苹果叶片不定芽再生过程的差异表达基因鉴定与分析  

Identification and Analysis of Differentially Expressed Genes in Adventitious Shoot Regeneration in Leaves of Apple

作　　者：刘锴 何闪闪 张彩霞[1] 张利义[1] 卞书迅 袁高鹏 李武兴[1] 康立群 丛佩华[1] 韩晓蕾[1] LIU Kai;HE ShanShan;ZHANG CaiXia;ZHANG LiYi;BIAN ShuXun;YUAN GaoPeng;LI WuXing;KANG LiQun;CONG PeiHua;HAN XiaoLei(Research Institute of Pomology,Chinese Academy of Agricultural Sciences/Key Laboratory of Horticultural Crop Germplasm Resources Utilization,Ministry of Agriculture and Rural Areas/National Apple Breeding Center,Xingcheng 125100,Liaoning)

机构地区：[1]中国农业科学院果树研究所/农业部园艺作物种质资源利用重点实验室/国家苹果育种中心,辽宁兴城125100

出　　处：《中国农业科学》2021年第16期3488-3501,共14页Scientia Agricultura Sinica

基　　金：中央级公益性科研院所基本科研业务费专项(Y2019XK09);国家现代农业产业技术体系建设专项(CARS-27);中国农业科学院科技创新工程(CAAS-ASTIP-2016-RIP-02)。

摘　　要：【目的】筛选分析‘GL-3’苹果叶片不定芽再生过程中的差异表达基因(differentially expressed gene,DEG),进一步解析苹果叶片不定芽再生的潜在分子机制,为提高苹果的遗传转化效率提供理论参考。【方法】‘GL-3’苹果继代组培苗叶片外植体接种在再生培养基上,分别于0、3、7、14和21 d后取样并提取RNA,构建mRNA文库后采用Illumina Nova seq平台进行测序。筛选出各时间点的DEGs,根据GO(Gene ontology)和KEGG(Kyoto encyclopedia of genes and genomes)注释结果以及官方分类,使用R软件中的phyper函数对筛选到的DEGs进行GO和KEGG富集分析;利用BLAST软件进行基因比对注释;重点分析植物再生相关的激素、酶、转录因子、多胺等DEGs;采用qRT-PCR对DEGs进行定量验证。【结果】再生培养基上培养3、7、14和21 d的苹果叶片外植体与对照组相比,分别筛选到5250、4937、6852、6493个DEGs,4个时间点共有的DEGs有3027个。DEGs的GO功能富集显示,4个时间点筛选到的共有DEGs中上调表达的DEGs主要与氧化还原过程、细胞外围、蛋白激酶活性和有机环化合物结合等功能有关;下调表达的DEGs主要与单细胞代谢过程、钙离子结合、光合膜和类囊体部分等功能有关。DEGs的KEGG通路富集分析显示,4个时间点筛选到的共有DEGs中上调表达的DEGs主要富集在磷酸戊糖途径、植物激素信号转导、植物-病原菌相互作用和内质网蛋白质加工等途径中;下调表达的DEGs主要富集在α-亚麻酸代谢、苯丙烷生物合成、碳代谢和光合作用等途径中。对与植物离体叶片再生相关的激素、酶、转录因子和多胺等相关DEGs的表达模式进行分析发现,这些DEGs大部分呈上调表达趋势。经qRT-PCR验证后,所检测基因的表达趋势与转录组测序结果一致。【结论】通过对苹果叶片不定芽再生过程中不同时间点的基因表达谱进行检测和对比分析,获得了大量与苹果叶片不【Objective】In this study,the differentially expressed genes(DEGs)in adventitious shoot regeneration of‘GL-3’apple leaves were screened.The potential mechanism of adventitious shoot regeneration of apple leaves was analyzed,which will contribute to develop an efficient genetic transformation system for apple.【Method】The explants of‘Gl-3’apple were cultured on regeneration medium.Samples were taken for RNA extraction and construction of mRNA library at 3,7,14 and 21 d post culture,respectively,further sequenced on the Illumina Nova seq platform.On the basis of the Kyoto Encyclopedia of Gene and Genome(KEGG)and Gene ontology(GO),the terms and pathway enrichment were then analyzed using the Phyper function with R software.Gene annotation was performed by using BLAST software.The DEGs related to plant regeneration,such as hormones,enzymes,transcription factors(TFs)and polyamines were analyzed,the expression levels of DEGs were verified by qRT-PCR.【Result】Compared with the control group,5250,4937,6852 and 6493 DEGs were identified at 3,7,14 and 21 d post culture,respectively,and 3027 DEGs were shared in all four points.GO functional enrichment analysis showed that the up-regulated DEGs shared in all four points were mainly related to oxidation reduction process,cell periphery,protein kinase activity and organic cyclic compound binding,while the down-regulated DEGs were mainly related to single organism metabolic process,calcium ion binding,photosynthetic membrane and thylakoid part.KEGG pathway enrichment analysis indicated that the up-regulated DEGs shared in all four points were significantly enriched in pentose phosphate pathway,plant hormone signal transduction,plant pathogen interaction and protein processing in endoplasmic reticulum,while the down-regulated DEGs were significantly enriched in alpha linolenic acid metabolism,phenylpropanoid biosynthesis,carbon metabolism and photosynthesis.In addition,the DEGs encoding transcription factors,enzymes,and components of hormone biosynthesis and sig

关 键 词：苹果 不定芽再生 RNA-SEQ 差异表达基因 影响因子 

分 类 号：S661.1[农业科学—果树学]