黄藤2个NAC基因的分子特征及其SSR分子标记开发

Molecular Characteristics and SSR Marker Development of Two NAC Genes from Daemonorops jenkinsiana

【目的】棕榈藤是重要的森林植物,纤鞭是其重要的攀援器官,也是重要的分类依据。研究黄藤NAC(NAM,ATAF和CUC)转录因子基因的分子特征并开发SSR分子标记,以期为棕榈藤的分子育种和辅助分类提供参考依据。【方法】以黄藤为材料,借助转录组数据,采用同源克隆的方法从黄藤中分离NAC基因,采用生物信息学方法进行基因结构、蛋白性质与结构分析和SSR位点预测,采用实时定量PCR技术分析基因的组织表达特性,利用PAGE电泳和测序技术分析SSR分子标记在不同棕榈藤样品中的通用性和多态性。【结果】从黄藤叶片中获得了2个NAC同源基因DjNAC3(Gen Bank登录号:KU556738)和DjNAC4(Gen Bank登录号:KX579750),二者的开放阅读框长度分别为729 bp和1 326 bp,对应的基因组序列为850 bp和1 441 bp,均包含2个外显子和1个内含子。DjNAC3和DjNAC4编码的蛋白分别为242 aa和441 aa。蛋白结构分析表明,DjNAC3和DjNAC4具有典型的NAC转录因子结构特征,属于NAC家族的CUC亚家族,但二者之间的相似系数仅为23.6%,表明它们在黄藤生长发育过程中可能具有不同的功能。DjNAC3和DjNAC4在不同组织中的表达模式存在明显差异,DjNAC3在发育成熟纤鞭中的表达丰度最高,叶片中的表达丰度最低,而DjNAC4则在发育成熟的钩刺中表达丰度最高,而发育初期的钩刺中最低。在DjNAC3和DjNAC4的基因组序列中分别包含1个SSR位点,其中前者的SSR位点位于内含子区域,为(TA)6,后者的SSR位点位于第1个外显子区域,为(GCA)5。根据DjNAC3和DjNAC4中SSR位点旁侧序列设计引物,以黄藤和另外20个不同棕榈藤样品的基因组DNA为模板进行扩增,PAGE电泳结果分析表明,引物具有较高的通用性,且扩增产物具有多态性。6个样品的测序结果证实,用DjNAC3设计引物的扩增产物测序获得的SSR位点序列存在着一定的差异,既包括SSR类型变异、重复次数变化等多态性,又有SSR位点缺失的现象;而根据DjNAC4设计引物扩增获得的SSR位点序列差异主要为重复次数的变化。【结论】黄藤DjNAC3和DjNAC4基因的基因结构、表达模式、SSR位点等均存在明显的差异,这表明它们在黄藤生长发育中可能具有不同的功能,二者所包含的SSR分子标记具有通用性和多态性,可以作为分子标记应用于棕榈藤的辅助分类和分子辅助育种。

【Objective 】 Rattan is one of the important forest plants,cirri and flagella are important organs for its climbing habit,which are also the important basis for rattan classification.In order to provide a basis for molecular breeding and SSR-assisted classification of rattan,study on the molecular characteristics of NAC （NAM,ATAF and CUC）transcription factor genes in Daemonorops jenkinsiana and the development of SSR markers was performed in this paper.【Method】With the aid of transcriptome data,the sequences of NAC homologue genes in D.jenkinsiana were isolated by PCR method.The analyses of gene structure,protein properties and structure as well as the prediction of SSR locus in NAC genes were conducted using bioinformatics method.The tissue specific expression of NAC genes in different tissues were analyzed using real-time quantitative PCR （qPCR）.To evaluate the universality and polymorphism of the developedSSR markers,PAGE electrophoresis and sequencing analyses were used on the basis of products amplified with different rattan samples.【Result 】 Two homologous genes,DjNAC3 （Gen Bank No.KU556738） and DjNAC4 （Gen Bank No.KX579750）,were obtained from leaves of D.jenkinsiana,of which open reading frame （ORF） were 729 bp and 1 326 bp,respectively.The genomic sequence corresponding to the ORFs of DjNAC3 and DjNAC4 were 850 bp and 1 441 bp,which all contained two exons and one intron.The proteins encoded by DjNAC3 and DjNAC4 were 242 aa and 441 aa respectively.Protein structure analysis showed that DjNAC3 and DjNAC4 had the typical structural features of NAC transcription factors,which were belonged to the CUC subfamily of NAC family.However,the similarity coefficient between DjNAC3 and DjNAC4 was only 23.6%,indicating that they might have different functions in the growth process of D.jenkinsiana.The expression patterns of DjNAC3 and DjNAC4 in different tissues were obviously different.DjNAC3 was expressed in developed cirri with the highest level,and the lowest in leaves,while that of DjNAC4 was the highest abundance in developed barbs and the lowest in early developed barbs.The SSR loci were detected in the genomic sequences of DjNAC3 and DjNAC4,the SSR locus of （TA）6was located in the intron region of DjNAC3 and that of （GCA）5was in the first exon of DjNAC4.SSR primers were designed according to the flanking sequences of SSR loci in DjNAC3 and DjNAC4.Genomic DNAs of D.jenkinsiana and other 20 different rattan samples were selected as templates for amplification.PAGE electrophoresis analysis showed that the primers had universality and polymorphism in the samples.The sequencing result of amplification products from six different templates further confirmed the polymorphism such as variation of SSR type,the number change of repetitions,and SSR locus missing,which were found in the sequences generated by the primer pair designed based on DjNAC3.The main difference in the sequences generated by the primer pair designed according to DjNAC4 was the number change of repetitions.【Conclusion 】 There are significant differences between DjNAC3 and DjNAC4,such as gene structures,gene expression patterns,and SSR locus,which means that DjNAC3 and DjNAC4 might play different roles in the growth and development of D.jenkinsiana.The universality and polymorphism of SSR markers developed from DjNAC3 and DjNAC4 indicate that they can serve as molecular markers for rattan classification and molecular-assisted breeding.