中国农业科学 ›› 2021, Vol. 54 ›› Issue (16): 3488-3501.doi: 10.3864/j.issn.0578-1752.2021.16.011
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()
收稿日期:
2020-09-19
接受日期:
2021-01-07
出版日期:
2021-08-16
发布日期:
2021-08-24
通讯作者:
韩晓蕾
作者简介:
刘锴,E-mail: 基金资助:
LIU Kai(),HE ShanShan,ZHANG CaiXia,ZHANG LiYi,BIAN ShuXun,YUAN GaoPeng,LI WuXing,KANG LiQun,CONG PeiHua,HAN XiaoLei()
Received:
2020-09-19
Accepted:
2021-01-07
Online:
2021-08-16
Published:
2021-08-24
Contact:
XiaoLei HAN
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。
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.
表1
差异表达基因的定量引物验证"
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 |
表2
极显著差异表达基因的功能分析"
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 |
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