基于第二代转录组、第三代全长转录组测序条件下草石蚕基因表达特性的初步研究

Preliminary Study on Gene Expression Characteristics of Stachys sieboldii based on Second-Generation and Third-Generation Whole Transcriptome Sequencing Conditions

以宁夏贺兰县地方草石蚕(Stachys sieboldii)品种为试材,探讨应用第三代测序技术获得草石蚕全长转录本信息,应用第二代测序技术获得3个不同发育阶段草石蚕叶片和块茎的转录组信息,对测序结果进行转录组水平分析,筛选特有差异基因,并进行GO和KEGG富集分析,开展草石蚕基因表达特性的初步研究。结果表明,第三代测序后Polymerase read的数据量为50.82 G,FLNC序列的reads数为525593个转录本;在KEGG等7大数据库的基因功能注释中均注释成功的转录本数目为6857个,至少有1个数据库注释成功的转录本数目为14078个;与NR数据库比对注释后,草石蚕与同为唇形目的芝麻(Sesamum indicum)基因序列相似性最高,相似基因个数为9149个;与GO数据库比对注释后,生物学过程、细胞成分与分子功能中注释到基因个数最多的分别是新陈代谢过程5093个、细胞2004个、键联结合6645个;与KEGG数据库比对注释后,在细胞转化、环境信息处理、遗传信息处理、新陈代谢和有机系统功能中注释到基因数最多的分别是运输和分解代谢409个、信号传导729个、转化626个、碳水化合物代谢601个、内分泌系统297个。第二代测序后,ZLZ_2_S_3和ZLZ_2_S_1差异基因比较中的上调基因数最高,为2303个;ZLZ_2_L_3和ZLZ_2_S_3差异比较中的下调基因数最高,为2033个。叶片3个时期ZLZ_2_L_1、ZLZ_2_L_2与ZLZ_2_L_3组合之间比较差异基因总数为13610个,共有差异基因数为10203个;ZLZ_2_L_3独有的差异基因数最多,为437个。块茎3个时期ZLZ_2_S_1、ZLZ_2_S_2与ZLZ_2_S_3的组合之间比较差异基因总数为13732个,共有的差异基因数为11370个,ZLZ_2_S_3独有的差异基因数最多,为412个。ZLZ_2_S_3、ZLZ_2_S_2聚类图显示高表达基因的数值范围主要为0~2,显著高于其他处理,可以聚为一类。3个不同时期叶片的差异基因主要集中在光合作用生物碳固定、内质网中蛋白质加工以及乙醛酸和二羧酸代谢等通路上;3个不同时期块茎的差异基因主要集中在淀粉和蔗糖代谢、糖解与糖代谢合成等通路上。以上结论将为今后研究草石蚕的生物学特性、阐明特有表型差异机制提供参考,为提升草石蚕的基础理论研究水平提供技术支撑。

Stachys sieboldii,a local variety from Helan County in Ningxia,was used as the experimental material to explore the application of third-generation sequencing technology to obtain the full-length transcriptome information of Stachys sieboldii.Second-generation sequencing technology was used to obtain the transcriptome information of leaves and tubers at three different developmental stages of Stachys sieboldii.The sequencing results were analyzed at the transcriptome level,specific differentially expressed genes were screened,and GO and KEGG enrichment analyses were conducted to conduct a preliminary study on the gene expression characteristics of Stachys sieboldii.The results showed that the data volume of Polymerase read after third-generation sequencing was 50.82 G,and the number of reads of FLNC sequences was 525593 transcripts.Among the gene function annotations in the seven major databases,the number of annotated transcripts was 6857,and the number of transcripts with at least one successful annotation in the database was 14078.After aligning with the NR database,the gene sequence of Stachys sieboldii showed the highest similarity to that of Sesamum in-dicum,which also belongs to the Lamiales order,with 9149 similar genes.After aligning with the GO database,the numbers of genes annotated in biological processes,cellular components,and molecular functions were 5093,2004 and 6645,respectively.After aligning with the KEGG database,the numbers of genes annotated in cell transformation,environmental information processing,genetic information processing,metabolism,and organic system functions were 409,729,626,601 and 297,respectively.After second-generation sequencing,the highest number of upregulated genes in the comparison between ZLZ_2_S_3 and ZLZ_2_S_1 was 2303.The highest number of downregulated genes in the comparison between ZLZ_2_L_3 and ZLZ_2_S_3 was 2033.The total number of differentially expressed genes between the three stages of leaves(ZLZ_2_L_1,ZLZ_2_L_2,and ZLZ_2_L_3)was 13610,with a total of 10203 differentially expressed genes.ZLZ_2_L_3 had the highest number of unique differentially expressed genes,with 437.The total number of differentially expressed genes between the three stages of tubers(ZLZ_2_S_1,ZLZ_2_S_2,and ZLZ_2_S_3)was 13732,with a total of 11370 differentially expressed genes.ZLZ_2_S_3 had the highest number of unique differentially expressed genes,with 412.Cluster analysis showed that ZLZ_2_S_3 and ZLZ_2_S_2 had a higher expression level of genes mainly distributed between 0 and 2,significantly higher than other treatments,and could be clustered into one category.The differentially expressed genes in the three stages of leaves mainly concentrated in pathways such as photosynthesis,carbon fixation in photosynthetic organisms,protein processing in the endoplasmic reticulum,and glyoxylate and dicarboxylate metabolism.The differentially expressed genes in the three stages of tubers mainly concentrated in pathways such as starch and sucrose metabolism,glycolysis/gluconeogenesis,and pyruvate metabolism.These conclusions will provide references for future studies on the biological characteristics of Stachys sieboldii and elucidate the mechanisms of unique phenotypic differences,thereby providing technical support for the improvement of the basic theoretical research level of Stachys sieboldii.