利用iTRAQ技术和转录组筛选芍药属远缘杂交不亲和基因

doi:10.3864/j.issn.0578-1752.2020.06.015

Abstract

Abstract:

【Objective】Distant hybrid breeding is the main method of cultivar improvement and breeding in tree peony and herbaceous peony, while cross-incompatibility is an important restriction for breeding rapid development. Based on the previous researches, the analysis on different protein of stigma in pollen-stigma interaction and transcriptome was further explored. The mechanism of cross-incompatibility between tree peony and herbaceous peony was revealed, so as to provide the theoretical support for hybridized breeding.【Method】The stigmas of combinations Paeonia lactiflora ‘Fenyunu’ × P. lactiflora ‘Fenyunu’ and P. lactiflora ‘Fenyunu’ × P. ostii ‘Fengdanbai’ were harvested at 24 h after pollination, which were used as materials for isobaric tags and analysis for relative and absolute quantitation (iTRAQ) and transcriptome, respectively. Bioinformatics was analyzed on the data of protein and transcriptome. Quantitative Real-time PCR (qRT-PCR) technique was used to validate the expression data of selected differentially expressed genes (DEGs). 【Result】iTRAQ was used to analyze DEPs of stigma of distant hybrid between tree peony and herbaceous peony, and the result showed that 685 DEPs were belonged to 188 pathways, in which 18 pathways were significantly enriched. There were four pathways with obvious difference in protein, including RNA degradation, mitogen-activated protein kinase (MAPK) signaling pathway, calcium signaling pathway, and phosphatidylinositol signaling system. In RNA degradation pathway, enolase, DnaK (HSP70), and GroEL (HSP60) were all down-regulated. In calcium signaling pathway, calmodulin (CALM) was down-regulated, while adenine nucleotide translocase (ANT) was up-regulated. In MAPK signaling pathway, Glyoxalase (GloI) was down-regulated. In phosphatidylinositol signaling system, CALM was also down-regulated. 6 genes were selected randomly to confirmed their expression by qRT- PCR, and the result showed that the expression profiles of the selected genes was in agreement with the results from protein analysis, and they were all down-regulated. A total of 52 998 annotated Unigenes were obtained by transcriptome sequencing, accounting for 40.37% of all Unigenes. Based on the RPKM (Reads Per Kilobase per Million) of six samples, 16 224 DEGs were obtained, among which13 61 were up-regulated, and 2 863 were down-regulated. Based on pathway enrichment analysis of DEGs, it indicated that the level of enrichment of DEGs in “Oxidative phosphorylation”, “ABC transporters” and “Biosynthesis of secondary metabolites” pathways were more significant and reliable than that of selfing. The genes related with incompatibility of distant hybridization were CalS-5, CalS-12 (Callose enzyme), and SPL, which were up regulating expression, but ABCF that was down regulating expression. 【Conclusion】In the data of transcriptome and protein, 6 proteins and 4 genes were closely related to incompatibility of distant hybridization. These proteins and genes might play an important role in incompatibility of distant hybridization.

Key words: Paeonia suffruticosa, Paeonia lactiflora, iTRAQ, transcriptome, distant hybridization

Dan HE,DongBo XIE,JiaoRui ZHANG,SongLin HE,ChaoMei LI,YunBing ZHENG,Zheng WANG,YiPing LIU,Yan LI,JiuXing LU. Selected Related Genes about Incompatibility of Distant Hybridization in Paeonia by iTRAQ Analysis and Transcriptome[J].Scientia Agricultura Sinica, 2020, 53(6): 1234-1246.

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References 33

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差异蛋白登记号 DEP Accessions	基因ID Gene ID	引物序列（5′-3′） Primer sequence (5′-3′)
gi\|327424468_1	JI445505	F:TGCTCTCACCTCTCTCCCAAAC
gi\|327424468_1	JI445505	R:GCCTTGCCTTGACGCTCTTG
gi\|327436891_2	JI457928	F:GTGCTTCAAGGTGAGAGAGAGTTC
gi\|327436891_2	JI457928	R:GCCGAGACAGATAGGATACCATTG
gi\|327427673_4	JI448710	F:CGTGTTTGTAGGATTCGTTACTGC
gi\|327427673_4	JI448710	R:ACGCCATCGGTAGTTGCTTTC
gi\|327426876_5	JI447913	F:TTAGCACCAGCACCTTTGAACAG
gi\|327426876_5	JI447913	R:ACCGTCCGAAGAAGAATGATGATG
gi\|327427197_1	JI448234	F:GGACCAAAACGGCTTCATTTCTG
gi\|327427197_1	JI448234	R:CTCGTCTACTTCCTCATCTGTCAG
gi\|327433480_5	JI454517	F:CTTCAGGTCCATAGCCCATCATTG
gi\|327433480_5	JI454517	R:GTGTCAAGTAATGCTCCGAGTAGG

通路 Pathway	差异蛋白数 DEPs	通路编号 Pathway ID
RNA降解 RNA degradation	14 (2.05%)	ko03018
MAPK信号途径 MAPK signaling pathway	9 (1.32%)	ko04010
钙信号途径 Calcium signaling pathway	4 (0.59%)	ko04020
磷脂酰肌醇信号系统 Phosphatidylinositol signaling system	1 (0.18%)	ko04070

通路 Pathway	差异蛋白名称 DEP name	注释的差异基因 Different genes with pathway annotation	通路编号 Pathway ID
RNA降解RNA degradation	烯醇酶Enolase,热休克蛋白 DnaK,病菌抗原GroEL	7	ko03018
MAPK信号途径MAPK signaling pathway	乙二醛酶Ⅰ GloI	2	ko04010
钙信号途径Calcium signaling pathway	腺苷酸转运酶 ANT,钙调蛋白 CALM	2	ko04020
磷脂酰肌醇信号系统Phosphatidylinositol signaling system	钙调蛋白 CALM	1	ko04070

基因ID Gene ID	KEGG	GO注释 GO pathway	Swissprot	基因名称 Gene name	表达量 Expression
CL268_All	K11000/胼胝质酶 Callose enzyme	GO:0006075/1,3-β-D-葡聚糖生物合成过程 Biosynthesis of 1,3-β-D-glucan GO:0003843/1,3-β-D-葡聚糖合成酶活性 1,3-β-D-glucan synthetase activity GO:0016021/膜的整体组成部分 Integral part of membrane GO:0000148/1,3-β-D-葡聚糖合成酶复合物 1,3-β-D-glucan synthetase complex	胼胝质合酶5 Callose synthase 5	CalS-5	上调 Up
Unigene36776_All	K11000/胼胝质酶 Callose enzyme		胼胝质合酶12 Callose synthase 12	CalS-12	上调 Up
CL8637_All	ko6158/ATP结合盒 ATP-binding cassette	GO:0046686/对镉离子的反应 Reaction to cadmium ion GO:0005524/ATP结合 ATP binding GO:0005829/细胞质 Cytoplasm GO:0042742/防御对细菌的反应 Defense against bacterial reactions GO:0016887/ATP酶活性 ATP enzyme activity	ABC transporter F family member 3	ABCF	下调 Down
CL7449_All	ko04075/植物激素信号转导 Phytohormone signal transduction ko01001/蛋白激酶 Protein kinase	GO:0046872/结合重金属离子 Binding heavy metal ions GO:0003677/DNA结合核 DNA binding nucleus	Squamosa promoter- binding-like protein 16	SPL	上调 Up

Selected Related Genes about Incompatibility of Distant Hybridization in Paeonia by iTRAQ Analysis and Transcriptome

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