香蕉MAP3K基因的克隆及表达

Cloning and expression analysis of a MAP3K gene in banana

为研究一个受低温诱导的香蕉MAP3K基因的序列特征以及其与靶定它的miRNA(miR408d)和lncRNA(MSTRG.4851)的表达特征,以‘天宝蕉’为材料,采用反转录PCR(RT-PCR)技术克隆该基因,分析其核苷酸和编码蛋白序列特性,基于长链非编码RNA(lncRNA)和小RNA(sRNA)测序数据分析MAP3K-miR408d-MSTRG.4851三者的表达趋势,并利用实时荧光定量PCR技术(qRT-PCR)验证它们在低温胁迫下的表达情况.结果显示:MAP3K编码序列(CDS)长为2 262 bp,可编码一个分子式为C3566H5625N1047O1090S32,氨基酸大小为753 aa,分子量为81 631.17,等电点为9.33的不稳定亲水性蛋白.MAP3K含有保守的PKinase结构域和MEKK结构域,不含跨膜结构和信号肽,与小果野蕉MAP3K亲缘关系最近.亚细胞定位预测结果显示MAP3K主要定位于外膜.蛋白互作预测结果显示MAP3K与MAPKK A亚族的3个MAP2K蛋白存在互作.MAP3K启动子含有大量的光响应和多种激素响应等元件.与组学测序结果一致,qRT-PCR结果显示MAP3K的表达受低温显著诱导,而靶定它的miRNA和lncRNA受低温抑制.本研究表明MAP3K的表达受miRNA和lncRNA调控,可能在香蕉响应低温胁迫过程中发挥重要作用.(图7表3参32)

To investigate the sequence characteristics of a low-temperature inducible banana MAP3K gene and to explore the expression patterns of the gene together with the miRNA (miR408d) and lncRNA (MSTRG.4851)targeting it,the MAP3K gene was cloned from‘Tianbaojiao’banana using reverse transcription PCR (RT-PCR).The sequence characteristics of the gene and its encoded proteins were analyzed bioinformatically.Based on long noncoding RNA (lncRNA) and small RNA (sRNA) sequencing data,the expression patterns of MAP3K-miRNA408d-lncRNA were studied and confirmed using quantitative real-time PCR (qRT-PCR).The results showed that the coding sequence (CDS) of MAP3K was 2 262 bp,which encoded an unstable hydrophilic protein with the molecular formula C3566H5625N1047O1090S32,amino acid size of 753 aa,molecular weight of81 631.17,and isoelectric point of 9.33.MAP3K was of conserved PKinase and MEKK domain that lacks a transmembrane structure and a signal peptide.The relationship between MAP3K and Musa acuminata MAP3K was the closest.The subcellular localization prediction results showed that MAP3K was mainly localized in the outer membrane.Protein-protein interaction prediction results showed that MAP3K could interact with three MAP2K proteins belonging to subfamily A of the MAPKK gene family.The MAP3K promoter contained many light-and hormone-responsive elements.qRT-PCR results showed that the expression of MAP3K was low temperature-inducible,but the expression of the miRNA and lncRNA targeting it was significantly induced by low temperature,which was consistent with the lncRNA and sRNA sequencing data.The results obtained in this study indicated that the expression of MAP3K is regulated by miRNAs and lncRNAs and might contribute greatly to the low-temperature stress response of banana.