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

摘要/Abstract

摘要：

【目的】远缘杂交育种是目前牡丹、芍药品种改良和育种的主要方法,而远缘杂交不亲和一直是制约其快速发展的主要因素。本研究从牡丹、芍药远缘杂交授粉后不亲和应答相关的柱头差异蛋白与转录组方面深入研究,揭示牡丹、芍药远缘杂交不亲和的分子机理,为杂交育种提供理论依据。【方法】以芍药‘粉玉奴’自交、芍药‘粉玉奴’与牡丹‘凤丹白’杂交为供试材料,在授粉后24 h采取柱头,分别进行同位素标记相对定量（iTRAQ）和转录组技术分析。对所获得的蛋白和转录组数据进行生物信息学分析,并对其中可能与远缘杂交不亲和相关的基因进行定量PCR验证。【结果】利用iTRAQ技术分析牡丹、芍药远缘杂交后柱头中蛋白质的表达差异,共鉴定到685个差异蛋白,富集到了188条通路,其中显著富集的Pathway有18条。与不亲和授粉相关代谢通路有RNA降解、钙信号途径、丝裂原活化蛋白激酶（mitogen-activated protein kinase signaling pathway,MAPK）信号途径、磷脂酰肌醇信号系统。在RNA降解代谢通路中,烯醇酶（Enolase）、热休克蛋白DnaK（HSP70）及病菌抗原（GroEL）均表达下调。在钙信号途径中,钙调蛋白（CALM）表达下调,腺苷酸转运酶（adenine nucleotide translocase,ANT）表达量增加,表达上调。MAPK信号途径中,乙二醛酶Ⅰ（GloI）表达下调。磷脂酰肌醇信号系统中的钙调蛋白（CALM）表达下调。随机选取与差异蛋白相关的6个基因进行qRT-PCR验证,结果显示,6个基因的表达与蛋白质水平趋势相一致,均表达下调。通过转录组测序,共获得了52 998个有注释信息的Unigene,占所有Unigene的40.37%。基于6组样品的RPKM（Reads Per Kilobase per Million）值,共筛选到16 224个差异基因。其中上调基因13 361,下调基因2 863个。对差异基因进行Pathway显著富集分析,杂交与自交相比,不亲和差异表达的基因主要富集在氧化磷酸化代谢、ABC转运蛋白、次级代谢产物等通路。与远缘杂交不亲和相关且发生显著变化的基因有CalS-5、CalS-12（胼胝质酶）和SPL（squamosa promoter binding protein-like）表达上调,ABCF（ABC transporter family protein）表达下调。【结论】在转录组和蛋白数据共注释到6个蛋白、4个基因与植物不亲和性密切相关,这些蛋白与基因可能在远缘杂交不亲和方面发挥着重要作用。

关键词: 牡丹, 芍药, iTRAQ, 转录组, 远缘杂交

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

贺丹,谢栋博,张佼蕊,何松林,李朝梅,郑云冰,王政,刘艺平,栗燕,逯久幸. 利用iTRAQ技术和转录组筛选芍药属远缘杂交不亲和基因[J]. 中国农业科学, 2020, 53(6): 1234-1246.

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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链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2020.06.015

https://www.chinaagrisci.com/CN/Y2020/V53/I6/1234

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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