秋茄KcBURP基因及KcC2H2型锌指蛋白基因的克隆及功能分析
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摘要
秋茄(Kandelia candel(L.)Druce)是红树的一种,属于非泌盐型红树,长期生活在海边,具有很强的耐盐性。前期研究显示,秋茄苗木KcBURP基因与KcC2H2型锌指蛋白基因在盐胁迫下表达明显上调,提示这两个基因可能对秋茄耐盐性有所贡献。
本文以秋茄为材料,利用前期Microarry分析所获得的KcBURP及KcC2H2型锌指蛋白基因的cDNA序列设计引物,通过RT-PCR,获得这两个基因cDNA的开放读码框(ORF)。序列分析结果显示KcBURP基因的ORF长1131bp,编码一个等电点为9.07、分子量为39.8kD、由375个氨基酸组成的多肽蛋白。KcC2H2型锌指蛋白基因ORF长738bp,由245氨基酸组成,蛋白分子量约26.1kD,理论等电点为8.65。
将秋茄KcBURP基因构建到植物表达载pCAMBIA-2300中,转化烟草。选取10个卡那霉素抗性株系进行基因组PCR鉴定,数据表明其中的8个株系为转基因株系。PT-PCR的结果显示在2个转基因株系中KcBURP得到过表达。150 mM NaCl处理后,野生型明显萎蔫,过表达转基因植株长势良好。进一步对野生型和转基因烟草进行光合作用测定,结果显示100mM盐处理显著降低了野生型烟草的净光合速率,而对转基因植株叶片的光合作用影响较小。盐浓度达到200mM时,转基因植株及野生型烟草净光合速率都明显降低,盐胁迫解除后,转基因烟草光合作用的恢复情况明显好于野生型烟草。说明KcBURP的过表达提高了烟草的抗盐性。另外,将KcC2H2型锌指蛋白基因全长cDNA构建到pK7WG2D上,转化烟草,选取8个株系进行基因组PCR鉴定,初步鉴定获得转基因植株。
Kandelia candel is one of salt-tolerant mangrove species and habitats in seaside area. Preliminary studies have shown that KcBURP and KcC2H2 zinc finger gene of Kandelia candel seedlings were significantly up-regulated under salt stress, suggesting that they contributed to salt tolerance of Kandelia candel.
In the presented study, the cDNA coding for the open reading frames (ORF) of KcBURP and KcC2H2 zinc finger genes of Kandelia candel were obtained by a PCR approach using the primers designed from the sequence information of Microarry cDNA. Sequencing analysis indicate that the ORF of KcBURP has a size of 1131bp, encoding for a protein of 375 amino acid with an isoelectric point of 9.07 and a molecular weight of 39886 Da.The ORF of KcC2H2 zinc finger gene has a size of 738bp, encoding for a protein of 245 amino acid with an isoelectric point of 8.65 and a molecular weight of 26174Da.
The gene construct of the KcBURP under the control of constitutive promoter pCAMBIA-2300 was transformed into tobacco. Ten individual KcBRP-transformed tobacco plants were identified by PCR. The PCR result show that eight of ten plants were KcBURP-positive. While there were only two plants showed over expression when the KcBURP-transformed tobacco plants were tested by RT-PCR. Salt tolerance examinations show that the KcBURP-transformed tobacco plants grew better than the wildtype under 150 mM NaCl. Furthermore, net photosynthetic rate of KcB URP-transformed tobacco plants were higher than the wildtype under 100 and 200 mM of NaCl saline, although the leaf photosynthesis was reduced by salt treatment. It is noting that the photosynthetic rate of KcBURP-transformed tobacco plants recovered rapidly than the'wildtype after the salt relief. Our result suggest that KcBURP gene is beneficial for plant salt tolerance. The gene construct of KcC2H2 the under the control of constitutive promoter pK7WG2D was transformed into tobacco. We identified seven KcC2H2-transformed tobacco plants by means of PCR test.
本文以秋茄为材料,利用前期Microarry分析所获得的KcBURP及KcC2H2型锌指蛋白基因的cDNA序列设计引物,通过RT-PCR,获得这两个基因cDNA的开放读码框(ORF)。序列分析结果显示KcBURP基因的ORF长1131bp,编码一个等电点为9.07、分子量为39.8kD、由375个氨基酸组成的多肽蛋白。KcC2H2型锌指蛋白基因ORF长738bp,由245氨基酸组成,蛋白分子量约26.1kD,理论等电点为8.65。
将秋茄KcBURP基因构建到植物表达载pCAMBIA-2300中,转化烟草。选取10个卡那霉素抗性株系进行基因组PCR鉴定,数据表明其中的8个株系为转基因株系。PT-PCR的结果显示在2个转基因株系中KcBURP得到过表达。150 mM NaCl处理后,野生型明显萎蔫,过表达转基因植株长势良好。进一步对野生型和转基因烟草进行光合作用测定,结果显示100mM盐处理显著降低了野生型烟草的净光合速率,而对转基因植株叶片的光合作用影响较小。盐浓度达到200mM时,转基因植株及野生型烟草净光合速率都明显降低,盐胁迫解除后,转基因烟草光合作用的恢复情况明显好于野生型烟草。说明KcBURP的过表达提高了烟草的抗盐性。另外,将KcC2H2型锌指蛋白基因全长cDNA构建到pK7WG2D上,转化烟草,选取8个株系进行基因组PCR鉴定,初步鉴定获得转基因植株。
Kandelia candel is one of salt-tolerant mangrove species and habitats in seaside area. Preliminary studies have shown that KcBURP and KcC2H2 zinc finger gene of Kandelia candel seedlings were significantly up-regulated under salt stress, suggesting that they contributed to salt tolerance of Kandelia candel.
In the presented study, the cDNA coding for the open reading frames (ORF) of KcBURP and KcC2H2 zinc finger genes of Kandelia candel were obtained by a PCR approach using the primers designed from the sequence information of Microarry cDNA. Sequencing analysis indicate that the ORF of KcBURP has a size of 1131bp, encoding for a protein of 375 amino acid with an isoelectric point of 9.07 and a molecular weight of 39886 Da.The ORF of KcC2H2 zinc finger gene has a size of 738bp, encoding for a protein of 245 amino acid with an isoelectric point of 8.65 and a molecular weight of 26174Da.
The gene construct of the KcBURP under the control of constitutive promoter pCAMBIA-2300 was transformed into tobacco. Ten individual KcBRP-transformed tobacco plants were identified by PCR. The PCR result show that eight of ten plants were KcBURP-positive. While there were only two plants showed over expression when the KcBURP-transformed tobacco plants were tested by RT-PCR. Salt tolerance examinations show that the KcBURP-transformed tobacco plants grew better than the wildtype under 150 mM NaCl. Furthermore, net photosynthetic rate of KcB URP-transformed tobacco plants were higher than the wildtype under 100 and 200 mM of NaCl saline, although the leaf photosynthesis was reduced by salt treatment. It is noting that the photosynthetic rate of KcBURP-transformed tobacco plants recovered rapidly than the'wildtype after the salt relief. Our result suggest that KcBURP gene is beneficial for plant salt tolerance. The gene construct of KcC2H2 the under the control of constitutive promoter pK7WG2D was transformed into tobacco. We identified seven KcC2H2-transformed tobacco plants by means of PCR test.
引文
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[2]侯佩臣.高盐胁迫下红树植物-秋茄的表达谱微阵列分析及差异表达基因的功能解析.(博士论文),2010
[3]廖宝文,郑松发,陈玉军等.红树林湿地恢复技术的研究进展[J].生态科学,2005,24(1)：61-65
[4]林鹏.中国红树林生态系[M].北京：科学出版社,1997
[5]唐玉林,李小杰,钟昀廷,等.利用酵母表达体系研究SALI3-2蛋白功能[J].深圳大学学(理工版)2007,24(3)：324-330
[6]余玉雯,孙海丹,郑易之,等.大豆耐盐相关基因的分离及功能鉴定[J].深圳大学学报(理工版)2004,21(4)：324-330
[7]申艳红,陈晓静.改良热硼酸小量法提取番木瓜果肉总RNA[J].中国南方果树2009,38(2)：7-8
[8]郑磊,刘关君,杨传平,等.西伯利亚蓼rd22基因的克隆与序列分析[J].植物研究,2007,(2)：212-217
[9]Banzai T, Sumiya K, Hanagata N et al. Molecular cloning and characterization of genes encoding BURP domain-containing protein in the mangrove, Bruguiera gymnorrhiza[J]. Trees-Structure and Function,2002,16(2-3):87-93
[10]Basmati L.Confers abiotic stress tolerance[J].Transgenic Research,2008,17:281-291
[11]Bassuener R,Baeumlein H,Huth A,et al.Abundant embryonic mRNA in field bean (Vicia faba L.) codes for a new class of seed proteins:cDNA cloning characterization of the primary translation product[J].Plant Mol Biol,1988,11(3):321-334
[12]Batchelor AK,Boutilier K,Miller SS,at el.SCBl,a BURP-domain protein gene,from developing soybean seed coats[J]. Planta,2002,215:523-532
[13]Benjamin L.Gene Ⅶ.OxfordUniversityPress.20 (X):654-656.
[14]Bernard P,Couturier M.Cell killing by the F plasmid CcdB protein involves poisoning of DNA-topoisomerase Ⅱ complexes[J].Molecular Biology,1992,226 (3):735-745
[15]Bowman JL,Sakai H and Jack T.SUPERMAN a regulator of floral homeotic genes in Arabidopsis [J].Development.1992,114:599-615
[16]Bushman W,Thompson JF,Vargas L,et al.Control of directionality in lambda site specific recom-bination[J].Science,1985,230(4278):906-911
[17]Camal A,Mica E and Gambino G.Cloning and characterization of small non-coding RNAs from grape[J]. The Plant Journal,2009,59:750-763
[18]Dathan N,Zaccacro L and Esposito S,The arabidopsis SUPERMAN protein is able to specifically bind DNA through its single Cys2-His2 zinc finger motif[J].Nucleic acids Research,2000,30 (22): 4945-49511
[19]Datta N,Lafayette PR,Kroner PA,et al.Isolation and characterization of three families of auxin down-regulated cDNA clones[J].Plant Mol Biol,1993,21 (5):859-169
[20]Doyle JJ and Doyle JL.A rapid DNA isolation procedure for small quantities of fresh leaf tissue [J]. Phytochemical Bulletin,1987,19 (1):11-151
[21]Ezawa S,Tada Y.Identification of salt tolerance genes from the mangrove plant Bruguiera gymnor-
hiza using Agrobacterium functional screening[J]. Plant Science,2009,176 (2):272-278
[22]Hanana M, Deluc L and Fouquet R.Identification and characterization of 'rd22' dehydration responsive gene in grapevine (Vitis vinifera L.). C. R[J]. Biologies,2008,331 (3):569-578
[23]Hattori J,Boutilier KA,van MM,et al.A conserved BURP domain defines a novel group of plant proteins with unusual primary structures[J].Mol Gen Genet,1998,259 (4):424-428
[24]Huang J,Zhang HS and CAO YJ.Cloning and sequencing analysis of a novel C2H2 zine finger protein cDNA from rice.NanjingAgri Univ[J].2002,25:110-11
[25]Huang W, Fang XD, Lin QF, et al.Identification and expression analysis of a full-length cDNA encoding a Kandelia candel tonoplast intrinsic protein[J]. Sheng Wu Gong Cheng Xue Bao,2003, 19 (2):147-52
[26]Kapoor S,Kobayashi A and takatsuji H.Silencing of the tapetum-specific zinc finger gene TAZ1 cause premature degenenetation of tapetum and pollen abortion in petunia[J].The Plant Cell,2002, 14:2354-2367
[27]Kobayashi A,Sakmoto A,Kuho K,et al.Seven zine-finger transcription factors are expressed sequentially during thed evelopment of anthers in Petunia[J]. The Plant Journal,1998,13(4): 571-576
[28]Kim JC,Lee SH and Cheon YH.A novel cold-inducible zine finger Protein from Soybean SCOF-1, enhances cold tolerance in transgenic Plants[J]. The Plant Journal,2001,25 (3):247-259
[29]Lippuner V,Cyert MS and Gasser CS.Two classes of Plant cDNA clones differentially eomplement yeast calcineurin mutants and increase salt tolerance of wild-type yeast [J]. Biological Chemistry, 1996,271:12859-12866.
[30]Landy A.Dynamic,structural and regulatory aspects of lambda Site-specif recombination[J].Ann Rev Biochem,1989,58:913-941
[31]Miller J,Melachlan AD and Klug A.Repetitive zine-binding domains in the protein transecription Factor IIIA from Xeno Pusoocytes[J].EMBO J,1985,4:1609-1614
[32]Miyama M and Hanagata N.Microarray analysis of 7029 gene expression patterns in burma mangrove under high-salinity stress[J]. Plant Science,2007,172 (5):948-957
[33]Miyama M,Shimizu H,Sugiyama M,et al. Sequencing and analysis of 14,842 expressed sequence tags of burma mangrove, Bruguiera gymnorrhiza[J]. Plant Science,2006,171(2):234-241
[34]Nguyen PD,Ho CL,Harikrishna JA,et al.Functional screening for salinity tolerant genes from Acanthus ebracteatus Vahl using Escherichia coli as a host[J].Trees-Structure and Function,2007, 21 (5):515-520.
[35]Prashanth SR,Sadhasivam V and Parida A.Overexpression of cytosolic copper/zinc superoxide dismutase from a mangrove plant Avicennia mayina in indica Rice var Pusa Basmati-1 confers abiotic stress tolerance[J]. Transgenic Research,17:281-291
[36]Ragland M and Soliman KM.Sali5-4a and Sali3-2,Two genes induced by aluminum in soybean roots[J]. Plant Physiol,1997,114:395
[37]Sakai H,krizek BA,Jacobsen SE,et al.Regulation of SUP expression identifies multiple regulators involved in Arabidopsis floral meristem development [J].The Plant Cell,2000,12:1607-1318
[38]Sakai H,Medrano LJ and Meyerowitz EM. Role of SUPERMAN in maintaining Arabidopsis floral whorl boundaries[J].Nature,1995,378:199-203
[39]Sakamoto A,Araki T,Meshi T,et al.Expression of a subset of the Arabidopsis Cys2His2-type zinc-finger protein gene family under water stress. Gene,2000,248 (1-2):23-32
[40]Sugihara K, Hanagata N, Dubinsky Z,et al.Molecular characterization of cDNA encoding oxygen evolving enhancer protein I increased by salt treatment in the mangrove Bruguiera gymnorrhiza[J]. The Plant Physiol,2000,41 (11):1279-128
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