unbalance of inorganic ion absorbing and damages the functions of plasma membrane.Now many salt-inducible genes and upstream sequences have been cloned, and there was certainly progress on Molecular mechanism of plant biotic and abiotic stress signaling. Some salt-inducible genes have been transformed into plants, and it was evidenced that exogenous gene can enforce the ability to tolerate salt stress.
The nucleosome consists of an octamer of two copies of the four core histones H2A, H2B, H3 and H4 in eukaryotes;many histone post-translational modifications can serve a distinct function in transcriptonal regulation (e.g.methylation,acetylation,phosp-horylation),and that indicated they could play a key role on the molecular mechanisms of salt-stress tolerance. RH3.2A was cloned through RT-PCR from salt-treated rice seedlings by Qiu Sheng Ping.The mRNA expression analysis of RH3.2A revealed that RH3.2A gene was upregulated by salt stress in rice roots and ABA treatment in seedlings. The potential role of RH3.2A during salt stress was discussed.
For investigating the Shan Rong 3 wheat (salt-tolerant wheat) transcriptional profiling salt stress,RNA samples were collected at different hours mixly influence dye-labeled complimentary DNA were used to hybridize with the whole genome wheat gene chip. Data analysis indicated that most of the histone genes was down-regulated simultaneously but interestingly,there were great changes of different expressed histone genes,even individual histone genes were up-regulated. In Jinan 177,22 histone genes were upregulated by salt stress in leaves and 9 histone genes were downregulated by salt stress in leaves; 9 histone genes were upregulated by salt stress in roots and 124 histone genes were downregulated by salt stress in roots.In Shan rong 3,5 histone genes were upregulated by salt stress in leaves and 122 histone genes were downregulated by salt stress in leaves; 37 histone genes were upregulated by salt stress in roots and 20 histone genes were downregulated by salt stress in roots. This were further verified to be true by Real Time PCR. Generally speaking,Real Time PCR analysis indicated that five cloned histone genes expressed the same as gene chip's data.At last we have chosen one of them named I-H2B to Transform of Arabidopsis.Now,we have TO seeds for investigating its function in plant.
引文
1.(美)J.萨姆布鲁克,D.W.拉塞尔著.分子克隆实验指南.科学出版社,2002
2.(美)N.C.琼斯,P A.帕夫纳著.生物信息学算法导论.化学工业出版社,2007
3.Alizadeh Ash A.,Michael B.Eisen,R.Eric Davis.Distinct types of diffuse large B-cell lymphoma identified by gene expression profiling.Nature 2000,403,503-511
4.Baer,Rhodes.Eukaryotic RNA polymerase Ⅱ binds to nucleosome cores from transcribed genes.Nature 1983,301,482-488
5.Bittner M,Meltzer RChen Y,el al.Molecular classification of cutaneous malignant melanoma by gene expression profiling.Nature,2000,406(6795):536-40
6.Brian D.Strahl,C.David Allis.The language of covalent histone modifications.Nature,2000,403,41-45
7.Chinnusamy V and Zhu.ICEI:a regulator of cold-induced transcriptome and freezing tolerance in Arabidopsis.Genes & Development,2003,Vol.17,1043-1054
8.Dan Wells and Celeste McBride.A comprehensive compilation and alignment of histones and histone genes.Nucleic Acids Res.1989,17:311-346
9.Dou Y,Gorovsky MA.Phosphorylation of linker histone Hl regulates gene expression in vivo by creating a charge patch.Moll Cell,2000,116:225-231
10.Gomez-Gadenas A,Verhey SD,Holappa LD,et al.An abscisic acid-induced protein kinase,PKABAl,mediates abscisic acid-suppressed gene expression in barley aleurone layers.Plant Biology,1999,Vol.96,1767-1772
11 Hayes JJ,Clark DJ,Wolfe AP.1991b.Histone contributions to the structure of DNA in the nucleosome.Proc Natl AcadS ci USA,1991,88:6829-6833
12.Hayes JJ,Bashkin J,Tullius TD,Wolfe AP 1991a.The histone core exerts a dominant constraint on the structure of DNA in a nucl eosome.Biochemistry,1991,30:8434-8440.
13 Hayes JJ,Tullius TD,Wolfe AP.The structure of DNA in a nucleosome.Proc Nall AcadSci USA,1990,87:7405-7409
14. Hyung-in Choi, Jung-hee Hong, Jin-ok Ha,el al. ABFs,a Family of ABA-responsive Element Binding Factors. J Biol Chem,2000, Vol.275, 1723-1730
15. Jae H,Ko,Seung H,Yang,Kyung H,Han. Upregulation of an Arabidopsis RING-H2 gene, XERICO, confers drought tolerance through increased abscisic acid biosynthesis.Plant Journal,2006, 47(3):343-55
16. Jose R.Dinneny, TerriA.Long,Jean Y.WangCell. Identity Mediates the Response of Arabidopsis Roots to Abiotic Stress. Science,2008,942,320
17. Joachim,Kilian,Dion W. The AtGenExpress global stress expression data set: protocols, evaluation and model data analysis of UV-B light, drought and cold stress respons.Plant Journal,2007, 50(2):347-63
18. Reeck GR,Swanson E,Teller DC. The evolution of histones.J Mol Evolve,1978, 10(4):30 9-17
19. Rosario GM,Jose ML,Jose MB,et al. The Arabidopsis HAL2-like gene family includes a novel sodium-sensitive phosphatase. Plant Journal, 1999, Vol.17 373-383
20. Spellman Paul T,Gavin Sherlock,Michael Q. Zhang. Comprehensive Identification of Cell Cycle-regulated Genes of the Yeast Saccharomyces cerevisiae by Microarray Hybridization.Mol Biol Cell,1998,9(12): 3273-3297
21. Tavazoie Saeed,Jason D.Hughes,Michael J.Campbell. Systematic determination of genetic network architecture.Nature Genetics, 1999,22, 281 - 285
22. Thomas J,Allis CD. Translating the histone code.Epigenetics,2001, 293:1074-1080
23. Thomas J,Allis CD. Translating the histone code.Epigenetics,2001,Vol. 293
24. Tokunori H,Mihoko A,Yasuo K,et al. ACGT-containing abscisic acid response element(ABRE) and coupling element 3(CE3) are functionally equivalent. Plant Journal, 1999,19(6),679-689
25. Tokunori H,Mihoko A,Yasuo K,et al. ACGT containing abscisic acid response element and coupling elememnt 3 are functionally equiva -lent.PlantJournal,1999,19
26.Urao T,Katagiri T.Two genes that encode Ca2' dependent protein kinase are induced by drought and high salt stress in Arabidopsis theliana.MolGenet,1994,244
27.Wells D,Brown D.Histone,and histone gene compilation and alignment update.NucleicAcidsRes,1991,19:2173-2188
28.Wells D,McBride C.A comprehensive compilation and alignment of histones and histone genes.Nucleic Acids Res,1989,17 Suppl:311-346
29.Wuilmart C,Wyns L.An evolutionaty scheme for the histones as derived from a study of internal repetitions and homologies among the different classes.J Theor Bio1,1977,65(2):231-252
30.Wuilmart C,Wyns L.An evolutionaty scheme for the histones as derived from a study of internal repetitions and homologies among the different classes.J Theor Biol,1977,65(2):231-52
31.Ye W.,Heidemann,Estrin.An energy-efficient MAC protocol for wireless sensor networks.INFOCOM2002,Vol.3,1567-1576
32.Zhang MQ.Promoter analysis of co-regulated genes in the yeast genome.Comput Chem,1999,23(3-4):233-50
33.Zhu JK.Genetic analysis of salt tolerance in Arabidopsis thaliana:evidence of a critical role for potassium nutrition.PlantCell,1998,Vol.10
34.Zhu JK,Hans JB,et al.Plant cellular and molecular responses to high salinity.Plant Mol Bio,2000,Vol.51:463-499
35.Zhu JK.Salt and drought stress signal transduction in plants.Annu Rev Plant Biol,2002,Vol.53:247-273
36.Zhu JK.Genetic analysis of plant salt tolerance using Arabidopsis.Plant Physiol,2000,Vol.124
37.李瑶主编.基因芯片技术.化学工业出版社,2004
38.刘强,张贵友,陈受宜.植物转录因子的结构与调控作用科学通报,2000.45:1465-1474
39.刘祖洞,江绍慧编.遗传学.人民教育出版社,1982
40.邱生平,黄骥等.水稻H3.2型组蛋白基因RH3.2A的克隆与盐胁迫下的表达分析.遗传学报,2006,33(9):833-840
41.孙啸,陆祖宏,谢建明编著.生物信息学基础.清华大学出版社,2005
42.孙啸,谢建明,周庆等编著.R语言及Bioconductor在基因组分析中的应用科学出版社,2006
43.王希庆,陈柏君,印莉萍.植物中的MYB转录因子.生物技术通报,2003,2003,22.25
44.王亚馥,戴灼华主编.遗传学.高等教育出版社,1999
45.吴耀荣,谢旗.ABA与植物胁迫抗性.植物学通报,2006,23(5):511-518
46.薛哲勇,支大英,夏光敏等.根癌农杆菌介导AtNHXl基因转化小麦.山东大学学报,2003,38(1):106-109
47.杨洪强,梁小娥.蛋白激酶与植物逆境信号转导.植物生理学通讯,2001年03期
48.张劲松,谢灿,吴晓雪等.烟草两组分信号系统基因NTHK2.科学通报,2000,,2000,45(20):2190-2195
49.赵可夫,范海编著.盐生植物及其对盐渍生境的适应生理.科学出版社,2005
50.赵可夫,范海,Harris PJC等.盐胁迫下外源ABA对玉米幼苗耐盐性的影响.植物学报,1995,37(4):295-300
51.赵可夫,李法曾.中国盐生植物.科学出版社,1999
2.(美)N.C.琼斯,P A.帕夫纳著.生物信息学算法导论.化学工业出版社,2007
3.Alizadeh Ash A.,Michael B.Eisen,R.Eric Davis.Distinct types of diffuse large B-cell lymphoma identified by gene expression profiling.Nature 2000,403,503-511
4.Baer,Rhodes.Eukaryotic RNA polymerase Ⅱ binds to nucleosome cores from transcribed genes.Nature 1983,301,482-488
5.Bittner M,Meltzer RChen Y,el al.Molecular classification of cutaneous malignant melanoma by gene expression profiling.Nature,2000,406(6795):536-40
6.Brian D.Strahl,C.David Allis.The language of covalent histone modifications.Nature,2000,403,41-45
7.Chinnusamy V and Zhu.ICEI:a regulator of cold-induced transcriptome and freezing tolerance in Arabidopsis.Genes & Development,2003,Vol.17,1043-1054
8.Dan Wells and Celeste McBride.A comprehensive compilation and alignment of histones and histone genes.Nucleic Acids Res.1989,17:311-346
9.Dou Y,Gorovsky MA.Phosphorylation of linker histone Hl regulates gene expression in vivo by creating a charge patch.Moll Cell,2000,116:225-231
10.Gomez-Gadenas A,Verhey SD,Holappa LD,et al.An abscisic acid-induced protein kinase,PKABAl,mediates abscisic acid-suppressed gene expression in barley aleurone layers.Plant Biology,1999,Vol.96,1767-1772
11 Hayes JJ,Clark DJ,Wolfe AP.1991b.Histone contributions to the structure of DNA in the nucleosome.Proc Natl AcadS ci USA,1991,88:6829-6833
12.Hayes JJ,Bashkin J,Tullius TD,Wolfe AP 1991a.The histone core exerts a dominant constraint on the structure of DNA in a nucl eosome.Biochemistry,1991,30:8434-8440.
13 Hayes JJ,Tullius TD,Wolfe AP.The structure of DNA in a nucleosome.Proc Nall AcadSci USA,1990,87:7405-7409
14. Hyung-in Choi, Jung-hee Hong, Jin-ok Ha,el al. ABFs,a Family of ABA-responsive Element Binding Factors. J Biol Chem,2000, Vol.275, 1723-1730
15. Jae H,Ko,Seung H,Yang,Kyung H,Han. Upregulation of an Arabidopsis RING-H2 gene, XERICO, confers drought tolerance through increased abscisic acid biosynthesis.Plant Journal,2006, 47(3):343-55
16. Jose R.Dinneny, TerriA.Long,Jean Y.WangCell. Identity Mediates the Response of Arabidopsis Roots to Abiotic Stress. Science,2008,942,320
17. Joachim,Kilian,Dion W. The AtGenExpress global stress expression data set: protocols, evaluation and model data analysis of UV-B light, drought and cold stress respons.Plant Journal,2007, 50(2):347-63
18. Reeck GR,Swanson E,Teller DC. The evolution of histones.J Mol Evolve,1978, 10(4):30 9-17
19. Rosario GM,Jose ML,Jose MB,et al. The Arabidopsis HAL2-like gene family includes a novel sodium-sensitive phosphatase. Plant Journal, 1999, Vol.17 373-383
20. Spellman Paul T,Gavin Sherlock,Michael Q. Zhang. Comprehensive Identification of Cell Cycle-regulated Genes of the Yeast Saccharomyces cerevisiae by Microarray Hybridization.Mol Biol Cell,1998,9(12): 3273-3297
21. Tavazoie Saeed,Jason D.Hughes,Michael J.Campbell. Systematic determination of genetic network architecture.Nature Genetics, 1999,22, 281 - 285
22. Thomas J,Allis CD. Translating the histone code.Epigenetics,2001, 293:1074-1080
23. Thomas J,Allis CD. Translating the histone code.Epigenetics,2001,Vol. 293
24. Tokunori H,Mihoko A,Yasuo K,et al. ACGT-containing abscisic acid response element(ABRE) and coupling element 3(CE3) are functionally equivalent. Plant Journal, 1999,19(6),679-689
25. Tokunori H,Mihoko A,Yasuo K,et al. ACGT containing abscisic acid response element and coupling elememnt 3 are functionally equiva -lent.PlantJournal,1999,19
26.Urao T,Katagiri T.Two genes that encode Ca2' dependent protein kinase are induced by drought and high salt stress in Arabidopsis theliana.MolGenet,1994,244
27.Wells D,Brown D.Histone,and histone gene compilation and alignment update.NucleicAcidsRes,1991,19:2173-2188
28.Wells D,McBride C.A comprehensive compilation and alignment of histones and histone genes.Nucleic Acids Res,1989,17 Suppl:311-346
29.Wuilmart C,Wyns L.An evolutionaty scheme for the histones as derived from a study of internal repetitions and homologies among the different classes.J Theor Bio1,1977,65(2):231-252
30.Wuilmart C,Wyns L.An evolutionaty scheme for the histones as derived from a study of internal repetitions and homologies among the different classes.J Theor Biol,1977,65(2):231-52
31.Ye W.,Heidemann,Estrin.An energy-efficient MAC protocol for wireless sensor networks.INFOCOM2002,Vol.3,1567-1576
32.Zhang MQ.Promoter analysis of co-regulated genes in the yeast genome.Comput Chem,1999,23(3-4):233-50
33.Zhu JK.Genetic analysis of salt tolerance in Arabidopsis thaliana:evidence of a critical role for potassium nutrition.PlantCell,1998,Vol.10
34.Zhu JK,Hans JB,et al.Plant cellular and molecular responses to high salinity.Plant Mol Bio,2000,Vol.51:463-499
35.Zhu JK.Salt and drought stress signal transduction in plants.Annu Rev Plant Biol,2002,Vol.53:247-273
36.Zhu JK.Genetic analysis of plant salt tolerance using Arabidopsis.Plant Physiol,2000,Vol.124
37.李瑶主编.基因芯片技术.化学工业出版社,2004
38.刘强,张贵友,陈受宜.植物转录因子的结构与调控作用科学通报,2000.45:1465-1474
39.刘祖洞,江绍慧编.遗传学.人民教育出版社,1982
40.邱生平,黄骥等.水稻H3.2型组蛋白基因RH3.2A的克隆与盐胁迫下的表达分析.遗传学报,2006,33(9):833-840
41.孙啸,陆祖宏,谢建明编著.生物信息学基础.清华大学出版社,2005
42.孙啸,谢建明,周庆等编著.R语言及Bioconductor在基因组分析中的应用科学出版社,2006
43.王希庆,陈柏君,印莉萍.植物中的MYB转录因子.生物技术通报,2003,2003,22.25
44.王亚馥,戴灼华主编.遗传学.高等教育出版社,1999
45.吴耀荣,谢旗.ABA与植物胁迫抗性.植物学通报,2006,23(5):511-518
46.薛哲勇,支大英,夏光敏等.根癌农杆菌介导AtNHXl基因转化小麦.山东大学学报,2003,38(1):106-109
47.杨洪强,梁小娥.蛋白激酶与植物逆境信号转导.植物生理学通讯,2001年03期
48.张劲松,谢灿,吴晓雪等.烟草两组分信号系统基因NTHK2.科学通报,2000,,2000,45(20):2190-2195
49.赵可夫,范海编著.盐生植物及其对盐渍生境的适应生理.科学出版社,2005
50.赵可夫,范海,Harris PJC等.盐胁迫下外源ABA对玉米幼苗耐盐性的影响.植物学报,1995,37(4):295-300
51.赵可夫,李法曾.中国盐生植物.科学出版社,1999