苹果叶片不定芽再生过程的差异表达基因鉴定与分析

doi:10.3864/j.issn.0578-1752.2021.16.011

摘要/Abstract

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5 250、4 937、6 852、6 493个DEGs,4个时间点共有的DEGs有3 027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定芽再生相关的基因,研究结果为深入探讨苹果离体叶片再生机理提供了理论依据。

关键词: 苹果, 不定芽再生, RNA-Seq, 差异表达基因, 影响因子

Abstract:

【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, 5 250, 4 937, 6 852 and 6 493 DEGs were identified at 3, 7, 14 and 21 d post culture, respectively, and 3 027 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 signaling pathways were analyzed. The results of qRT-PCR showed that most of these DEGs were up-regulated, which was consistent with data of RNA-Seq. 【Conclusion】Through the detection and comparative analysis of large-scale gene expression profiles in adventitious shoot of ‘GL-3’ apple leaves at different time points, a number of genes related to adventitious shoot regeneration of apple leaves were obtained, which could provide a basis for further study on the mechanism of apple leaves in vitro regeneration.

Key words: apple, leaves regeneration, RNA-Seq, differentially expressed genes, impact factors

刘锴,何闪闪,张彩霞,张利义,卞书迅,袁高鹏,李武兴,康立群,丛佩华,韩晓蕾. 苹果叶片不定芽再生过程的差异表达基因鉴定与分析[J]. 中国农业科学, 2021, 54(16): 3488-3501.

LIU Kai,HE ShanShan,ZHANG CaiXia,ZHANG LiYi,BIAN ShuXun,YUAN GaoPeng,LI WuXing,KANG LiQun,CONG PeiHua,HAN XiaoLei. Identification and Analysis of Differentially Expressed Genes in Adventitious Shoot Regeneration in Leaves of Apple[J]. Scientia Agricultura Sinica, 2021, 54(16): 3488-3501.

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基因ID Gene ID	基因名称 Gene name	正向引物 Forward primer	反向引物 Reverse primer
MD02G1100200	SAUR72	AATTTCAGGAGACCTCCGCC	CGTTGATCGTGTCGTCGTCT
MD10G1121700	LAX2	AACACAACTTCCGCTCCTCC	CCGGGCGTGGAGTAATCAAA
MD05G1052100	SAUR14	TTCCGTCTTCCGGCTGTAAT	TCTCGCCAACATAAACCGCA
MD15G1208300	CKX3	ATACAACCCAGCAGCCGTTT	CCTCTGGTGGAATGACCGTG
MD01G1164700	MSD1	GCGCTCCGATTAGGTCTTCA	TGGATCTGCATGATCTCGCC
MD03G1014400	PA2	ACGGAAGCGGAGATACGTTG	CTGATCGGTCTGGAGAAGCC
MD10G1103500	LBD38	CCGTTTTGTTGTTGCCGAGT	CAACGCAATGCAAGAGTGCT
MD17G1073700	RAV2	TTGACCAAGGGATGGACACG	TGAACCGCAGACTATCGACG
MD13G1252700	PLT2	CCTCAGTTTGCTACTGCCGA	AACCACGGAGGAAACTAGCG
MD03G1118500	IFL	TGCGGCAGTACCCATTGATT	AGGCTTCCACATGACAGCAA
MD08G1077100	ANT	AATCCGTCCTCGTTGGTGAC	CCGCAGAGGTTGGACTTACA
MD01G1162100	PRX52	GCCTTGCAAGATGCACAACA	AGGTTGTCGTCATTTCCGCT

基因ID Gene ID	GO功能 GO function	KEGG途径 KEGG_pathway
MD00G1137700	植物细胞壁 Plant-type cell wall;胞间连丝 Plasmodesma
MD13G1023200	防御反应 Defense response;对生物刺激的反应 Response to biotic stimulus
MD15G1208300	细胞分裂素代谢过程Cytokinin metabolic process;氧化还原过程Oxidation- reduction process;膜的整体成分 Integral component of membrane;氧化还原酶活性 Oxidoreductase activity;细胞分裂素脱氢酶活性 Cytokinin dehydrogenase activity;黄素腺嘌呤二核苷酸结合 Fflavin adenine dinucleotide binding	玉米素生物合成 Zeatin biosynthesis
MD08G1161200	磷酸化信号转导系统 Phosphorelay signal transduction system;胞内受体 Intracellular	植物激素信号转导 Plant hormone signal transduction
MD11G1093400	氧化还原过程 Oxidation-reduction process;氧化还原酶活性Oxidoreductase activity	托烷、哌啶和吡啶生物碱的生物合成 Tropane, piperidine and pyridine alkaloid biosynthesis
MD04G1089800	果胶分解过程 Pectin catabolic process;膜的整体成分 Integral component of membrane;果胶裂解酶活性 Pectate lyase activity;金属离子结合 Metal ion binding	戊糖和葡萄糖醛酸转换 Pentose and glucuronate interconversions
MD03G1192000	-	-
MD12G1226800	磷酸化信号转导系统 Phosphorelay signal transduction system;细胞分裂素激活信号通路 Cytokinin-activated signaling pathway;磷酸化 Phosphorylation;氧化还原过程 Oxidation-reduction process;细胞核 Nucleus;细胞质 Cytoplasm;组氨酸磷酸转移激酶活性 Histidine phosphotransfer kinase activity;2-烯醛还原酶活性 2-Alkenal reductase activity;蛋白质组氨酸激酶结合 Protein histidine kinase binding	植物激素信号转导 Plant hormone signal transduction