甜高粱WRKY转录因子基因的克隆与表达分析

Cloning and Expression Analysis of WRKY Transcription Factor Gene in Sweet Sorghum

【目的】克隆甜高粱WRKY基因,分析其序列特征和表达谱,为甜高粱抗旱育种提供基因来源。【方法】以甜高粱品种M81-E幼苗总RNA为模板,利用RT-PCR技术克隆甜高粱WRKY基因,通过生物信息学软件分析其序列特征,采用实时荧光定量PCR检测基因表达情况,获得2条WRKY基因,命名为SbWRKY1(Genebank登录号:KY231904)和SbWRKY2(Genebank登录号:KY231905)。【结果】SbWRKY1 cDNA序列全长1086 bp,包括1个1059 bp的开放阅读框,编码352个氨基酸。SbWRKY2 cDNA序列全长750 bp,包括1个717 bp的开放阅读框,编码238个氨基酸。2个基因编码的蛋白均具有WRKY核心序列"WRKYGQK"和"C-X4-5-C-X22-23-H-X1-H"锌指结构模型。SbWRKY1蛋白分子式为C_(1628)H_(2619)N_(509)O_(504)S_(16),预测蛋白质分子量为37.89 kDa,等电点(PI)为9.78。SbWRKY2蛋白分子式为C_(1131)H_(1752)N_(326)O_(344)S_(21),预测蛋白质分子量为26.09 kDa,等电点(PI)为8.43。系统进化树分析表明,SbWRKY1蛋白与高粱、玉米和谷子WRKY蛋白亲缘关系较近,SbWRKY2蛋白与高粱、芒草和玉米WRKY蛋白亲缘关系较近。荧光定量PCR分析表明,随着干旱胁迫时间延长,SbWRKY1和SbWRKY2呈上调表达趋势。【结论】SbWRKY1和SbWRKY2基因可能在甜高粱应对干旱胁迫中发挥一定作用。

【 Objective】 The objective of this study was to cloneSKr genes from sweet sorghum and analyzed their sequence characteristics and expression patterns, which would provided the source of gene in sweet sorghum breeding for drought resistance. [ Method ] Taken seed-lings RNA of sweet sorghum cultivar M81-E as tested materials, WRKY genes from sweet sorghum by RT-PCR were cloned, their characteris-tics of sequences with bioinformatics softwares and their expression patterns by real-time PCR were analyzed, and two WRKY genes from sweet sorghum were cloned, which were named SbWRKYl （Genebank accession number： KY231904） and SbWRKY2 （ Genebank accession number： KY231905 ）？ 【 Result】 The cDNA full length of was 1086 bp with a 1059 bp open reading frame,which encoded 352 a-mino acids. The cDNA full length of SbWRKY2 was 750 bp with a 717 bp open reading frame, which encoded 238 amino acids. The two genes encoding proteins had WRKY core sequence ‘ WRKYGQK’ and zinc finger structure model ‘ C-X4-5-C-X22-23 -H-Xl-H ’ ？ The pre-dicted protein molecular formula of SbWRKYl was C162gH26i9N5〇 9 0 5 0 4 8^ , the molecular mass was 37. 89 kDa, and the PI was 9. 78. The predicted protein molecular formula of SbWRKY2 was C1131H1752 N326 0 344 S21 , the molecular mass was 26. 09 kDa, and the PI was 8 . 43. Phylogenetic analysis indicated that SbWRKYl protein was close to WRKY proteins of Sorghum bicolor, Zea mays and Setaria italica, andSbWRKY2 protein was close to WRKY proteins of Sorghum bicolor, Miscanthus lutarioriparius and Zea mays. Real-time PCR results showed that the SbWRKYl and SbWRKYl gene were up-regulated expression with the time of drought stress.【 Conclusion】 These results demonstratedthat SbWRKYl and SbWRKY2 might play a role in response todrought stress in sweet sorghum.