紫花苜蓿β-1,3-葡聚糖酶基因同源克隆与序列变异分析
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摘要
β-1,3-葡聚糖酶是植物重要的防卫蛋白之一。采用同源克隆法,从16个紫花苜蓿品种的集群DNA中分离到3个苜蓿β-1,3-葡聚糖酶基因的部分基因组编码序列,分别对应于蒺藜苜蓿3个不同的β-1,3-葡聚糖酶基因,并命名为β-1,3-葡聚糖酶基因1、β-1,3-葡聚糖酶基因2和β-1,3-葡聚糖酶基因3。其中,β-1,3-葡聚糖酶基因2和苜蓿β-1,3-葡聚糖酶基因3及其蒺藜苜蓿对应基因与包括β-1,3-葡聚糖酶基因1在内的其他豆科同类基因遗传距离较大。序列变异分析显示,β-1,3-葡聚糖酶基因2的SNP位点间平均距离为255bp,DNA和氨基酸序列均非常保守,表明其在进化中受到了强烈的选择压力。β-1,3-葡聚糖酶基因1的SNP位点间平均距离虽然较小(31bp),但其全部21个SNP位点均为同义SNP,同时,测序片段仅发现了10个单倍型,远远小于其理论单倍型数目(2~(21))。这表明β-1,3-葡聚糖酶基因1的氨基酸序列高度保守,在功能上非常重要,少量单倍型的存在是为了调节其表达,以使苜蓿适应多种内、外部环境条件。
Beta-1,3-glucanase genes are one of the most important defensive proteins in plant.This study aimed to isolate the genomic coding sequences of beta-1,3-glucanase genes by a homology-based cloning method using the bulked DNA from 16 cultivars as materials in alfalfa(Medicago sativa).Consequently,the part genomic coding sequences of a total of 3 beta-1,3-glucanase genes were isolated,and each of these 3 genes had a corresponding homologous beta-1,3-glucanase gene from Medicago truncatula.They were named as beta-1,3-glucanase gene 1,beta-1,3-glucanase gene 2 and beta-1,3-glucanase gene 3,respectively.Cluster analysis showed that beta-1,3-glucanase gene 2,beta-1,3-glucanase gene 3,and the corresponding genes of these two genes in Medicago truncatula clearly diverged from beta-1,3-glucanase genes from other Fabaceae plants and beta-1,3-glucanase gene 1 found.Sequence vibration analysis showed beta-1,3-glucanase gene 2 had an high average inter-SNP distance with a value of 225 bp,indicating this gene was very conserved and thus suffered from a strong evolutionary selection pressure.beta-1,3-glucanase gene 1 possessed an low average inter-SNP distance with a value of 31 bp,but all of 21 SNP sites from this gene were synonymous.Furthermore,only a total of 10 haplotypes were detected for the fragment sequenced from beta-1,3-glucanase gene 1,which was far less the theoretical haplotype number(2~(21)).This indicated that beta-1,3-glucanase gene 1 also was very conservative and had a very important function in alfalfa genome.Preserving a little of haplotypes could help to regulate the expression of this gene,therefore enhance the adaptability of alfalfa to a variety of internal and external environmental conditions.
Beta-1,3-glucanase genes are one of the most important defensive proteins in plant.This study aimed to isolate the genomic coding sequences of beta-1,3-glucanase genes by a homology-based cloning method using the bulked DNA from 16 cultivars as materials in alfalfa(Medicago sativa).Consequently,the part genomic coding sequences of a total of 3 beta-1,3-glucanase genes were isolated,and each of these 3 genes had a corresponding homologous beta-1,3-glucanase gene from Medicago truncatula.They were named as beta-1,3-glucanase gene 1,beta-1,3-glucanase gene 2 and beta-1,3-glucanase gene 3,respectively.Cluster analysis showed that beta-1,3-glucanase gene 2,beta-1,3-glucanase gene 3,and the corresponding genes of these two genes in Medicago truncatula clearly diverged from beta-1,3-glucanase genes from other Fabaceae plants and beta-1,3-glucanase gene 1 found.Sequence vibration analysis showed beta-1,3-glucanase gene 2 had an high average inter-SNP distance with a value of 225 bp,indicating this gene was very conserved and thus suffered from a strong evolutionary selection pressure.beta-1,3-glucanase gene 1 possessed an low average inter-SNP distance with a value of 31 bp,but all of 21 SNP sites from this gene were synonymous.Furthermore,only a total of 10 haplotypes were detected for the fragment sequenced from beta-1,3-glucanase gene 1,which was far less the theoretical haplotype number(2~(21)).This indicated that beta-1,3-glucanase gene 1 also was very conservative and had a very important function in alfalfa genome.Preserving a little of haplotypes could help to regulate the expression of this gene,therefore enhance the adaptability of alfalfa to a variety of internal and external environmental conditions.
引文
[1]Dzhavakhiya V G,Ozeretskovskaya O L,Zinovyeva S V.Immune response[A].In:Y Dyakov & OL Ozeretskovskaya(Eds.).Comprehensive and molecular phytopathology[M].Netherlands:Elsevier,2007:265-314.
[2]Vanloon L C,Vanstrien E A.The families of pathogenesis related proteins,their activities,and comparative an analysis of PR1 type proteins [J].Physiological and Molecular Plant Pathology,1999,55:85-97.
[3]Mohammadi M,Karr A L.β-1,3-Glucanase and chitinase activities in soybean root nodules [J].Journal of Plant Physiol,2002,159:245-256.
[4]Gupta P,Ravi I,Sharma V.Induction of β-1,3-glucanase and chitinase activity in the defense response of Eruca sativa plants against the fungal pathogen Alternaria brassicicola[J].Journal of Plant Interactions,2013,8(2):155-161.
[5]Salim A P,Saminaidu K,Marimuthu M,et al.Defense responses in tomato landrace and wild genotypes to early blight pathogen Alternaria solani infection and accumulation of pathogenesis-related proteins[J].Archives of Phytopathology Plant Protection,2011,44:1147-1164.
[6]Aggarwal R,Purwar S,Kharbikar L,et al.Induction of a wheat β-1,3-glucanase gene during the defense response to Bipolaris sorokiniana[J].Acta Phytopathologica et Entomologica Hungarica,2011,46:39-47.
[7]Mendis H C,Queiroux C,Brewer T E,et al.The succinoglycan endoglycanase encoded by exoK is required for efficient symbiosis of sinorhizobium meliloti 1021 with the host plants Medicago truncatula and Medicago sativa(Alfalfa)[J].Molecular Plant Microbe Interactions,2013,26(9):1089-1105.
[8] Balasubramanian V,Vashisht D,Cletus J,et al.Plant β-1,3-glucanases:their biological functions and transgenic expression against phytopathogenic fungi[J].Biotechnology Letters,2012,34:1983-1990.
[9]Baldridge G D,O’Neill N R,Samac D A.Alfalfa(Medicago sativa L.) resistance to the root-lesion nematode,Pratylenchus penetrans:defense-response gene mRNA and isoflavonoid phytoalexin levels in roots[J].Plant Molecular Biology,1998,38(6):999-1010.
[10]Salles I I,Blount J W,Dixon R A,et al.Phytoalexin induction and beta-1,3-glucanase activities in Colletotrichum trifolii infected leaves of alfalfa(Medicago sativa L.)[J].Physiological and Molecular Plant Pathology,2002,61(2):89-101.
[11] Capomaccio S,Barone P,Reale L,et al.Isolation of genes from female sterile flowers in Medicago sativa[J].Sex Plant Reprod,2009,22(2):97-107.
[12]李思经.诱导性β-1,3-葡聚糖酶在苜蓿中的组成性表达减轻了由卵菌病原体大雄疫霉引起的病害但不能减轻含有几丁质真菌的病害[J].生物技术通报,1997(3):53.
[13]丁守彦.不同苜蓿品种对镰刀菌的抗性鉴定及诱导抗性研究[D].兰州:甘肃农业大学,2010.
[14]高萍.丛枝菌根真菌和根瘤菌对苜蓿根腐病和叶斑病的防病促生作用[D].兰州:兰州大学,2014.
[15]孙涛.SA诱导苜蓿抗霜霉病的研究[D].兰州:甘肃农业大学,2006.
[16]张振亚,裴翠明,马进.基于转录组和蛋白质组关联研究技术筛选紫花苜蓿耐盐相关候选基因[J].植物生理学报,2016,52(3):317-324.
[17]戴怡龄.苜蓿Ⅰ类chi、酸性glu的克隆和农杆菌介导的转化过程的超微结构研究[D].兰州:兰州大学,2005.
[18]Dellaporta S,Wood J,Hicks J.A plant DNA minipreparation Ver.II[J].Plant Molecular Biology Reporter,1983(1):19-21.
[19]Hall T A.BioEdit:a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT[J].Nucleic acids symposium series,1999,41:95-98.
[20]Tamura K,Peterson D,Peterson N,et al.MEGA5:molecular evolutionary genetics analysis using maximum likelihood,evolutionary distance,and maximum parsimony methods[J].Molecular Biology and Evolution,2011,28:2731-2739.
[21]洪绂曾,卢欣石,高洪文.苜蓿科学[M].北京:中国农业出版社,2009:230-255.
[22]桂枝,肖婷,皮永硕,等.紫花苜蓿PGIP同源基因克隆与序列核苷酸多态性分析[J].草业科学,2015,32(10):1603-1611.
[23]Onaga S,Taira T.A new type of plant chitinase containing LysM domains from a fern(Pteris ryukyuensis):roles of LysM domains in chitin binding and antifungal activity[J].Glycobiology,2008,18:414-423.
[24]Ozeretskovskaya O L.Vertical pathosystem:resistance genes and their products[A].In Y Dyakov & OL Ozeretskovskaya(Eds.).Comprehensive and molecular phytopathology[M].Netherlands:Elsevier,2007:217-245.
[25]Bonanomi A,Wiemken A,Boller T,et al.Local induction of a mycorrhiza-specific class III chitinase gene in cortical root cells of Medicago truncatula containing developing or mature arbuscules[J].Plant Biology,2001,3(2):194-199.
[26]Cletus J,Balasubramanian V,Vashisht D,et al.Transgenic expression of plant chitinases to enhance disease resistance[J].Biotechnol Letters,2013,35:1719-1732.
[27]Li X H,Wei Y L,Moore K J,et al.Associat ion mapping of biomass yield and stem composition in a tetraploid alfalfa breeding population[J].Plant Genome-US,2011,4(1):24-35.
[28]Yu L X,Zheng P,Zhang T J,et al.Genotyping-by-sequencing based genome-wide association studies on Verticillium wilt resistance in autotetraploid alfalfa(Medicago sativa L)[J].Molecular Plant Pathology,2017,18(2):187-194.
[29]Muller M H,Poncet C,Prosperi JM,et al.Domestication history in the Medicago sativa species complex:inferences from nuclear sequence polymorphism[J].Molecular Ecology,2006,15:1589-1602.
[30]桂枝,皮永硕,袁庆华,等.苜蓿多聚半乳糖醛酸酶抑制蛋白2(MsPGIP2)基因多态性分析[J].草地学报,2016,24(5):1073-1079.
[31]Gao J,Gui Z,Wang Y,et al.Polymorphism of polygalacturonase-inhibiting protein genes by fluorescence-based PCR single-strand conformation profiling andsequencing in autotetraploid alfalfa(Medicago sativa) populations[J].Euphytica,2015,206(3):567-577.
[2]Vanloon L C,Vanstrien E A.The families of pathogenesis related proteins,their activities,and comparative an analysis of PR1 type proteins [J].Physiological and Molecular Plant Pathology,1999,55:85-97.
[3]Mohammadi M,Karr A L.β-1,3-Glucanase and chitinase activities in soybean root nodules [J].Journal of Plant Physiol,2002,159:245-256.
[4]Gupta P,Ravi I,Sharma V.Induction of β-1,3-glucanase and chitinase activity in the defense response of Eruca sativa plants against the fungal pathogen Alternaria brassicicola[J].Journal of Plant Interactions,2013,8(2):155-161.
[5]Salim A P,Saminaidu K,Marimuthu M,et al.Defense responses in tomato landrace and wild genotypes to early blight pathogen Alternaria solani infection and accumulation of pathogenesis-related proteins[J].Archives of Phytopathology Plant Protection,2011,44:1147-1164.
[6]Aggarwal R,Purwar S,Kharbikar L,et al.Induction of a wheat β-1,3-glucanase gene during the defense response to Bipolaris sorokiniana[J].Acta Phytopathologica et Entomologica Hungarica,2011,46:39-47.
[7]Mendis H C,Queiroux C,Brewer T E,et al.The succinoglycan endoglycanase encoded by exoK is required for efficient symbiosis of sinorhizobium meliloti 1021 with the host plants Medicago truncatula and Medicago sativa(Alfalfa)[J].Molecular Plant Microbe Interactions,2013,26(9):1089-1105.
[8] Balasubramanian V,Vashisht D,Cletus J,et al.Plant β-1,3-glucanases:their biological functions and transgenic expression against phytopathogenic fungi[J].Biotechnology Letters,2012,34:1983-1990.
[9]Baldridge G D,O’Neill N R,Samac D A.Alfalfa(Medicago sativa L.) resistance to the root-lesion nematode,Pratylenchus penetrans:defense-response gene mRNA and isoflavonoid phytoalexin levels in roots[J].Plant Molecular Biology,1998,38(6):999-1010.
[10]Salles I I,Blount J W,Dixon R A,et al.Phytoalexin induction and beta-1,3-glucanase activities in Colletotrichum trifolii infected leaves of alfalfa(Medicago sativa L.)[J].Physiological and Molecular Plant Pathology,2002,61(2):89-101.
[11] Capomaccio S,Barone P,Reale L,et al.Isolation of genes from female sterile flowers in Medicago sativa[J].Sex Plant Reprod,2009,22(2):97-107.
[12]李思经.诱导性β-1,3-葡聚糖酶在苜蓿中的组成性表达减轻了由卵菌病原体大雄疫霉引起的病害但不能减轻含有几丁质真菌的病害[J].生物技术通报,1997(3):53.
[13]丁守彦.不同苜蓿品种对镰刀菌的抗性鉴定及诱导抗性研究[D].兰州:甘肃农业大学,2010.
[14]高萍.丛枝菌根真菌和根瘤菌对苜蓿根腐病和叶斑病的防病促生作用[D].兰州:兰州大学,2014.
[15]孙涛.SA诱导苜蓿抗霜霉病的研究[D].兰州:甘肃农业大学,2006.
[16]张振亚,裴翠明,马进.基于转录组和蛋白质组关联研究技术筛选紫花苜蓿耐盐相关候选基因[J].植物生理学报,2016,52(3):317-324.
[17]戴怡龄.苜蓿Ⅰ类chi、酸性glu的克隆和农杆菌介导的转化过程的超微结构研究[D].兰州:兰州大学,2005.
[18]Dellaporta S,Wood J,Hicks J.A plant DNA minipreparation Ver.II[J].Plant Molecular Biology Reporter,1983(1):19-21.
[19]Hall T A.BioEdit:a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT[J].Nucleic acids symposium series,1999,41:95-98.
[20]Tamura K,Peterson D,Peterson N,et al.MEGA5:molecular evolutionary genetics analysis using maximum likelihood,evolutionary distance,and maximum parsimony methods[J].Molecular Biology and Evolution,2011,28:2731-2739.
[21]洪绂曾,卢欣石,高洪文.苜蓿科学[M].北京:中国农业出版社,2009:230-255.
[22]桂枝,肖婷,皮永硕,等.紫花苜蓿PGIP同源基因克隆与序列核苷酸多态性分析[J].草业科学,2015,32(10):1603-1611.
[23]Onaga S,Taira T.A new type of plant chitinase containing LysM domains from a fern(Pteris ryukyuensis):roles of LysM domains in chitin binding and antifungal activity[J].Glycobiology,2008,18:414-423.
[24]Ozeretskovskaya O L.Vertical pathosystem:resistance genes and their products[A].In Y Dyakov & OL Ozeretskovskaya(Eds.).Comprehensive and molecular phytopathology[M].Netherlands:Elsevier,2007:217-245.
[25]Bonanomi A,Wiemken A,Boller T,et al.Local induction of a mycorrhiza-specific class III chitinase gene in cortical root cells of Medicago truncatula containing developing or mature arbuscules[J].Plant Biology,2001,3(2):194-199.
[26]Cletus J,Balasubramanian V,Vashisht D,et al.Transgenic expression of plant chitinases to enhance disease resistance[J].Biotechnol Letters,2013,35:1719-1732.
[27]Li X H,Wei Y L,Moore K J,et al.Associat ion mapping of biomass yield and stem composition in a tetraploid alfalfa breeding population[J].Plant Genome-US,2011,4(1):24-35.
[28]Yu L X,Zheng P,Zhang T J,et al.Genotyping-by-sequencing based genome-wide association studies on Verticillium wilt resistance in autotetraploid alfalfa(Medicago sativa L)[J].Molecular Plant Pathology,2017,18(2):187-194.
[29]Muller M H,Poncet C,Prosperi JM,et al.Domestication history in the Medicago sativa species complex:inferences from nuclear sequence polymorphism[J].Molecular Ecology,2006,15:1589-1602.
[30]桂枝,皮永硕,袁庆华,等.苜蓿多聚半乳糖醛酸酶抑制蛋白2(MsPGIP2)基因多态性分析[J].草地学报,2016,24(5):1073-1079.
[31]Gao J,Gui Z,Wang Y,et al.Polymorphism of polygalacturonase-inhibiting protein genes by fluorescence-based PCR single-strand conformation profiling andsequencing in autotetraploid alfalfa(Medicago sativa) populations[J].Euphytica,2015,206(3):567-577.
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