灰毡毛忍冬MADS-box基因家族鉴定与CMB1基因克隆

Identification of the MADS-box gene family and cloning of the CMB1 gene in Lonicera macranthoides

目的鉴定灰毡毛忍冬Lonicera macranthoides MADS-box家族基因并进行生物信息学分析与表达模式验证,克隆湘蕾型和野生型灰毡毛忍冬MADS-box家族成员CMB1全长。方法基于转录组数据,利用在线工具对灰毡毛忍冬MADS-box进行生物信息学分析,并利用qRT-PCR验证MADS-box基因在不同品种中的表达模式,通过RT-PCR、RACE技术克隆湘蕾型和野生型灰毡毛忍冬CMB1基因全长。结果28个灰毡毛忍冬MADS-box蛋白长度为89~359 aa,碱性蛋白占比85.7%,不稳定蛋白占比96.4%,亲水性蛋白占比96.4%,均定位于细胞核,均为无跨膜结构的非分泌蛋白。转录组显示,与野生型比较,湘蕾型中28个MADS-box基因有17.86%表达下调,MIKCC型基因中有18.75%表达下调。克隆得到在两个品种的灰毡毛忍冬花中高度特异性表达的CMB1基因全长,均包含一个738 bp的ORF,编码245个氨基酸。CMB1基因在两个品种的花中表达量存在显著差异(P<0.01),且与茎、叶相比,在花中高度特异性表达(P<0.01)。结论基于灰毡毛忍冬转录组数据,鉴定了28个MADS-box家族基因,克隆得到湘蕾型与野生型灰毡毛忍冬CMB1基因全长,为进一步研究灰毡毛忍冬花发育、优良表型形成的分子机制提供研究基础与理论依据。

Objective To identify the MADS-box gene family of Lonicera macranthoides,analyze its bioinformatics,validate the expression patterns,and clone the full-length sequences of CMB1,a member of the MADS-box family of Lonicera macranthoides of Xianglei and wild types.Methods Based on the transcriptome data,the bioinformatics of Lonicera macranthoides MADS-box was analyzed with online tools,the expression patterns of MADS-box genes in different cultivars were verified using qRT-PCR,and the full-length sequences of Lonicera macranthoides CMB1 gene of the Xianglei and wild types were cloned by RT-PCR and RACE techniques.Results The sequence lengths of 28 MADS-box proteins of Lonicera macranthoides ranged from 89 aa to 359 aa,with alkali proteins accounting for 85.7%,unstable proteins accounting for 96.4%,and hydrophilic proteins accounting for 96.4%,which were localized in the nucleus,and were non-secretory proteins without transmembrane structure.The transcriptome showed that compared with the wild type,17.86%of the 28 MADS-box genes in the Xianglei type and 18.75%of the genes of MIKCC type were downregulated.The full-length sequences of CMB1 genes,which were highly specifically expressed in two varieties of flowers of Lonicera macranthoides,were cloned.Both of them contained a 738 bp ORF,encoding 245 amino acids.There was a significant difference in expression level of CMB1 gene between the two varieties of flowers(P<0.01),and it was highly and specifically expressed in flowers compared to that in stems and leaves(P<0.01).Conclusion Based on the transcriptome data of Lonicera macranthoides,28 MADS-box family genes were identified,and the full-length sequences of CMB1 gene of Xianglei and wild types were cloned,which provides the research foundation and theoretical basis for the further study of molecular mechanism of development and superior phenotype formation of Lonicera macranthoides.