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52998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16224个差异基因。其中上调基因13361,下调基因2863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。
【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 52998 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,16224 DEGs were obtained,among which1361 were up-regulated,and 2863 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.
作者
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
HE Dan;XIE DongBo;ZHANG JiaoRui;HE SongLin;LI ChaoMei;ZHENG YunBing;WANG Zheng;LIU YiPing;LI Yan;LU JiuXing(College of Forestry,Henan Agricultural University,Zhengzhou 450002;Colleg of Horticulture Landscape Architecture,Henan Institute of Science and Technology,Xinxiang 453000,Henan)
出处
《中国农业科学》
CAS
CSCD
北大核心
2020年第6期1234-1246,共13页
Scientia Agricultura Sinica
基金
国家自然科学基金(31600568,31870698)
河南农业大学科技创新基金(KJCX2015A03,KJCX2018A05)
河南省科技攻关项目(192102110062,202102110234)
河南省高等学校重点科研项目(19A220003)。
关键词
牡丹
芍药
ITRAQ
转录组
远缘杂交
Paeonia suffruticosa
Paeonia lactiflora
iTRAQ
transcriptome
distant hybridization
