不同糖源对葡萄试管苗蛋白激酶相关基因表达的影响
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摘要
【目的】探究不同外源糖对葡萄试管苗生长发育及蛋白激酶基因转录调控的影响,应用转录组测序挖掘蛋白质磷酸化过程中的基因,为葡萄蛋白激酶相关基因功能的验证奠定一定基础。【方法】在基本培养基中分别添加2%的蔗糖、葡萄糖和果糖,以无糖为对照,分别命名为S20、G20、F20和CK,经过37 d培养后,测定不同处理的地上和地下鲜重,并采用Illumina HiSeq~(TM) 2000对各处理叶片进行转录组测序,通过综合生物信息学分析(参考基因组比对、差异基因(DEGs)筛选、COG(Cluster of Orthologous Groups of proteins)注释、GO(Gene Ontology)注释等)筛选出蛋白激酶相关基因,通过qRT-PCR分析该蛋白激酶相关基因的表达特性。【结果】F20、G20和S20处理下的葡萄(‘红地球’)试管苗与CK相比,地上鲜重具有明显差异,且F20最高,而G20地下鲜重最高。SNP统计发现,转换是主要的变异类型,颠换次之,且发生在基因间区的SNP数量最多,其次是下游;剪接位点供体和同义终止发生的基因数量最少且相等。4个样品中共获得了2 633个差异基因,3个处理与CK相比,共有差异基因180个且被聚类为3组,第一组中127个基因仅在CK中高表达,第二组19个基因仅在G20下高表达,而第三组34个基因在3个处理下表达模式不尽相同。这些共有的差异基因在COG中注释到了26个基因并分在11个功能类别中,且主要注释在一般的功能类别中。在GO分类中,共有的基因分别被注释在分子功能、生物学过程和细胞组分的14、22和13个功能类别中。共筛选出7种蛋白激酶,分别为葡萄糖激酶(Glucokinase,GK)、丝裂原活化蛋白激酶(Mitogen-activated protein kinases,MAPKs)、钙调蛋白激酶(Calcineurin protein kinase,CBL)、蛋白磷酸酶2(Protein phosphatase 2,PP2A)、己糖激酶(Hexokinase,HXK)、组氨酸蛋白激酶(Histidine protein kinase,HPK)和酪氨酸激酶(Tyrosine kinase,TK),其不同激酶的基因在不同处理中具有各自的表达模式,经qRT-PCR验证,选择的20个差异基因中有17个基因表达与转录组测序结果相一致。【结论】在葡萄试管苗培养中,果糖较葡萄糖和蔗糖相比对生长较好。测序得出180个差异基因对3种不同糖均作出响应,这些基因在COG数据库中主要富集在膜酯转运和代谢、次级代谢物和碳水化合物的合成、转运和分解;GO中大多注释在蛋白激酶和氧化还原酶的活性中;筛选出了7种蛋白激酶,这些差异基因在数量、功能分类和代谢通路上对糖的响应各不相同。
【Objective】 To explore the effects of different exogenous sugars on the growth and development of grape plantlets and the regulation of protein kinase gene transcription, the candidate genes were tapped in the process of protein phosphorylation by using transcription, which made a foundation for the verification of grape protein kinase-related gene function.【Method】 Sucrose(2%), glucose(2%) and fructose(2%) were added to the basic medium, and the free-sugar treatment was as control, which were named as S20, G20, F20 and CK, respectively. After 37 days of culture, the fresh weight of the leaf-stem and root under different treatments was determined. Transcriptome sequencing of each treated foliages was performed by using Illumina HiSeq~(TM) 2000, and a series of protein kinases related genes were screened by integrated bioinformatics analysis, including reference genomic alignment,differentially expressed gene(DEGs) screening, COG(Cluster of Orthologous Groups of proteins) annotation, GO(Gene Ontology)annotation, etc., and the expression characteristic of these genes were further analyzed by qRT-PCR. 【Result】 Compared with CK,‘Red Globe' grape plantlets under F20, G20 and S20 treatments exhibited significant differences in the fresh weight of leaf-stem, and the highest was obtained by F20 treatment, while the weight of fresh root under G20 was the highest. The SNP statistics found that the Transition was the main type of mutation, the second was Transversion. The highest number of SNPs that occurred in the Intergenic, and the next was the Upstream. Splice_Site_Donor and Synonymous_Stop events occurred with the least number of genes and equal. A total of 2 633 deferentially expressed genes were obtained in the 4 samples. The Venn diagram showed that there were a total of 180 differential genes under the 3 treatments compared with CK, and these genes were clustered into 3 groups. In the first group, 127 genes were only highly expressed under CK. The 19 genes of the second group were only highly expressed under G20,while the expression patterns of the 34 genes in third group were different under three treatments. The common 180 differential genes were annotated with 26 genes in the COG database to 11 functional categories, and these DEGs were mainly enriched in general functional categories. In the annotation of GO, the common genes were annotated in 14, 22 and 13 functional categories of molecular function, biological process and cellular component, respectively. Seven kinds of protein kinases were screened by this sequencing, including Glucokinase(GK), Mitogen-activated protein kinases(MAPKs), Calcineurin protein kinase(CBL), Protein phosphatase 2(PP2 A), Hexokinase(HXK), Histidine protein kinase(HPK) and Tyrosine kinase(TK), and these different protein kinases genes showed their own expression patterns among different treatments. By qRT-PCR analysis, 17 out of 20 screened genes expression were consistent with the transcriptome sequencing results. 【Conclusion】 Compared with glucose and sucrose, fructose was the best sugar during grape culture process. The sequencing results showed that 180 DEGs all responded to three different sugars.In the COG annotation, these genes were mainly enriched in membrane ester transport and metabolism, the synthesis, transport and decomposition of secondary metabolites and carbohydrates. In the GO databases, the most of common DEGs were annotated in the activities of protein kinases and oxidoreductases. Seven protein kinases were identified, which were selectively in responses to different exogenous sugars in quantity, functional, category and metabolic pathways, and had their own choice of expression specificity.
【Objective】 To explore the effects of different exogenous sugars on the growth and development of grape plantlets and the regulation of protein kinase gene transcription, the candidate genes were tapped in the process of protein phosphorylation by using transcription, which made a foundation for the verification of grape protein kinase-related gene function.【Method】 Sucrose(2%), glucose(2%) and fructose(2%) were added to the basic medium, and the free-sugar treatment was as control, which were named as S20, G20, F20 and CK, respectively. After 37 days of culture, the fresh weight of the leaf-stem and root under different treatments was determined. Transcriptome sequencing of each treated foliages was performed by using Illumina HiSeq~(TM) 2000, and a series of protein kinases related genes were screened by integrated bioinformatics analysis, including reference genomic alignment,differentially expressed gene(DEGs) screening, COG(Cluster of Orthologous Groups of proteins) annotation, GO(Gene Ontology)annotation, etc., and the expression characteristic of these genes were further analyzed by qRT-PCR. 【Result】 Compared with CK,‘Red Globe' grape plantlets under F20, G20 and S20 treatments exhibited significant differences in the fresh weight of leaf-stem, and the highest was obtained by F20 treatment, while the weight of fresh root under G20 was the highest. The SNP statistics found that the Transition was the main type of mutation, the second was Transversion. The highest number of SNPs that occurred in the Intergenic, and the next was the Upstream. Splice_Site_Donor and Synonymous_Stop events occurred with the least number of genes and equal. A total of 2 633 deferentially expressed genes were obtained in the 4 samples. The Venn diagram showed that there were a total of 180 differential genes under the 3 treatments compared with CK, and these genes were clustered into 3 groups. In the first group, 127 genes were only highly expressed under CK. The 19 genes of the second group were only highly expressed under G20,while the expression patterns of the 34 genes in third group were different under three treatments. The common 180 differential genes were annotated with 26 genes in the COG database to 11 functional categories, and these DEGs were mainly enriched in general functional categories. In the annotation of GO, the common genes were annotated in 14, 22 and 13 functional categories of molecular function, biological process and cellular component, respectively. Seven kinds of protein kinases were screened by this sequencing, including Glucokinase(GK), Mitogen-activated protein kinases(MAPKs), Calcineurin protein kinase(CBL), Protein phosphatase 2(PP2 A), Hexokinase(HXK), Histidine protein kinase(HPK) and Tyrosine kinase(TK), and these different protein kinases genes showed their own expression patterns among different treatments. By qRT-PCR analysis, 17 out of 20 screened genes expression were consistent with the transcriptome sequencing results. 【Conclusion】 Compared with glucose and sucrose, fructose was the best sugar during grape culture process. The sequencing results showed that 180 DEGs all responded to three different sugars.In the COG annotation, these genes were mainly enriched in membrane ester transport and metabolism, the synthesis, transport and decomposition of secondary metabolites and carbohydrates. In the GO databases, the most of common DEGs were annotated in the activities of protein kinases and oxidoreductases. Seven protein kinases were identified, which were selectively in responses to different exogenous sugars in quantity, functional, category and metabolic pathways, and had their own choice of expression specificity.
引文
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