【作者】 张慧；

【导师】 田汇；

【作者基本信息】 西北农林科技大学 ， 植物营养学， 2022， 硕士

【摘要】 丛枝菌根(Arbuscular mycorrhiza,AM)真菌与小麦形成互惠共利的菌根共生体,一方面AM真菌从植物根部获取其生长所需的碳水化合物,另一方面可帮助植物从土壤中吸收矿质营养和水分,从而促进植物生长,增强其抗逆性。小麦作为一种菌根作物,AM真菌可以促进小麦对矿质养分的吸收与利用、增强其抗逆性。本研究拟探究不同育成年代、株高、麦芒类型、生活习性、主栽区域的小麦品种菌根侵染率差异,筛选出极端差异品种,并利用高通量测序技术明确菌根侵染过程中关键基因对菌根侵染的响应,比较菌根共生参与基因对菌根侵染响应的差异状况,这对于理解小麦菌根共生形成的分子机制具有重要意义。本研究以291份小麦品种为试验材料,于2017-2018和2018-2019年在西北农林科技大学的网室内进行两个生长季节的盆栽试验,使用Claroideoglomus etunicatum,Rhizophagus intraradices,Funneliformis mosseae,Acaulospora delicata四种菌剂进行混合接种,测定并计算小麦成熟期根系的菌根侵染率,并筛选出菌根侵染率差异显著的小麦品种(小红麦(低侵染)和扬麦14(高侵染)),后通过转录组分析比较两个品种中菌根共生参与基因(转录因子、转运蛋白、酶类和其他基因)的表达对菌根侵染的响应,旨在探究小麦菌根侵染率基因型差异的分子机制。论文主要得出以下结果:1.在两个生长季,小麦品种间的AM侵染率显著不同。2017-2018生长季的AM侵染率范围为23.5%-91.7%;2018-2019生长季的AM侵染率为4.2%-90.7%。两个生长季的平均侵染率都在53%左右,并且两个生长季的菌根侵染率最大值都达到了90%以上。扬麦14的菌根侵染率显著高于小红麦,二者侵染率差异可达7倍以上。2.在两个生长季中,不同生活习性以及不同麦芒类型的小麦品种间菌根侵染率不存在显著性差异。在2017-2018生长季,矮秆小麦品种的平均菌根侵染率较中杆小麦品种高7%;源自北美的小麦菌根侵染率(平均值为60.9%)显著高于源自西亚(平均值为47.5%)的小麦品种。在2018-2019生长季,矮秆小麦品种菌根侵染率(平均值为55.31%)显著高于高杆(平均值为49.04%);1970年之前育成的小麦品种AM侵染率显著低于1991年后育成的小麦品种;源自澳大利亚的小麦菌根侵染率(平均值为65.5%)显著高于来自西亚(平均值为41.9%)和南亚(平均值为42.0%)地区的小麦品种。3.在极端低侵染率小麦品种小红麦根中共筛选到6401个差异表达基因(DEG),其中表达上调基因4328个,表达下调基因2073个;在极端高侵染率小麦品种扬麦14中共筛选到8279个差异表达基因,其中表达上调基因5562个,表达下调基因2717个。GO(Gene Ontology)富集分析发现,这些DEG主要定位在细胞膜系统中,参与防御反应和脱落酸反应相关生物学过程,行使铁离子结合、蛋白丝氨酸/苏氨酸激酶活性、氧化还原酶活性和单加氧酶活性等分子功能;KEGG(Kyoto Encyclopedia of Genes and Genomes)富集分析表明,在低侵染品种小红麦中,DEG主要参与氮代谢、二萜生物合成以及脂肪酸生物合成途径。在高侵染品种扬麦14中,DEG主要参与氮代谢、二萜生物合成、脂肪酸生物合成、磷酸肌醇代谢以及类胡萝卜素生物合成途径。4.通过转录组分析比较了两个极端侵染品种中菌根共生参与基因(转录因子、转运蛋白、酶类和其他基因)对菌根侵染的响应,检测到GRAS、AP2和MYB等3类转录因子家族基因响应菌根侵染,绝大多数被菌根侵染上调表达,仅有2个MYB转录因子基因在两品种小麦中对菌根侵染的响应表现出明显差异,Myb转录因子基因Traes CS7D02G470000在小红麦中表达上调,在扬麦14中不表达,而另外一个MYB转录因子基因Traes CS5D02G411800在扬麦14中表达上调,在小红麦中则不表达。5.检测到编码ABC转运蛋白、磷酸盐转运蛋白、蔗糖转运蛋白和铵转运蛋白的基因响应菌根侵染,其中19个ABC转运蛋白基因对菌根侵染的响应强烈(差异倍数≥4)。ABC转运蛋白编码基因Traes CS4A02G384800和Traes CSU02G223100在扬麦14中表达上调,且差异倍数在32倍以上,而它们在小红麦中则对菌根侵染不响应或弱响应。6.检测到12类酶编码基因响应菌根侵染,其中24个基因的表达差异倍数在16倍以上,3个E3泛素连接酶基因,1个枯草杆菌蛋白酶家族基因、6个细胞色素P450家族基因以及2个丝氨酸羧肽酶编码基因在两个小麦品种中对菌根侵染的响应状况明显不同,所有这些基因的差异表达倍数均在25倍以上。7.检测到3类106个其它菌根共生参与基因也显著响应菌根侵染,其中3个F-box家族基因在两个小麦品种中对菌根侵染的响应情况不同,基因Traes CS7D02G518400在小红麦中受菌根侵染强烈下调;在扬麦14中基因Traes CS5D02G367000和Traes CS7A02G063600均被上调,且差异表达倍数在16倍以上。本研究共鉴定到57个在两个小麦品种中对菌根侵染响应明显差异的基因,这些基因可能在影响小麦菌根侵染率基因型差异的过程中发挥关键作用。综上所述,本研究所调查的291个小麦品种中,多数品种的菌根侵染率高于50%,菌根侵染率高于70%的品种主要包括扬麦14、ENESCO和INDIANO MOCHO 6296等,低于30%的品种主要有小红麦、冀麦6号、北京837和扬麦2号等;春性小麦和冬性小麦之间、有芒小麦和无芒小麦之间具有相似的菌根侵染率;株高的降低有利于小麦根系被AM真菌侵染;源自南美与西亚的小麦品种较其他地区菌根侵染率低;Traes CS7D02G113900、Traes CS4A02G412100和Traes CS2D02G029500等3个基因可能在影响小麦菌根侵染率方面发挥关键作用。更多还原

【Abstract】 Arbuscular mycorrhiza(AM)fungi can form mutually beneficial mycorrhizal symbionts with wheat.On the one hand,AM fungi obtain carbohydrates for their growth from plant roots,and on the other hand,they help plants obtain mineral nutrients and water from the soil,thereby promoting plant growth and enhancing its resistance to stress.AM fungi can promote the absorption and utilization of mineral nutrients in wheat and enhance its resistance to stress.This study amied to:(1)explore the genotypic variations of AM colonization rates of wheat with different growth habits,release years,origins,awn types and plant heights;(2)identify the wheat varieties with extreme AM colonization rates,and then investigate the responses of the genens involved in mycorrhizal symbiosis to AM colonization at the transcriptomic level.This is of great significance for understanding the molecular mechanisms behind the development of wheat-AM symbiosis.In this study,291 wheat varieties were used as experimental materials,and pot experiments were carried out in the net house of Northwest A&F University in the year 2017-2018 and 2018-2019.Pots were inoculated with a mixture AM inoculum of Claroideoglomus etunicatum,Rhizophagus intraradices,Funneliformis mosseae and Acaulospora delicata,and the AM colonization rate of wheat roots was determined at the mature stage.The wheat varieties with extreme AM colonization rate(XHM,low root colonization and YM14,high root colonization)were screened out.The responses of the genes(transcription factors,transporters,enzymes and other genes)involved in mycorrhizal symbiosis to AM colonizaiton in the two varieties were investigated by transcriptome analysis.The main results of the paper are as follows:1.AM colonization rates were significantly different among the 291 wheat cultivars in the two growth seasons.In 2017-2018,the AM colonization rate ranged from 23.5% to 91.7%;and the range was 4.2%-90.7% in 2018-2019.The average colonization rates in both growth seasons were around 53%,and the maximum colonization rate in both growing seasons reached over 90%.The mycorrhizal colonization rate of YM14 was significantly higher than that of XHM,and the difference could be more than 7 times between the two varities.2.In the two growth seasons,there was no significant difference in mycorrhizal colonization rate among wheat varieties with different living habits and different awn types.In 2017-2018,the average mycorrhizal colonization rate of dwarf wheat varieties was 7%higher than that of medium-stem wheat varieties;the mycorrhizal colonization rate of wheat from North America(average of 60.9%)was significantly higher than that from West Asia(average 47.5%)of wheat varieties.In 2018-2019,the mycorrhizal colonization rate of dwarf wheat varieties(average 55.31%)was significantly higher than that of high-stem wheat varieties(average 49.04%);and the AM colonization rate of the wheat varieties released before1950 was significantly lower than that of the wheat varieties released after 1991;mycorrhizal colonization rate of wheat from Australia(average 65.5%)was significantly higher than that of the wheat varieties from West Asia(average 41.9%)and South Asia(average 42.0%).3.A total of 6,401 differentially expressed genes(DEGs)were obtianed in the wheat cultivar XHM,including 4,328 up-regulated genes and 2,073 down-regulated genes.A total of 8,279 DEGs were found in the wheat variety YM14,including 5,562 up-regulated genes and 2,717 down-regulated genes.GO(Gene Ontology)enrichment analysis showed that these DEGs were mainly located in the cell membrane system,involved in defense responses and abscisic acid response-related biological processes,and exercised iron ion binding,protein serine/threonine kinase activity,oxidoreductase activity and monooxygenase activity;KEGG(Kyoto Encyclopedia of Genes and Genomes)enrichment analysis showed that the DEGs were mainly involved in nitrogen metabolism,diterpene biosynthesis and fatty acid biosynthesis pathway in XHM.In YM14,the DEGs were mainly involved in nitrogen metabolism,diterpene biosynthesis,fatty acid biosynthesis,phosphoinositide metabolism and carotenoid biosynthesis pathway.4.Three types of transcription factor genes including GRAS,AP2 and MYB were found to respond to mycorrhizal colonization,and most of them were up-regulated by AM colonization.Only two MYB transcription factor genes have distinct different responses to mycorrhizal colonization between the two varieties of wheat.The MYB gene Traes CS7D02G470000 was up-regulated in XHM but not in YM14,while the gene Traes CS5D02G411800,which was also annotated as MYB transcription factor,was upregulated in YM14 but not in XHM.5.Genes encoding ABC transporter,phosphate transporter,sucrose transporter an d ammonium transporter were detected to response to mycorrhizal colonization,of wh ich 19 ABC transporter genes responded most strongly to mycorrhizal colonization(L og2(Fold change)≥2).The ABC transporter-encoding genes Traes CS4A02G384800 and Traes CSU02G223100 were up-regulated in YM14,and the difference was more than32 times,while they did not or weakly responded to mycorrhizal colonization in XH M.6.We detected 12 types of enzyme-encoding genes could respond to AM colonizaiton,of which 24 genes had more than 16-fold difference in expression,another three E3 ubiquitin ligase genes,one subtilisin family gene,six cytochrome P450 family genes and two serine carboxypeptidase genes in the two wheat varieties were distinct different,and the differential expression fold was more than 25 times.7.The expression of 106 other genes involved in mycorrhizal symbiosis also responded to mycorrhizal colonization.Among them,three F-box family genes had different responses to mycorrhizal colonization in the two wheat varieties.The gene Traes CS7D02G518400 was strongly down-regulated in XHM after mycorrhizal colonization;and the genes Traes CS5D02G367000 and Traes CS7A02G063600 were both up-regulated in YM14,and the differential expression fold was more than 16-fold.A total of 57 genes showed different responses to mycorrhizal colonization between the two wheat varieties,which may play key roles in influencing the genotypic differences of AM colonization in wheat.In conclusion,among the 291 wheat cultivars investigated in this study,the mycorrhizal colonization rate of most cultivars is higher than 50%,and the cultivars with mycorrhizal colonization rate higher than 70% mainly include YM14,ENESCO and INDIANO MOCHO6296,etc.The varieties with less than 30% are mainly wheat XHM,JIMAI6,BEIJING837 and YANGMAI2,etc.;there are similar bacteria between spring wheat and winter wheat,and between awn wheat and awnless wheat.The root colonization rate;the reduction of plant height is conducive to the colonization of wheat roots by AM fungi;the mycorrhizal colonization rate of wheat varieties from South America and West Asia is lower than that of other regions;Traes CS7D02G113900,Traes CS4A02G412100 and Traes CS2D02G029500 three genes may affect the mycorrhizal colonization of wheat play a key role.更多还原