我国小麦秆锈菌兼Ug99监测新体系建立及其品种抗病基因分析
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摘要
小麦秆锈病是对小麦生产最具毁灭性的真菌病害，我国处在小麦秆锈病的特定流行区，秆锈病流行曾多次使我国小麦生产蒙受毁灭性损失。由于全球普遍引入抗秆锈病基因Sr31，保证了世界范围小麦秆锈病持续控制长达近40年。对Sr31具有高度致病力的小种Ug99（TTKSK)及其变异体的出现对包括我国在内的世界小麦生产造成了新的严重威胁。针对我国小麦秆锈病及Ug99的防控方面亟待解决的理论与关键技术问题，开展秆锈菌小种监测（部分工作配合国际小麦秆锈锈菌小种监测），中国的抗病品种和种质资源的筛选，抗病品种的基因检测和新抗性基因挖掘和中国重要辅助鉴别寄主Minn2761抗性相关的蛋白质组学研究。取得的主要进展如下。
1．国内首次研究确定了能够对Ug99及其变异体具有区分作用的新鉴别寄主体系，并用五辅音字母对新监测的菌株命名。由于近几年全国小麦秆锈病发生罕见的轻，2009-2010年从全国重要秆锈病流行麦区采集共得到75个菌株。共鉴定出8个不同小种，其出现频率分别为:21C3CTHTM为42.7%、34MKGRM为14.7%、21C3CPHTM为13.3%和21C3CTHTP为9.3%、34MKGTM为6.7%、21C3CFHTM为5.3%、21C3CTHRM和34MKGRP分别为4.0%。说明21C3系统仍然是我国发生最普遍和出现频率最高的小种类群，但34系统出现频率有所上升由16.9%上升到25.4%，暂未发现Ug99。利用47个小麦抗秆锈病单基因系对上述8个小种的毒力谱进行了测定，结果表明：毒力频率100%的基因有:Sr7a，7b，8a，9a，9b，9d，9f，9g，12，13，14，15，16，18，19，20，23，24，25，27，28，29，32，34，Tmp，Dp-2；80%以上的有：Sr6；50%以上都有：Sr10，11，17，Wld-1，GT；30%以上的有：Sr5；10%～30%的有: Sr38；毒力频率为0的基因有:Sr9e，21，22，26，30，31，33，35，36，37，并与Ug99及其变异小种的毒力谱进行了比较，结果发现对我国小种具有良好抗性的基因Sr9e，21，30，31和38等均能被Ug99克服，对我国优势小种和Ug99均具有抗性的基因为Sr22，26，33，35和37等，为今后筛选和利用兼抗中、外秆锈菌小种的有效抗病基因提供了可靠依据。
2.利用我国当前流行小种鉴定了57份国际抗Ug99小麦材料和1418份国内小麦品种（系）。国际材料中有51份材料对我国主要小种类型具有良好的抗性；在我国448份小麦种植品种中，对全部供试小种类型表现抗性的有147份，占32.8％。；长江中下游麦区的105份材料中只有1份对全部供试小种抗性表现免疫，其他都为高感品系；黄淮麦区的236份小麦高代系中对全部供试小种类型表现抗性的有64份，占27.6％；在东北春麦区供试的629份高代系中有562份表现为免疫至高抗，其中442份表现免疫，说明东北麦区育成的小麦品种对秆锈病的抗性较好。
3.开展小麦品种中重要抗秆锈基因的分子检测分析研究：对现有报道的抗秆锈病基因标记的实用性进行反复验证后，选取了重要抗病标记Sr26（兼抗Ug99）、Sr31和Sr36对我国小麦品种含有的抗秆锈性基因进行检测，通过对448份小麦品种的检测结果表明：在黑龙江的垦九10号和北京的保丰104中检测到Sr26；在多达70份的小麦品种检测到含有Sr31，其中河南13份；河北7份；四川9份，黑龙江4份；辽宁、山东5份；北京、陕西4份；湖北、甘肃和贵州2份；安徽、云南各1份小麦品种；引自国外品种4份及其他品种9份；在检测中尚未测到含Sr36的品种。其中，关于国内品种含有的Sr26以前鲜见报道。
4.国内首次.利用mRNA差异显示技术（DDRT-PCR）对我国重要辅助鉴别寄主明尼2761的与抗性相关基因进行了研究。筛选出24对引物能够在辅助鉴别寄主明尼2761接菌/未接菌和感病亲本萨其尔接菌/未接菌之间揭示出多态性，根据基因表达与否和表达量，获得9种不同差异表达的多态性条带类型，分别表示不同状态下测试材料的表达状况。通过对获得的cDNA差异片段进行测序，在NCBI网站上BLAST同源性比较分析，在GenBank数据库中并未找到与其具有同源性的已知序列。
5.建立了适合小麦叶片蛋白的高通量、高分辨率和高重复性的双向电泳技术体系，确定了双向电泳最佳条件：以改进的三氯乙酸/丙酮法提取小麦叶片总蛋白，裂解缓冲液为9M尿素，2M硫脲，4%CHAPS，1%DTT，1%TBP，0.5%IPG buffer，IEF等点聚焦最终控制在10000V，99999vh，SDS-PAGE胶浓度为12%。
6.首次对辅助鉴别寄主明尼2761进行双向电泳(2-DE)分离分析，结果显示：在明尼2761和萨其尔分别接菌/未接菌对比中，分别出现7个和6个差异表达蛋白点。经过比较共发现差异点11个。通过基质辅助激光解析电离飞行时间质谱技术(MALDI-TOF-TOF)获得了13个蛋白点的肤质量指纹图谱(PMF)，Mascot数据库搜索比对后，鉴定了8个蛋白质点，根据蛋白质功能的不同分为三类，第一类是与植物防卫相关的蛋白：ascorbate peroxidase(66)和glutathione synthetase (247)；第二类是与能量代谢相关蛋白：ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit RuBisCO largesubunit(152)；第三类是与光合作用相关的蛋白：23kDa oxygen evolving protein ofphotosystem II(437，524)；第四类是未知功能蛋白: ADP-ribosylation factor(498)，unknown(147,648)。它们分别参与了植物自身的防卫反应、能量代谢、光合作用等多个生理生化过程，这些上调或下调的蛋白都是复杂的蛋白调控网络中的一部分，在植物的抗病反应中协同起着重要作用。
Wheat stem rust caused by Puccinia graminis f.sp. tritici is a catastrophic wheat diseasebecause of its ability to cause complete annihilation of wheat crops over wide areas. China isin special wheat stem rust epidemic region and had suffered from several times of devastatingwheat losses due to the rust. For nearly four decades this disease has been under controlinternationally by widespread use of the1BL.1RS translocation with Sr31.Race variation isinternal reasons for wheat rust pandemic and resistance loss. Resurgence of raceUg99(TTKSK) is such a case. The race is highly virulent on Sr31and causes severe threat towheat production in China and the world. the urgent researches would be included in thefollowing aspects Race survey (a section of international wheat stem rust surveillance),Chinese resistant variety and germplasm screening, Chinese resistant variety gene detectionand new resistant gene mining, and Chinese important complementary host: Minn2761resistance related proteomics studies. The main progresses were as follows.
1. The new sets of wheat stem rust differential single gene lines and five-letter racenomenclature system which could distinguish new races Ug99and its mutants were used toidentify75isolates in the year2009-2010. Eight pathotypes, namely,21C3CTHTM,34MKGRM,21C3CPHTM,21C3CTHTP,34MKGTM,21C3CFHTM,21C3CTHRM and34MKGRP, were identified with frequencies of42.7%,14.7%,13.3%,9.3%,6.7%,5.3%,4.0%and4.0%, respectively. Only a few races were found, without finding new pathotypeUg99. Race21C3still remained predominant. The virulence spectra of eight race pathotypeswere determined on47single gene lines. The virulence frequencies for Sr7a,7b,8a,9a,9b,9d,9f,9g,12,13,14,15,16,18,19,20,23,24,25,27,28,29,32,34,Tmp,Dp-2were100%; thosefor Sr6, Sr10,11,17, Wld-1, GT, between50%-80%; those for Sr5and Sr38, between10%-30%; and those for Sr9e，21，22，26，30，31，33，35，36，37，0. It was indicatedthat Sr5,9e,11,21,30and31were highly resistant to Chinese wheat stem rust races currentlybut susceptible to Ug99and only Sr22,26,33,35and37were resistant to both Chinese racesand Ug99and could be utilized in breeding for resistance.
2. Using current main races of wheat stem rust in China, the resistance of57abroadUg99-resistant wheat cultivars and1418Chinese cultivars (lines) was appraised. It wasindicated that51wheat materials resistant to Chinese stem rust races and Ug99;52.0%of448cultivars from China also had good resistance, only1in105materials from Jiangsu was resistant to all races tested,64in236breeding lines from Shandong, resistant to all racestested;562in629wheat lines from Heilongjiang were highly resistant and442of them wereimmune.
3. Molecular detection for stem rust resistant genes carried in448Chinese commercialcultivars was conducted by using validated closely linked stem rust resistant gene markers,such as those of available STS and SSR markers of Sr26(resistant to Ug99), Sr31and Sr36.The result was indicated that among those cultivars, Kenjiu10grown in HeilongjiangProvince and Baofeng104grown in Beijing were detected to contain Sr26, about70cultivarswere shown to carry the Sr31and no Sr36-carrying varieties were met with.
4. For the first time in China, the mRNA differential display was used to study wheatstem rust resistance related protein of Chinese complementary differential: Minne2761.Twenty-four pairs of primers could amplify differential expression bands among Minn2761and Thatcher byDDRT-PCR analysis. Polymorphic bands were grouped into nine different types and four types of themwere supposed to correspond to disease-resistant genes. After sequencing cDNA differentialfragments and Blast homology analysis in the GenBank database it was indicated that therewas no known similar sequences found.
5. A high-throughput, high-resolution and high-repeatability2-DE system was established for wheatleaf proteins. the proteins were extracted by using the TCA/acetones, the elements of lysis buffer werecomposed of urea:9mol/L, Thiourea:2mol/L,4%CHAPS，1%DTT，1%TBP and0.5%IPGbuffer. IEF condition was10000vh(17cm), SDS-PAGE was performed in12%polyacrylamide gels.
6. The2-D maps of inoculated and un-inoculated Minn2761and Thatcher by of34MKGwere analyzed. The analysis result was shown that there were seven and six differentiallyexpressed protein spots, respectively, there were eleven differentially expressed protein spotsin inoculated Minne2761and Thatcher. Analysis of13interested protein spots was conductedwith MALDI-TOF-TOF. Among them,8proteins spots were obtained by Mascot databasesearching and inter-comparison preliminary identification. These protein spots could beclassified into three groups: group1was included in ascorbate peroxidase(66) and glutathionesynthetase (247), which is thought to relate to host defense; group2,ribulose-1,5-bisphosphate carboxylase/oxygenase large subunitRuBisCO large subunit(152), which isthought to relate to energy metabolism; group3,23kDa oxygen evolving protein ofphotosystem II(437,524) and group4was included in ADP-ribosylation factor(498),unknown(147,648).All of those up-or down-regulated proteins were probably parts of network of resistance of development-related proteins had close connection with plant defense responses,energy metabolism, cell signal transduction and transcriptional regulation.
1．国内首次研究确定了能够对Ug99及其变异体具有区分作用的新鉴别寄主体系，并用五辅音字母对新监测的菌株命名。由于近几年全国小麦秆锈病发生罕见的轻，2009-2010年从全国重要秆锈病流行麦区采集共得到75个菌株。共鉴定出8个不同小种，其出现频率分别为:21C3CTHTM为42.7%、34MKGRM为14.7%、21C3CPHTM为13.3%和21C3CTHTP为9.3%、34MKGTM为6.7%、21C3CFHTM为5.3%、21C3CTHRM和34MKGRP分别为4.0%。说明21C3系统仍然是我国发生最普遍和出现频率最高的小种类群，但34系统出现频率有所上升由16.9%上升到25.4%，暂未发现Ug99。利用47个小麦抗秆锈病单基因系对上述8个小种的毒力谱进行了测定，结果表明：毒力频率100%的基因有:Sr7a，7b，8a，9a，9b，9d，9f，9g，12，13，14，15，16，18，19，20，23，24，25，27，28，29，32，34，Tmp，Dp-2；80%以上的有：Sr6；50%以上都有：Sr10，11，17，Wld-1，GT；30%以上的有：Sr5；10%～30%的有: Sr38；毒力频率为0的基因有:Sr9e，21，22，26，30，31，33，35，36，37，并与Ug99及其变异小种的毒力谱进行了比较，结果发现对我国小种具有良好抗性的基因Sr9e，21，30，31和38等均能被Ug99克服，对我国优势小种和Ug99均具有抗性的基因为Sr22，26，33，35和37等，为今后筛选和利用兼抗中、外秆锈菌小种的有效抗病基因提供了可靠依据。
2.利用我国当前流行小种鉴定了57份国际抗Ug99小麦材料和1418份国内小麦品种（系）。国际材料中有51份材料对我国主要小种类型具有良好的抗性；在我国448份小麦种植品种中，对全部供试小种类型表现抗性的有147份，占32.8％。；长江中下游麦区的105份材料中只有1份对全部供试小种抗性表现免疫，其他都为高感品系；黄淮麦区的236份小麦高代系中对全部供试小种类型表现抗性的有64份，占27.6％；在东北春麦区供试的629份高代系中有562份表现为免疫至高抗，其中442份表现免疫，说明东北麦区育成的小麦品种对秆锈病的抗性较好。
3.开展小麦品种中重要抗秆锈基因的分子检测分析研究：对现有报道的抗秆锈病基因标记的实用性进行反复验证后，选取了重要抗病标记Sr26（兼抗Ug99）、Sr31和Sr36对我国小麦品种含有的抗秆锈性基因进行检测，通过对448份小麦品种的检测结果表明：在黑龙江的垦九10号和北京的保丰104中检测到Sr26；在多达70份的小麦品种检测到含有Sr31，其中河南13份；河北7份；四川9份，黑龙江4份；辽宁、山东5份；北京、陕西4份；湖北、甘肃和贵州2份；安徽、云南各1份小麦品种；引自国外品种4份及其他品种9份；在检测中尚未测到含Sr36的品种。其中，关于国内品种含有的Sr26以前鲜见报道。
4.国内首次.利用mRNA差异显示技术（DDRT-PCR）对我国重要辅助鉴别寄主明尼2761的与抗性相关基因进行了研究。筛选出24对引物能够在辅助鉴别寄主明尼2761接菌/未接菌和感病亲本萨其尔接菌/未接菌之间揭示出多态性，根据基因表达与否和表达量，获得9种不同差异表达的多态性条带类型，分别表示不同状态下测试材料的表达状况。通过对获得的cDNA差异片段进行测序，在NCBI网站上BLAST同源性比较分析，在GenBank数据库中并未找到与其具有同源性的已知序列。
5.建立了适合小麦叶片蛋白的高通量、高分辨率和高重复性的双向电泳技术体系，确定了双向电泳最佳条件：以改进的三氯乙酸/丙酮法提取小麦叶片总蛋白，裂解缓冲液为9M尿素，2M硫脲，4%CHAPS，1%DTT，1%TBP，0.5%IPG buffer，IEF等点聚焦最终控制在10000V，99999vh，SDS-PAGE胶浓度为12%。
6.首次对辅助鉴别寄主明尼2761进行双向电泳(2-DE)分离分析，结果显示：在明尼2761和萨其尔分别接菌/未接菌对比中，分别出现7个和6个差异表达蛋白点。经过比较共发现差异点11个。通过基质辅助激光解析电离飞行时间质谱技术(MALDI-TOF-TOF)获得了13个蛋白点的肤质量指纹图谱(PMF)，Mascot数据库搜索比对后，鉴定了8个蛋白质点，根据蛋白质功能的不同分为三类，第一类是与植物防卫相关的蛋白：ascorbate peroxidase(66)和glutathione synthetase (247)；第二类是与能量代谢相关蛋白：ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit RuBisCO largesubunit(152)；第三类是与光合作用相关的蛋白：23kDa oxygen evolving protein ofphotosystem II(437，524)；第四类是未知功能蛋白: ADP-ribosylation factor(498)，unknown(147,648)。它们分别参与了植物自身的防卫反应、能量代谢、光合作用等多个生理生化过程，这些上调或下调的蛋白都是复杂的蛋白调控网络中的一部分，在植物的抗病反应中协同起着重要作用。
Wheat stem rust caused by Puccinia graminis f.sp. tritici is a catastrophic wheat diseasebecause of its ability to cause complete annihilation of wheat crops over wide areas. China isin special wheat stem rust epidemic region and had suffered from several times of devastatingwheat losses due to the rust. For nearly four decades this disease has been under controlinternationally by widespread use of the1BL.1RS translocation with Sr31.Race variation isinternal reasons for wheat rust pandemic and resistance loss. Resurgence of raceUg99(TTKSK) is such a case. The race is highly virulent on Sr31and causes severe threat towheat production in China and the world. the urgent researches would be included in thefollowing aspects Race survey (a section of international wheat stem rust surveillance),Chinese resistant variety and germplasm screening, Chinese resistant variety gene detectionand new resistant gene mining, and Chinese important complementary host: Minn2761resistance related proteomics studies. The main progresses were as follows.
1. The new sets of wheat stem rust differential single gene lines and five-letter racenomenclature system which could distinguish new races Ug99and its mutants were used toidentify75isolates in the year2009-2010. Eight pathotypes, namely,21C3CTHTM,34MKGRM,21C3CPHTM,21C3CTHTP,34MKGTM,21C3CFHTM,21C3CTHRM and34MKGRP, were identified with frequencies of42.7%,14.7%,13.3%,9.3%,6.7%,5.3%,4.0%and4.0%, respectively. Only a few races were found, without finding new pathotypeUg99. Race21C3still remained predominant. The virulence spectra of eight race pathotypeswere determined on47single gene lines. The virulence frequencies for Sr7a,7b,8a,9a,9b,9d,9f,9g,12,13,14,15,16,18,19,20,23,24,25,27,28,29,32,34,Tmp,Dp-2were100%; thosefor Sr6, Sr10,11,17, Wld-1, GT, between50%-80%; those for Sr5and Sr38, between10%-30%; and those for Sr9e，21，22，26，30，31，33，35，36，37，0. It was indicatedthat Sr5,9e,11,21,30and31were highly resistant to Chinese wheat stem rust races currentlybut susceptible to Ug99and only Sr22,26,33,35and37were resistant to both Chinese racesand Ug99and could be utilized in breeding for resistance.
2. Using current main races of wheat stem rust in China, the resistance of57abroadUg99-resistant wheat cultivars and1418Chinese cultivars (lines) was appraised. It wasindicated that51wheat materials resistant to Chinese stem rust races and Ug99;52.0%of448cultivars from China also had good resistance, only1in105materials from Jiangsu was resistant to all races tested,64in236breeding lines from Shandong, resistant to all racestested;562in629wheat lines from Heilongjiang were highly resistant and442of them wereimmune.
3. Molecular detection for stem rust resistant genes carried in448Chinese commercialcultivars was conducted by using validated closely linked stem rust resistant gene markers,such as those of available STS and SSR markers of Sr26(resistant to Ug99), Sr31and Sr36.The result was indicated that among those cultivars, Kenjiu10grown in HeilongjiangProvince and Baofeng104grown in Beijing were detected to contain Sr26, about70cultivarswere shown to carry the Sr31and no Sr36-carrying varieties were met with.
4. For the first time in China, the mRNA differential display was used to study wheatstem rust resistance related protein of Chinese complementary differential: Minne2761.Twenty-four pairs of primers could amplify differential expression bands among Minn2761and Thatcher byDDRT-PCR analysis. Polymorphic bands were grouped into nine different types and four types of themwere supposed to correspond to disease-resistant genes. After sequencing cDNA differentialfragments and Blast homology analysis in the GenBank database it was indicated that therewas no known similar sequences found.
5. A high-throughput, high-resolution and high-repeatability2-DE system was established for wheatleaf proteins. the proteins were extracted by using the TCA/acetones, the elements of lysis buffer werecomposed of urea:9mol/L, Thiourea:2mol/L,4%CHAPS，1%DTT，1%TBP and0.5%IPGbuffer. IEF condition was10000vh(17cm), SDS-PAGE was performed in12%polyacrylamide gels.
6. The2-D maps of inoculated and un-inoculated Minn2761and Thatcher by of34MKGwere analyzed. The analysis result was shown that there were seven and six differentiallyexpressed protein spots, respectively, there were eleven differentially expressed protein spotsin inoculated Minne2761and Thatcher. Analysis of13interested protein spots was conductedwith MALDI-TOF-TOF. Among them,8proteins spots were obtained by Mascot databasesearching and inter-comparison preliminary identification. These protein spots could beclassified into three groups: group1was included in ascorbate peroxidase(66) and glutathionesynthetase (247), which is thought to relate to host defense; group2,ribulose-1,5-bisphosphate carboxylase/oxygenase large subunitRuBisCO large subunit(152), which isthought to relate to energy metabolism; group3,23kDa oxygen evolving protein ofphotosystem II(437,524) and group4was included in ADP-ribosylation factor(498),unknown(147,648).All of those up-or down-regulated proteins were probably parts of network of resistance of development-related proteins had close connection with plant defense responses,energy metabolism, cell signal transduction and transcriptional regulation.
引文
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