木薯糖转运蛋白MeSWEET10-8基因克隆与功能分析

Cloning and functional analysis of cassava sugar transporter protein MeSWEET10-8 gene

  • 摘要: 【目的】 克隆木薯糖转运蛋白MeSWEET10-8基因,并对其进行功能分析,为探究MeSWEET10-8基因的糖转运调控机制及其与植物病原菌互作功能提供理论参考。【方法】 以木薯品种华南9号为材料,以接种木薯黄单胞菌(Xanthomonas axonopodis pv. manihotisXam)成熟叶片的cDNA为模板,PCR扩增MeSWEET10-8基因,对其进行生物信息学分析,并通过MEGA 11.0构建不同物种SWEET蛋白的系统发育树;结合亚细胞定位、糖转运试验、转录组测序(RNA-seq)分析基因功能;实时荧光定量PCR检测接种XamMeSWEET10-8基因表达量的影响。【结果】 从木薯感病的成熟叶片中克隆获得MeSWEET10-8基因的编码区(CDS)序列,长度为840 bp,与Phytozome数据库的参考序列(登录号:Manes.14G047700)完全一致,其编码蛋白的相对分子质量为31.81 kD,理论等电点(pI)为8.18,不包含信号肽,具有2个保守的MtN3_Slv功能域和7个跨膜结构域,启动子区包含5种光响应元件(Box 4、G-box、GA-motif、GT1-motif、TCT-motif)、1种水杨酸(SA)响应元件(TCA-element)、1种脱落酸(ABA)响应元件(ABRE)、1种茉莉酸甲酯(MeJA)响应元件(TGACG-motif)、2种赤霉素(GA)响应元件(CGTCA-motif、GARE-motif)、3种生物和非生物胁迫响应元件(ARE、LTR、TC-rich repeats)。系统发育分析结果显示,MeSWEET10-8蛋白与拟南芥AtSWEET10亲缘关系最近,与感病蛋白OsSWEET13和OsSWEET14同属于Clade Ⅲ分支。亚细胞定位和糖转运试验证明MeSWEET10-8蛋白定位于细胞膜上,具有转运木糖的功能。RNA-seq结果显示,MeSWEET10-8基因在木薯茎部组织中表达水平较高,叶柄和根顶端分生组织次之,在叶和贮藏根中则不表达。实时荧光定量PCR检测结果显示,MeSWEET10-8基因受Xam诱导,其相对表达量在接种前期逐渐升高,在72 h时达最高值,之后开始下降。【结论】 木薯MeSWEET10-8蛋白具有转运木糖的功能,在Xam的侵染下也被诱导表达,表明MeSWEET10-8蛋白参与植物与病原菌互作过程,且其可能对植物茎部发育起到重要的生理作用。

     

    Abstract: 【Objective】 The sugar transporter protein MeSWEET10-8 gene of cassava was cloned, and its function was analyzed to provide theoretical reference for exploring the sugar transport regulation mechanism of MeSWEET10-8 gene and its interaction function with plant pathogen. 【Method】 Using the cassava variety Huanan 9 as the research material, with the cDNA of cassava mature leaves inoculated with Xanthomonas axonopodis pv. manihotisXam) as the template, the MeSWEET10-8 gene was amplified by PCR. Its bioinformatics analysis was conducted, and the phylogenetic tree of SWEET proteins in different species was constructed using MEGA 11.0. The gene function was analyzed through subcellular localization, sugar transport experiments, and transcriptome sequencing (RNA-seq). The effects of inoculation with Xam on the expression of MeSWEET10-8 gene was detected by real-time fluorescence quantitative PCR. 【Result】 The coding sequence (CDS) of MeSWEET10-8 gene was cloned from the mature diseased leaves of cassava. The length of this sequence was 840 bp, which was exactly the same as the reference sequence (accession number:Manes.14G047700) in the Phytozome database. The encoded protein had a molecular weight of 31.81 kD,a theoretical isoelectric point (pI) of 8.18,and did not contain signal peptide. MeSWEET10-8 protein contained 2 conserved MtN3_Slv functional domains and 7 transmembrane domains. The promoter region contained five light-responsive elements (Box 4, G-box, GA-motif, GT1-motif, and TCT-motif), one salicylic acid (SA) responsive element (TCA-element), one abscisic acid (ABA) responsive element (ABRE), one methyl jasmonate (MeJA) responsive element (TGACG-motif), two gibberellin (GA) responsive elements (CGTCA-motif, GARE-motif), and three biological and abiotic stress response elements (ARE, LTR, TC-rich repeats). Phylogenetic analysis showed that MeSWEET10-8 protein had the closest genetic relationship with the AtSWEET10 of Arabidopsis thaliana, it located in the Clade III with pathogenic OsSWEET13 and OsSWEET14 proteins. Subcellular localization and sugar transport assay demonstrated that the MeSWEET10-8 protein was located on the cell membrane and had the function of transporting xylose. RNA-seq results showed that the MeSWEET10-8 gene was highly expressed in the stem tissues of cassava, followed by the petiole and root apical meristems, and no expression was detected in the leaves and storage roots. Real-time fluorescence quantitative PCR results indicated that MeSWEET10-8 gene was induced by Xam, and its relative expression gradually increased in the early stage of inoculation, reaching the highest value at 72 h, and then began to decline. 【Conclusion】 The cassava MeSWEET10-8 protein functions in transporting xyloses and is induced during Xam infection,indicating that MeSWEET10-8 protein is involved in the interaction between plants and pathogens, and may play a significant physiological role in plant stem development.

     

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