梨胶锈菌侵染西府海棠和山荆子转录组比较_王学府.pdf

分子植物育种，2023 年，第 21 卷，第 3 期，第 754-763 页Molecular Plant Breeding,2023,Vol.21,No.3,754-763研究报告Research Report梨胶锈菌侵染西府海棠和山荆子转录组比较王学府1狄红梅1曹秋芬2*1 晋中职业技术学院生物工程系,晋中,030602;2 山西农业大学生命科学学院,太原,030002*通信作者,摘要研究苹果属西府海棠和山荆子在梨胶锈菌侵染下的生理和分子响应机制，发掘抗病相关的功能基因，对研究苹果属植物的抗病分子机制有重要意义。对西府海棠及山荆子分别接种梨胶锈菌，进而对接种锈菌前后的西府海棠和山荆子进行高通量转录组测序，并利用生物信息学方法对基因表达和功能进行分析。离体叶片抗病性鉴定表明，山荆子对梨胶锈菌的抗性最强，西府海棠抗性最弱；高通量测序表明，有74.73%80.61%的 clean 数据比对到唯一的基因组位点，说明测序质量好，可信度高；采用 RPKM(Reads PerKilobase of exon model per Million mapped reads,每百万读段中来自于某个基因每千碱基上映射的平均读段数)法计算基因表达量，共筛选出山荆子和西府海棠接种梨胶锈菌后的 1 185 个差异表达基因。其中 687 个差异基因在基因本体联合会数据库(gene ontology,GO)中富集，主要功能有酶活性、代谢过程和防御反应等。有 320 个差异基因能归入京都基因与基因组百科全书(kyoto encyclopedia of genes and genomes,KEGG)通路，包括植物病原体相互作用、植物激素信号转导、泛素化信号通路等过程。其中，在 13 个富集在植物病原体互作通路的差异基因中，有 5 个表现为钙调素类蛋白功能，说明在钙信号通路的活跃是山荆子相较西府海棠对锈病有较强抗性的重要原因。山荆子和西府海棠抗锈病是一个高度复杂、多途径协同的过程，由蛋白质代谢、防御反应、激素调节、泛酸化等通路共同调控，其结果可为后期深入研究苹果属抗病分子机制奠定基础，为抗病育种提供理论依据。关键词西府海棠;梨胶锈菌;转录组;差异表达基因;功能ComparativeTranscriptomeofMalus micromalus and Malus baccata InfectedbyGymmosporangium haraeanumWang Xuefu1Di Hongmei1Cao Qiufen2*1 Jinzhong Vocational&Technical College,Department of Biotechnology Jinzhong,030602;2 College of Life Science,Shanxi Agricultural Universi-ty,Taiyuan,030002*Corresponding author,DOI:10.13271/j.mpb.021.000754AbstractThe objective of this study was to study the physiological and molecular response mechanisms ofMalus genus plants of Malus micromalus and Malus baccata infected by Gymmosporangium haraeanum,and to ex-plore the functional genes related to disease resistance,which is of great significance to study the disease resis-tance mechanisms of Malus genus plants.We inoculated M.micromalus and M.baccata with G.haraeanum,andthen carried out high-throughput transcriptome sequencing on M.micromalus and M.baccata before and after inoc-ulation with G.haraeanum,and analyzed the gene expression and function by bioinformatics method.In vitro leafdisease resistance evaluation showed that the resistance of M.baccata was the strongest,and that of M.micro-malus was the weakest.High throughput sequencing showed that 74.73%80.61%of clean data were compared tothe unique genome locus,indicating good sequencing quality and high reliability.The gene expression level was基金项目：本研究由山西省重点研发计划项目(201803D221003-8)资助引用格式：Wang X.F.,Di H.M.,and Cao Q.F.,2023,ComparativetranscriptomeofMalus micromalus and Malus baccata infectedbyGymmosporangiumharaeanum,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),21(3):754-763.(王学府,狄红梅,曹秋芬,2023,梨胶锈菌侵染西府海棠和山荆子转录组比较,分子植物育种,21(3):754-763.)calculated byRPKM(Reads Per Kilobase of exon model per Million mapped reads)method,and 1 185 differentiallyexpressed genes were screened after inoculation of M.baccata and M.micromalus.Among them,687 differentialgeneswereenrichedinGO(GeneOntology),whosemainfunctionswereenzymeactivity,metabolicprocessanddefen-seresponse.There are 320 DEGscould be included in the KEGG(Kyoto Encyclopedia ofGenesand Genomes)path-ways,including plant pathogen interaction,plant hormone signal transduction,ubiquitination signal pathway andotherprocesses.Amongthe13differentiallyenrichedgenesinplantpathogeninteractionpathways,5showed calmo-dulin-like protein function,indicating that the activity in calcium signaling pathway is an important reason for thestrongerresistanceofM.baccata toG.haraeanum comparedwith M.micromalus.G.haraeanum resistance ofM.bac-cata and M.micromalus is a highly complex and multi-pathway process,which is jointly regulated by protein meta-bolism,defense response,hormone regulation,panacidification and other pathways.The results can lay a foundationfor further research on molecular mechanism of resistance to apple,and provide a theoretical basis for breeding forresistance to disease.KeywordsMalus micromalus Makino;Gymmosporangium haraeanum Syd.;Transcriptome;Differentially ex-pressed gene;Function西府海棠(Malus micromalus Makino)又名海红果，属蔷薇科(Rosaceae)苹果属(Malus)，是中国常见的园林树木(刘源等,2018)。同属野生种主要有山荆子(Malus baccata)、湖北海棠(Malus hupehensis)、变叶海棠(Malus toringoides)、陇东海棠(Malus kansuensis)和三叶海棠(Malus sieboldii)等，在中国陕西、山西和内蒙古等省(区)的丘陵、中高海拔地区有零星分布。苹果属的野生种已经广泛地经人类驯化，大大减少了它的自然种群，很多品种濒临灭绝，亟需保护(Yangetal.,2019)。西府海棠能够忍耐-30 低温，抗涝抗旱，对环境的适应极强，养护简单，花色绚丽有“花中仙子”之称，因而被广泛应用于国内外的园林景观中(李瑶和承河元,2000)。近年来，中国西府海棠锈病发生情况极为严重，海棠锈病同时也会危害梨、苹果等经济作物，已经对园林经济乃至果树产业造成极大影响。西府海棠锈病的病原菌主要为梨胶锈菌(Gymmospo-rangium haraeanum Syd.)，梨胶锈菌属于担子菌亚门冬孢纲锈菌目胶锈菌属。春季气温在 1522时，冬孢子在柏科植物上遇雨水后萌发产生担孢子，借风雨传播到海棠、梨和苹果等蔷薇科植物上，夏季产生锈孢子，秋季重新传到桧柏嫩梢上过冬(王杰花等,2019)。目前，园林中主要依靠化学药剂防治西府海棠锈病，但化学药剂成本高，长期使用会造成农药残留、病原菌抗药性等问题(高源等,2018)。利用品种抗性是防治植物病害最基本、最经济、最有效的途径。但是西府海棠的抗锈病育种进展缓慢，目前还未发现对锈病有较强抗性的品种。因此，挖掘抗梨胶锈菌种质资源，明确抗锈病机理在中国苹果属园林植物抗锈病育种中具有重要意义。随着遗传图谱标记技术、转录组测序等分子生物学新技术的广泛应用，现有栽培品种已不能满足研究和育种的需要。与现有栽培种有亲缘关系的野生种具有广泛的遗传背景和优良的抗性基因。利用野生种作为育种材料，进行抗性基因挖掘和抗病种质创新已成为育种的重要突破方向。苹果属的野生种在抗病性上往往有优良的性状，苹果属中的山荆子对褐斑病菌有明显的抗性(王洁等,2012)，可用于进一步的抗病机理研究。利用组织染色技术发现山荆子在接种褐斑病菌后活性氧积累高于苹果(范涛等,2015)。武山变叶海棠、新疆野苹果、三叶海棠、滇池海棠、海棠花、野苹果 9 号、中熟红果子和腾冲三叶海棠等 8 个野生种对苹果褐斑病有较强的抗性(殷丽华,2013)。但是这些研究都集中在苹果相关病害上，国内外对西府海棠锈病相关分子机制的研究还是一个空白。近年来，高通量测序技术在生物学领域研究越来越广泛，转录组测序技术(RNA-seq)可以快