甘蔗泛素结合酶基因的克隆与表达
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摘要
为克隆鉴定甘蔗泛素结合酶基因(Sugarcane ubiquitin-conjugating enzyme,ScUBc E2)并探索其在激素信号通路和甘蔗与黑穗病互作过程中的作用,选择黑穗病菌胁迫下甘蔗抑制消减杂交文库(Suppression subtractive hybridization,SSH)中注释为泛素结合酶的差异表达EST序列为探针,结合电子克隆技术和RT-PCR技术,以甘蔗cDNA为模板进行泛素结合酶基因克隆.对克隆获得的序列进行生物信息学分析,并利用qRT-PCR技术分析该基因在甘蔗根、蔗髓、叶、芽中的组织特异性表达以及在黑穗病菌、茉莉酸甲酯(Methyl jasmonate,Me JA)、脱落酸(Abscisic acid,ABA)和水杨酸(Salicylic acid,SA)胁迫下的表达情况.最终克隆得到一条长度为699 bp的甘蔗泛素结合酶基因(ScUBc E2;Gen Bank accession number:KJ577594.1),该基因包含长度为447 bp的完整开放读码框,编码148个氨基酸.生物信息学分析结果显示,ScUBc E2编码的蛋白分子量(Mr)为16.507×10~3;无信号肽,为碱性不稳定的亲水蛋白;包含4个α螺旋、4个β折叠和一些无规则卷曲;第15位氨基酸为泛素化位点,74-89位氨基酸为活性位点;在进化过程中与高粱泛素结合酶基因的亲缘关系最近.qRT-PCR分析结果表明,ScUBc E2基因组成型表达,但在芽中的表达量最高;其表达在甘蔗感黑穗病基因型ROC22中受到黑穗病菌胁迫的抑制,在黑穗病抗病基因型YC05-179中则先被抑制,后被诱导;ScUBc E2基因受MeJA及SA诱导表达,对ABA胁迫的响应不明显.本研究表明,甘蔗ScUBc E2基因在抗病基因型和感病基因型甘蔗中存在不同表达模式,可能参与甘蔗与黑穗病菌的互作过程,有望为抗病育种分子标记提供潜在基因资源;同时,该基因受MeJA和SA外源激素胁迫后的表达模式,可为泛素-蛋白酶体途径及激素调控的信号转导在甘蔗与黑穗病菌互作过程中的作用提供一定的理论依据.
The objective of this study was to examine the biological characteristics of the sugarcane ubiquitin-conjugating enzyme(Sc UBc E2) gene, its role in the hormone signaling pathway, and the interaction between sugarcane and Sporisorium scitamineum. An EST was annotated as the ubiquitin-conjugating enzyme from a sugarcane suppression subtractive hybridization(SSH) library from the infection of S. scitamineum. This EST was subsequently used as a probe for in silicocloning, and the c DNA sequence of the sugarcane ubiquitin-conjugating enzyme gene was cloned using RT-PCR and was then sequenced. The bioinformatic characteristics of this gene was analyzed, and q RT-PCR was used to detect expression in sugarcane tissues, including the root, stem pith, leaf, and bud, and when treated with S. scitamineum, methyl jasmonate(Me JA), abscisic acid(ABA), and salicylic acid(SA). Finally, a ubiquitin-conjugating enzyme gene(Sc UBc E2, Gen Bank accession number: KJ577594.1) that was 699 bp was obtained. The sequence of Sc UBc E2 contained a complete open reading frame of 447 bp that encodes 148 amino acids. The bioinformatics analysis showed that Sc UBc E2 was 16.507 × 103 and an alkaline unstable hydrophilic protein with no signal peptide. It contained 4 alpha helix, 4 beta folding, and some random coils,its fifteenth amino acid was a ubiquitination site, and the 74 th-89 th amino acid was an active site. The phylogenetic analysis showed that ScUBc E2 had the closest genetic relationship to a homologous gene in sorghum. The qRT-PCR analysis indicated that the ScUBc E2 gene was constitutively expressed in sugarcane with the highest level in the buds. The expression of the ScUBc E2 gene was inhibited in the smut-susceptible genotype ROC22 and in the early smut-infected stage of the smut-resistant genotype YC05-179, but it was induced at the later smut-infected stage in the smut-resistant genotype YC05-179 when the sugarcane plant was infected by S. scitamineum. Moreover, the expression of the ScUBc E2 gene was induced by the treatment of MeJA and SA in the sugarcane tissue culture plantlets, but it was insensitive to ABA. This study showed that the ScUBc E2 gene was involved in the interaction of sugarcane and S. scitamineum. The different expression patterns of the ScUBc E2 gene and the smut-resistant and smut-susceptible genotypes suggest that the ScUBc E2 gene may serve as a molecular marker for disease resistance during breeding. The expression patterns of the ScUBc E2 gene under the stress of MeJA and SA could also provide a basis for understanding the role of the ubiquitin-proteasome pathway and hormone regulated signal transduction in the interaction of sugarcane and S. scitamineum.
The objective of this study was to examine the biological characteristics of the sugarcane ubiquitin-conjugating enzyme(Sc UBc E2) gene, its role in the hormone signaling pathway, and the interaction between sugarcane and Sporisorium scitamineum. An EST was annotated as the ubiquitin-conjugating enzyme from a sugarcane suppression subtractive hybridization(SSH) library from the infection of S. scitamineum. This EST was subsequently used as a probe for in silicocloning, and the c DNA sequence of the sugarcane ubiquitin-conjugating enzyme gene was cloned using RT-PCR and was then sequenced. The bioinformatic characteristics of this gene was analyzed, and q RT-PCR was used to detect expression in sugarcane tissues, including the root, stem pith, leaf, and bud, and when treated with S. scitamineum, methyl jasmonate(Me JA), abscisic acid(ABA), and salicylic acid(SA). Finally, a ubiquitin-conjugating enzyme gene(Sc UBc E2, Gen Bank accession number: KJ577594.1) that was 699 bp was obtained. The sequence of Sc UBc E2 contained a complete open reading frame of 447 bp that encodes 148 amino acids. The bioinformatics analysis showed that Sc UBc E2 was 16.507 × 103 and an alkaline unstable hydrophilic protein with no signal peptide. It contained 4 alpha helix, 4 beta folding, and some random coils,its fifteenth amino acid was a ubiquitination site, and the 74 th-89 th amino acid was an active site. The phylogenetic analysis showed that ScUBc E2 had the closest genetic relationship to a homologous gene in sorghum. The qRT-PCR analysis indicated that the ScUBc E2 gene was constitutively expressed in sugarcane with the highest level in the buds. The expression of the ScUBc E2 gene was inhibited in the smut-susceptible genotype ROC22 and in the early smut-infected stage of the smut-resistant genotype YC05-179, but it was induced at the later smut-infected stage in the smut-resistant genotype YC05-179 when the sugarcane plant was infected by S. scitamineum. Moreover, the expression of the ScUBc E2 gene was induced by the treatment of MeJA and SA in the sugarcane tissue culture plantlets, but it was insensitive to ABA. This study showed that the ScUBc E2 gene was involved in the interaction of sugarcane and S. scitamineum. The different expression patterns of the ScUBc E2 gene and the smut-resistant and smut-susceptible genotypes suggest that the ScUBc E2 gene may serve as a molecular marker for disease resistance during breeding. The expression patterns of the ScUBc E2 gene under the stress of MeJA and SA could also provide a basis for understanding the role of the ubiquitin-proteasome pathway and hormone regulated signal transduction in the interaction of sugarcane and S. scitamineum.
引文
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2 Ying M,Zhan Z,Wang W,Chen D.Origin and evolution of ubiquitinconjugating enzymes from Guillardia theta nucleomorph to hominoid[J].Gene,2009,447(2):72-85
3 Yao T,Ndoja A.Reg ulation of gene expression by the ubiquiti nproteasome system[J].Semin Cell Dev Biol,2012,23(5):523-529
4 Gray WM,Estelle I.Function of the ubiquitin-proteasome pathway in auxin response[J].Trends Biochem Sci,2000,25(3):133-138
5 Fr ied man J,Xue D.To live or die by the sword:the reg ulation of apoptosis by the proteasome[J].Dev Cell,2004,6(4):460-461
6 Vierstra RD.The ubiquitin/26S proteasome pathway,the complex last chapter in the life of many plant proteins[J].Trends Plant Sci,2003,8(3):135-142
7 Bae H,Kim WT.The N-terminal tetra-peptide(IPDE)short extension of the U-box motif in rice SPL11 E3 is essential for the interaction with E2and ubiquitin-ligase activity[J].Biochem Biophys Res Commun,2013,433(2):266-271
8 Ye Y,Rape M.Building ubiquitin chains:E2 enzymes at work[J].Nat Rev Mol Cell Biol,2009,10(11):755-764
9 Bahmani R,Kim D,Lee BD,Hwang S.Over-expression of tobacco UBC1 encoding a ubiquitin-conjugating enzyme increases cadmium tolerance by activating the 20S/26S proteasome and by decreasing Cd accumulation and oxidative stress in tobacco(Nicotiana tabacum)[J].Plant Mol Biol,2017,94(4-5):433-451
10 Cui F,Liu L,Zhao Q,Zhang Z,Li Q,Lin B,Wu Y,Tang S,Xie Q.Arabidopsis ubiquitin conjugase UBC32 is an ERAD component that functions in brassinosteroid-mediated salt stress tolerance[J].Plant Cell,2012,24(1):233-244
11 Chung E,Cho CW,So HA,Kang JS,Chung YS,Lee JH.Overexpression of Vr UBC1,a mung bean E2 ubiquitin-conjugating enzyme,enhances osmotic stress tolerance in Arabidopsis[J].PLo S ONE,2013,8(6):e66056
12 Zhou GA,Chang RZ,Qiu LJ.Overexpression of soybean ubiquitinconjugating enzyme gene Gm UBC2 confers enhanced drought and salt tolerance through modulating abiotic stress-responsive gene expression in Arabidopsis[J].Plant Mol Biol,2010,72(4-5):357-367
13 Wan X,Mo A,Liu S,Yang L,Li L.Constitutive expression of a peanut ubiquitin-conjugating enzyme gene in Arabidopsis confers improved water-stress tolerance through regulation of stress-responsive gene expression[J].J Biosci Bioeng,2011,111(4):478-484
14 Zhou B,Zeng L.Elucidating the role of highly homologous Nicotiana benthamiana ubiquitin E2 gene family members in plant immunity through an improved virus-induced gene silencing approach[J].Plant Methods,2017,13(1):59
15 Cheng C,Zhang C,Yao Q,Chen K.Ubiquitin-conjugating enzyme involved in the immune response caused by pathogens invasion[J].Int J Biol Biotech 2013,10(1):1-5
16 Millyard L,Lee J,Zhang C,Yates G,Sadanandom A.The ubiquitin conjugat i ng en z y me,Ta U4 reg u lates wheat defence agai n st t he phytopathogen Zymoseptoria tritici[J].Sci Rep,2016,6:35683
17 Jeon EH,Pak JH,Kim MJ,Kim HJ,Shin SH,Lee JH,Kim DH,Oh JS,Oh B-J,Jung HW,Chung YS.Ectopic expression of ubiquitinconjugating enzyme gene from wild rice,Og UBC1,confers resistance against UV-B radiation and Botrytis infection in Arabidopsis thaliana[J].Biochem Biophys Res Commun 2012,427(2):309-314
18 Unver T,Turktas M,Budak H.In planta evidence for the involvement of a ubiquitin conjugating enzyme(UBC E2 clade)in negative regulation of disease resistance[J].Plant Mol Biol Rep,2013,31(2):323-334
19 Ei n i O,Dog r a S,Selt h LA,D r y I B,R a nd les J W,Rez aia n M A.Interaction with a host ubiquitin-conjugating enzyme is required for the pathogenicity of a geminiviral DNA beta satellite[J].Mol Plant Microbe Interact,2009,22(6):737-746
20 Arruda P.Genetically modified sugarcane for bioenergy generation[J].Curr Opin Biotechnol,2012,23(3):315-322
21 Li YR,Yang LT.Sugarcane agriculture and sugar industry in China[J].Sugar Tech,2015,17(1):1-8
22 黄宁.黑穗病菌胁迫下甘蔗SSH文库构建及差异表达基因的克隆与分析[D].福建:福建农林大学,2014[Huang N.Construction of suppression subtractive hybridization libraries of sugarcane challenged by Sporisorium scitamineum and cloning / analysis of several differentially expressed genes[D].Fu Jian:Fujian Agriculture and Foresty University,2014]
23 苏炜华,黄宁,凌辉,刘峰,曾瑞金,苏亚春,吴期滨,高世武,阙友雄.甘蔗乙醇脱氢酶基因的克隆与表达[J].应用与环境生物学报,2017,23(3):474-481[Su WH,Huang N,Ling H,Liu Feng,Zeng RJ,Su YC,Wu QB,Gao SW,Que YX.Cloning and expression of Sc ADH from sugarcane[J].Chin J Appl Environ Biol,2017,23(3):474-481]
24 肖新换,黄珑,黄宁,张玉叶,凌辉,刘峰,苏炜华,阙友雄.甘蔗Sc BAK1基因及其可变剪接体的克隆与表达分析[J].应用与环境生物学报,2015,21(5):872-881[Xiao XH,Huang L,Huang N,Zhang Y Y,Ling H,Liu Feng,Su W H,Que YX.Cloning and expression analysis of Sc BAK1 gene and its alternative spliceosome in sugarcane[J].Chin J Appl Environ Biol,2015,21(5):872-881]
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