黄藤2个NAC基因的分子特征及其SSR分子标记开发

发布时间：2018-07-05 14:39

本文选题：黄藤 + NAC　；参考：《林业科学》2017年08期

【摘要】：【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。
[Abstract]:[objective] Rattan is an important forest plant, the whip is an important climbing organ and an important taxonomic basis. To study the molecular characteristics and develop SSR molecular markers of transcription factor gene of NAC (NAMU ATAF and CUC), in order to provide reference for molecular breeding and auxiliary classification of rattan. [methods] with the aid of transcriptome data, NAC gene was isolated by homologous cloning method. The gene structure, protein properties and structure analysis and SSR site prediction were analyzed by bioinformatics. The tissue expression characteristics of NAC gene were analyzed by real-time quantitative PCR. Page electrophoresis and sequencing techniques were used to analyze the versatility and polymorphism of SSR markers in different samples. [results] two NAC homologous genes DjNAC3 (Gen Bank accession number: KU556738) and DjNAC4 (Gen Bank accession number: KX579750) were obtained from leaves of Rhizoma lucifera. The length of the open reading frame was 729 BP and 1 326 BP, respectively, and the corresponding genomic sequence was 850 BP and 1 441 BP, respectively, containing two exons and one intron. DjNAC3 and DjNAC4 encoded a protein of 242aa and 441aa, respectively. The protein structure analysis showed that DjNAC3 and DjNAC4 had typical structural characteristics of NAC transcription factors and belonged to the CUC subfamily of NAC family. But the similarity coefficient between them was only 23.6B, which indicated that they might have different functions during the growth and development of Rhizoma vines. There were obvious differences in the expression patterns of DjNAC3 and DjNAC4 in different tissues. The expression of DjNAC3 was the most abundant in the mature whip. The expression abundance of DjNAC4 was the lowest in the leaves, but the highest in the mature and the lowest in the early stage of development. One SSR locus was found in the genomic sequence of DjNAC3 and one SSR locus was found in the intron region of DjNAC4. The SSR locus of the latter was (TA) 6, and the SSR locus of the latter was (GCA) 5. The primers were designed according to the SSR sequences in DjNAC3 and DjNAC4, and the genomic DNA of Rhizoma sinensis and 20 other samples were used as templates. The results of page electrophoresis showed that the primers had high versatility. The sequencing results of 6 samples confirmed that there were some differences in the sequence of SSR loci obtained by PCR products sequenced with DjNAC3 primers, including SSR type variation, repeat frequency variation and so on. The sequence difference of SSR locus based on DjNAC4 primer amplification was mainly the variation of repeat times. [conclusion] there were significant differences in gene structure, expression pattern and SSR loci of DjNAC3 and DjNAC4 genes. These results suggest that they may have different functions in the growth and development of Rattan, and the SSR molecular markers contained in them are versatile and polymorphic, which can be used as molecular markers for auxiliary classification and molecular assisted breeding of Rattan.
【作者单位】：国际竹藤中心国家林业局竹藤科学与技术重点开放实验室;河北农业大学园艺学院;
【基金】：“十二五”农村领域国家科技计划项目“竹藤种质资源创新利用研究”第一课题“竹藤优异种质创制创新与种苗培育标准化示范”(2015BAD04B01)
【分类号】：S792.91

【相似文献】