Identification and Analysis of Differentially Expressed Genes in Adventitious Shoot Regeneration in Leaves of Apple
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
Received:2020-09-19Accepted:2021-01-7 作者简介 About authors 刘锴,E-mail: liukai2429@163.com。
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. Keywords:apple;leaves regeneration;RNA-Seq;differentially expressed genes;impact factors
PDF (2082KB)元数据多维度评价相关文章导出EndNote|Ris|Bibtex收藏本文 本文引用格式 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 doi:10.3864/j.issn.0578-1752.2021.16.011 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 Acricultura Sinica, 2021, 54(16): 3488-3501 doi:10.3864/j.issn.0578-1752.2021.16.011
提取上述样品的总RNA,检测合格后委托北京贝瑞和康生物技术有限公司采用Illumina Nova seq测序平台进行二代双向测序。测序得到的数据经碱基识别分析后得到原始数据raw reads,过滤掉低质量、含N比例大于10%、接头污染的reads后得到适合分析的数据clean reads。使用比对软件BWA[16]将数据比对‘金冠’苹果基因组。
A:各时间点差异表达基因上下调数量统计;B:各时间点差异表达的散点图;C:各时间点差异表达基因韦恩图 Fig. 2The number of DEGs at each time point during adventitious bud regeneration of ‘Gl-3’ apple leaves
A: The number of up-regulated genes and down-regulated DEGs at each time point; B: Scattered plot of DEGs at each time point; C: The venn of DEGs at each time point
每一行代表一个差异表达基因,从左到右分别代表log2 (3 d FPKM/0 d FPKM)、log2 (7 d FPKM/0 d FPKM)、log2 (14 d FPKM/0 d FPKM)、log2 (21 d FPKM/0 d FPKM)。大于0表示上调,为红色;小于0表示下调,为绿色。下同 Fig. 5Heatmaps of DEGs involved in phytohormone signaling pathways, including IAA and CTK signaling pathways
Each horizontal row represents a DEG with its gene ID, and the vertical columns represent log2 (3 d FPKM/0 d FPKM), log2 (7 d FPKM/0 d FPKM), log2 (14 d FPKM/0 d FPKM), log2 (21 d FPKM/0 d FPKM) from left to right. Red for greater than 0 and up-regulated, green for less than 0 and down-regulated. The same as below
CONG PH, ZHANG CX, HAN XL, TIANY, ZHANG LY, LI WX. Current research situation and prospect of apple breeding in China China Fruits, 2018(6):1-5. (in Chinese) [本文引用: 2]
VELASCOR, ZHARKIKHA, AFFOURTITJ, DHINGRAA, CESTAROA, KALYANARAMANA, FONTANAP, BHATNAGAR SK, TROGGIOM, PRUSSD, SALVIS, PINDOM, BALDIP, CASTELLETTIS, CAVAIUOLOM, COPPOLAG, COSTAF, COVAV, DAL RIA, GOREMYKINV, et al. The genome of the domesticated apple (Malus × domestica Borkh.) Nature Genetics, 2010, 42(10):833-839. DOI:10.1038/ng.654URL [本文引用: 1]
CHANG YS, CHENG LL, WANG HB, HEP, LI HF, LI LG. Review of molecular marker and marker assisted breeding of apple Acta Horticulturae Sinica, 2017, 44(9):1658-1680. (in Chinese) [本文引用: 1]
JAMES DJ, PASSEY AJ, BARBARA DJ, BEVANM. Genetic transformation of apple (Malus pumila Mill.) using a disarmed Ti-binary vector Plant Cell Reports, 1989, 7(8):658-661. [本文引用: 1]
DAI HY, LI WR, HAN GF, YANGY, MA YE, LIH, ZHANG ZH. Development of a seedling clone with high regeneration capacity and susceptibility to Agrobacterium in apple Scientia Horticulturae, 2013, 164:202-208. DOI:10.1016/j.scienta.2013.09.033URL [本文引用: 3]
VIDALN, MALLÓNR, VALLADARESS, MEIJOMÍN AM, VIEITEZ AM. Regeneration of transgenic plants by Agrobacterium- mediated transformation of somatic embryos of juvenile and mature Quercus robur Plant Cell Reports, 2010, 29(12):1411-1422. DOI:10.1007/s00299-010-0931-8URL [本文引用: 1]
RICHARD LB, RALPHS, DELORESL. Adventitious shoot regeneration of pear (Pyrus spp.) genotypes. Plant Cell, Tissue and Organ Culture (PCTOC), 2012, 108(2):229-236. [本文引用: 1]
ZHAO ZY, FU RM, SHUI SQ, ZHANG XQ, HUANGY. Study on the regeneration of apple plantlets from the leaves Shaanxi Journal of Agricultural Sciences, 1992(6):18-19. (in Chinese) [本文引用: 1]
IIZASAS, IIZASAE, WATANABEK, NAGANOY. Transcriptome analysis reveals key roles of AtLBR-2 in LPS-induced defense responses in plants BMC Genomics, 2017, 18(1):995. DOI:10.1186/s12864-017-4372-4URL [本文引用: 1]
XIANG YN, HUANG RR, GU TT, GAN LJ. Analysis of RNA- Seq-based expression profiles during adventitious shoot regeneration in Arabidopsis thaliana Journal of Nanjing Agricultural University, 2018, 41(2):308-320. (in Chinese) [本文引用: 1]
CHEP, LALLS, NETTLETOND, HOWELL SH. Gene expression programs during shoot, root, and callus development in Arabidopsis tissue culture Plant Physiology, 2006, 141(2):620-637. DOI:10.1104/pp.106.081240URL [本文引用: 1]
MAYER K FX, SCHOOFH, HAECKERA, LENHARDM, JÜRGENSG, LAUXT. Role of WUSCHEL in regulating stem cell fate in the Arabidopsis shoot meristem Cell, 1998, 95(6):805-815. DOI:10.1016/S0092-8674(00)81703-1URL [本文引用: 2]
ZHANG TQ, LIANH, TANG HB, DOLEZALK, ZHOU CM, YUS, CHEN JH, CHENQ, LIU HT, LJUNGK, WANG JW. An intrinsic MicroRNA timer regulates progressive decline in shoot regenerative capacity in plants The Plant Cell, 2015, 27(2):349-360. DOI:10.1105/tpc.114.135186URL [本文引用: 1]
LIH, DURBINR. Fast and accurate short read alignment with Burrows-Wheeler transform Bioinformatics, 2009, 25(14):1754-1760. DOI:10.1093/bioinformatics/btp324URL [本文引用: 1]
OHY, DONOFRION, PAN HQ, COUGHLANS, BROWN DE, MENG SW, MITCHELLT, DEAN RA. Transcriptome analysis reveals new insight into appressorium formation and function in the rice blast fungus Magnaporthe oryzae Genome Biology, 2008, 9(5):R85. DOI:10.1186/gb-2008-9-5-r85URL [本文引用: 1]
LIVAK KJ, SCHMITTGEN TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method Methods (San Diego, Calif.), 2001, 25(4):402-408. DOI:10.1006/meth.2001.1262URL [本文引用: 1]
ATTAR, LAURENSL, BOUCHERON-DUBUISSONE, GUIVARC'HA, CARNEROE, GIRAUDAT-PAUTOTV, RECHP, CHRIQUID. Pluripotency of Arabidopsis xylem pericycle underlies shoot regeneration from root and hypocotyl explants grown in vitro The Plant Journal, 2009, 57(4):626-644. DOI:10.1111/tpj.2009.57.issue-4URL [本文引用: 1]
SUGIMOTOK, JIAO YL, MEYEROWITZ EM. Arabidopsis regeneration from multiple tissues occurs via a root development pathway Developmental Cell, 2010, 18(3):463-471. DOI:10.1016/j.devcel.2010.02.004URL [本文引用: 1]
DUCLERCQJ, SANGWAN-NORREELB, CATTEROUM, SANGWAN RS. De novo shoot organogenesis: from art to science Trends in Plant Science, 2011, 16(11):597-606. DOI:10.1016/j.tplants.2011.08.004URL [本文引用: 1]
ZHANG TQ, LIANH, ZHOU CM, XUL, JIAO YL, WANG JW. A two-step model for de novo activation of WUSCHEL during plant shoot regeneration The Plant Cell, 2017, 29(5):1073-1087. DOI:10.1105/tpc.16.00863URL [本文引用: 2]
WILLIAMS AC, FORD W CL. Functional significance of the pentose phosphate pathway and glutathione reductase in the antioxidant defenses of human sperm Biology of Reproduction, 2004, 71(4):1309-1316. DOI:10.1095/biolreprod.104.028407URL [本文引用: 1]
FAJKUSJ, FULNEČKOVÁJ, HULÁNOVÁM, BERKOVÁK, ŘÍHAK, MATYÁŠEKR. Plant cells express telomerase activity upon transfer to callus culture, without extensively changing telomere lengths Molecular and General Genetics MGG, 1998, 260(5):470-474. DOI:10.1007/s004380050918URL [本文引用: 1]
BHATIAR, DALTONS, ROBERTS LA, MORON-GARCIA OM, IACONOR, KOSIKO, GALLAGHER JA, BOSCHM. Modified expression of ZmMYB167 in Brachypodium distachyon and Zea mays leads to increased cell wall lignin and phenolic content Scientific Reports, 2019, 9(1):8800. DOI:10.1038/s41598-019-45225-9URL [本文引用: 1]
TAKEDAY, KOSHIBAT, TOBIMATSUY, SUZUKIS, MURAKAMIS, YAMAMURAM, RAHMAN MM, TAKANOT, HATTORIT, SAKAMOTOM, UMEZAWAT. Regulation of CONIFERALDEHYDE 5-HYDROXYLASE expression to modulate cell wall lignin structure in rice Planta, 2017, 246(2):337-349. DOI:10.1007/s00425-017-2692-xURL [本文引用: 1]
SHANGB, XUC, ZHANGX, CAOH, XINW, HUY. Very-long- chain fatty acids restrict regeneration capacity by confining pericycle competence for callus formation in Arabidopsis Proceedings of the National Academy of Sciences of the United States of America, 2016, 113(18):5101-5106. [本文引用: 1]
SUN BB, LIUJ, GE YC, SHENG LH, CHEN LQ, HU XM, YANG ZN, HUANGH, XUL. Recent progress on plant regeneration Chinese Science Bulletin, 2016, 61(36):3887-3902. (in Chinese) [本文引用: 1]
GAJ MD. Factors influencing somatic embryogenesis induction and plant regeneration with particular reference to Arabidopsis thaliana (L.) heynh Plant Growth Regulation, 2004, 43(1):27-47. DOI:10.1023/B:GROW.0000038275.29262.fbURL [本文引用: 1]
GRIENEISEN VA, XUJ, MARÉE A FM, HOGEWEGP, SCHERESB. Auxin transport is sufficient to generate a maximum and gradient guiding root growth Nature, 2007, 449(7165):1008-1013. [本文引用: 1]
GORDON SP, HEISLER MG, REDDY GV, OHNOC, DASP, MEYEROWITZ EM. Pattern formation during de novo assembly of the Arabidopsis shoot meristem Development (Cambridge, England), 2007, 134(19):3539-3548. DOI:10.1242/dev.010298URL [本文引用: 1]
ANGELA KS, SANG HL, JONATHAN PW, NATHALIEG, HIRONORII, DIRKI, WENDY AP, ANGUS SM, PAUL JO, WILLIAM MG. The SAUR19 subfamily of SMALL AUXIN UP RNA genes promote cell expansion The Plant Journal, 2012, 70(6):978-990. DOI:10.1111/tpj.2012.70.issue-6URL [本文引用: 1]
BARTRINAI, OTTOE, STRNADM, WERNERT, SCHMÜLLINGT. Cytokinin regulates the activity of reproductive meristems, flower organ size, ovule formation, and thus seed yield in Arabidopsis thaliana The Plant Cell, 2011, 23(1):69-80. DOI:10.1105/tpc.110.079079URL [本文引用: 1]
XU ZH, ZHANG XS, SU YH, HU YX, XUL, WANG JW. Plant cell totipotency and regeneration Science in China (Series C), 2019, 49(10):1282-1300. (in Chinese) [本文引用: 1]
FAN MZ, XU CY, XUK, HU YX. LATERAL ORGAN BOUNDARIES DOMAIN transcription factors direct callus formation in Arabidopsis regeneration Cell Research, 2012, 22(7):1169-1180. DOI:10.1038/cr.2012.63URL [本文引用: 1]
XU CY, CAO HF, ZHANG QQ, WANG HZ, XINW, XU EJ, ZHANG SQ, YU RX, YU DX, HU YX. Control of auxin-induced callus formation by bZIP59-LBD complex in Arabidopsis regeneration Nature Plants, 2018, 4(2):108-115. DOI:10.1038/s41477-017-0095-4URL [本文引用: 1]
LAUXT, MAYER KF, BERGERJ, JÜRGENSG. The WUSCHEL gene is required for shoot and floral meristem integrity in Arabidopsis Development (Cambridge, England), 1996, 122(1):87-96. DOI:10.1242/dev.122.1.87URL [本文引用: 1]
MENG WJ, CHENG ZJ, SANG YL, ZHANG MM, RONG XF, WANG ZW, TANG YY, ZHANG XS. Type-B ARABIDOPSIS RESPONSE REGULATORs specify the shoot stem cell niche by dual regulation of WUSCHEL The Plant Cell, 2017, 29(6):1357-1372. DOI:10.1105/tpc.16.00640URL [本文引用: 1]
HORSTMANA, WILLEMSENV, BOUTILIERK, HEIDSTRAR. AINTEGUMENTA-LIKE proteins: hubs in a plethora of networks Trends in Plant Science, 2014, 19(3):146-157. DOI:10.1016/j.tplants.2013.10.010URL [本文引用: 1]
KAREEMA, DURGAPRASADK, SUGIMOTOK, DU YJ, PULIANMACKAL AJ, TRIVEDI ZB, ABHAYADEV PV, PINONV, MEYEROWITZ EM, SCHERESB, PRASADK. PLETHORA genes control regeneration by a two-step mechanism Current Biology, 2015, 25(8):1017-1030. DOI:10.1016/j.cub.2015.02.022URL [本文引用: 1]
SHAFIA, GILLT, SREENIVASULUY, KUMARS, AHUJA PS, SINGH AK. Improved callus induction, shoot regeneration, and salt stress tolerance in Arabidopsis overexpressing superoxide dismutase from Potentilla atrosanguinea Protoplasma, 2015, 252(1):41-51. DOI:10.1007/s00709-014-0653-9URL [本文引用: 1]
TANGW, HARRIS LC, OUTHAVONGV, NEWTON RJ. Antioxidants enhance in vitro plant regeneration by inhibiting the accumulation of peroxidase in Virginia pine (Pinus virginiana Mill.) Plant Cell Reports, 2004, 22(12):871-877. [本文引用: 1]
SRIVASTAVAS, DWIVEDI UN. Plant regeneration from callus of Cuscuta reflexa-an angiospermic parasite- and modulation of catalase and peroxidase activity by salicylic acid and naphthalene acetic acid Plant Physiology & Biochemistry, 2001, 39(6):529-538. [本文引用: 1]
CHAI ML, JIA YF, CHENS, GAO ZS, WANG HF, LIU LL, WANG PJ, HOU DQ. Callus induction, plant regeneration, and long-term maintenance of embryogenic cultures in Zoysia matrella [L.] Merr. Plant Cell,Tissue and Organ Culture (PCTOC), 2011, 104(2):187-192. [本文引用: 1]
FLORES HE, GALSTON AW. Osmotic stress-induced polyamine accumulation in cereal leaves I. physiological parameters of the response Plant Physiology, 1984, 75(1):102-109. DOI:10.1104/pp.75.1.102URL [本文引用: 1]
SHOEBF, YADAV JS, BAJAJS, RAJAM MV. Polyamines as biomarkers for plant regeneration capacity: Improvement of regeneration by modulation of polyamine metabolism in different genotypes of indica rice Plant Science, 2001, 160(6):1229-1235. DOI:10.1016/S0168-9452(01)00375-2URL [本文引用: 1]
MUKHOPADHYAYA, CHOUDHURI MM, SENK, GHOSHB. Changes in polyamines and related enzymes with loss of viability in rice seeds Phytochemistry, 1983, 22(7):1547-1551. DOI:10.1016/0031-9422(83)80086-7URL [本文引用: 1]