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东北农业大学大豆研究所生物技术应用研究进展情况东北农业大学大豆研究所中国·哈尔滨陈庆山、李文滨东北农业大学大豆研究所生物技术研究概况大豆遗传图谱构建大豆功能基因分离与鉴定抗病基因分子标记重要农艺性状QTL分析大豆SMV抗病相关表达谱分析大豆功能基因转化研究大豆资源分析一、大豆遗传图谱构建1.1材料与方法群体构建Charleston×东农594F2:10RIL154SSR分析大豆总DNA的提取SSR引物-----SOYBASE引物扩增数据统计及分析遗传作图Mapmaker/EXP3.0/MapChart2.1MSD1.2研究结果——SSR引物及位点评价SSR引物扩增结果分析500对----绝大多数有稳定扩增产物194对----有多态多态率为38.8%。164对----群体扩增大多数产物只有一个片段，以引物名命名其座位名称SSR位点在RILs株系中的分布161SSR----整合到遗传图谱89（55.28%）SSR引物----符合1:1分离比72（44.72%）SSR引物---偏分离原因：一些引物在143个RILs群体中缺失较多一些RILs位点杂合个数较多15个sat_091、satt009、satt012、satt584、satt521重组自交系材料构建过程可能导致偏分离的产生RIL群体遗传结构分析群体的遗传结构----各株系亲本的基因型组成父母本对群体总体及各株系的遗传贡献率东农594----平均遗传贡献率为46%，对各株系分布在14-80%Charleston----平均遗传贡献率为53%，对各株系分布在20-86%RILs群体株系评价各株系的多样性指数（SHANNON）均值为4.93，分布在4.56-5.06，比较接近完全平衡时的5.04平均遗传相似系数为0.892，0.682-0.997，部分集中在0.95-0.98研究结果——RIL群体株系的评价大豆遗传图谱NEAUSRI—GMS1913.5cM20连锁群161SSR标记间平均距离为11.89cM每个连锁群长度变动在0.4cM-309.5cM之间连锁群上的标记数在2-28个之间研究结果——遗传图谱构建NEAUSRI-GMSsct_0670.0satt54521.4sat_08751.8satt20072.5satt16488.9satt042110.0satt449113.8satt155116.4satt300118.4satt522125.7satt571131.7satt587135.5satt276144.4sat_119157.0satt242167.3sat_105191.3satt270199.0satt390212.8satt218231.0GM1-A1sat_0360.0satt4689.5satt32749.2GM2-A2satt2330.0satt42625.8satt50944.4satt19758.3satt25161.4satt22971.1sat_09980.8sat_11391.7satt521131.5GM3-B1satt0940.0satt556satt47439.2satt27245.4satt16864.9satt02082.7sct_09486.1sat_08398.6GM4-B2satt1950.0sat_04211.9GM5-C1satt3720.0satt07644.2satt33471.3satt002109.7sat_092114.5satt460127.5satt202134.1satt134141.3satt289142.1satt277150.1satt243155.9satt341169.7sct_188179.0satt335183.3sat_120187.0sat_103192.9sct_033205.9sat_097231.6GM6-C2NEAUSRI-GMSsatt3580.0sat_00143.0sat_11450.6satt22651.7satt52853.2satt17563.0satt18283.9satt49594.7satt584107.5sat_124115.8sat_084125.9satt220138.5sat_112148.4satt373161.7sat_062188.1satt482203.1sat_106210.5satt370218.4satt383224.0satt402228.6satt267231.9satt254232.6satt515233.6satt203234.4satt198244.7satt273262.1satt502300.7satt342309.5GM7-D1asatt2710.0satt27415.8satt45917.4sat_06939.1sat_13586.9satt537100.1satt141101.7satt041108.5satt428124.0satt266148.7satt157161.5GM8-D1bsatt4130.0sat_0863.6sat_02214.1GM9-D2satt2630.0satt1510.9satt1177.5satt452satt35510.0satt23129.6satt04549.1satt4450.0satt25730.9satt55153.8satt02277.6GMABAB90.5sat_095100.7sat_091118.3GM10-Esatt2520.0satt26910.8GMRUBP19.6satt03021.9satt14623.9satt53853.5satt3310.0satt17315.4satt58131.0GM11-Fsatt1990.0satt5050.4sat_08811.1satt13814.2sat_09418.3satt28841.2satt01253.4satt39482.3satt570108.6GM12-GGM19-NGM20-ONEAUSRI-GMSsat_1170.0satt19120.9satt29346.3GM13-Hsatt009satt1520.0satt5302.9satt44025.4sct_18926.4satt33073.6satt29275.6GMGLPSI298.9GM14-Isatt4570.0satt244satt54723.4satt43130.7satt41453.8satt38061.0sat_09366.7sat_07693.3GM15-Jsatt3490.0satt5550.4GM16-Ksatt2780.0satt3982.9sat_1345.8sat_07119.2GM17-Lsatt1960.0satt15021.0sat_02056.0GM18-MNEAUSRI遗传图谱97/161SSR引物与国内外对应连锁群上的相同引物对应对应引物从GM2-A2、GM13-H、GM18-M的1个到GM7-D1a12个平均配对个数为4.85个对应个数少的连锁群主要是本研究中SSR引物较少的几个连锁群与国内外图谱比较二、大豆功能基因分离与鉴定大豆抗病相关基因的分离与鉴定大豆其它基因的分离疫霉根腐病菌培养种植绥农10接种样品TotalRNA提取RGA扩增与克隆RGA产物测序与鉴定全长基因获得――RACE方法大豆SR1基因的斑点杂交和Southern杂交大豆SR1的转录表达检测取样2.1.1研究方法2.1大豆抗病相关基因的分离与鉴定2.1.2研究结果—抗病基因同源序列的获得提取绥农10号叶片总RNA泳道1~4分别为接种后24、36、48、60h取样提取的绥农10号叶片总RNART-PCR扩增大豆抗病基因同源序列500bpM：DL2000marker，1：RT-PCR扩增片断，2：CKRT-PCRDegenerateoligonucleotideprimersdesignedaccordingtoNandRPS2andL6P1-P3cDNAfromSuinong101234M1228S18SRT-PCR扩增结果目的cDNA片段的克隆及鉴定克隆的鉴定-蓝白斑筛选1：转化后的大肠杆菌DH5α在100mg/mlAmp的LB培养基的生长；2：未转化的DH5α在100mg/mlAmp的LB培养基的生长；3：未转化的DH5α在无Amp的LB培养基的生长123克隆的鉴定-质粒酶切M11234567M2M1：λ-EcoT14Imarker；1~7：转化后质粒EcoRI酶切；M2：DL2000marker质粒酶切鉴定PCR产物的插入送交5个测序，有两个通读3.0kb500bpSR1全长cDNA的获得5´RACE的扩增结果——以RNEAU-1为耙序列M：DL2000marker，1：巢式PCR扩增产物，2：第一次PCR产物900bpM123'RACE扩增结果M：λ-EcoT14Imarker，1：巢式PCR产物，2：第一次PCR产物M122.5kb5'RACE和3'RACE与靶序列RNEAU-1的拼接将5'RACE序列、3'RACE序列与靶序列RNEAU-1进行了拼接，得到3574bp的拼接序列。35741全长cDNA序列的获得M13.5kbM：λ-EcoT14Imarker，1：RT-PCR扩增产物全基因测序3574bp全长cDNA序列3411bp的开放读码框72bp的5'非翻译区68bp的3'非翻译区20bp的多聚腺苷酸尾73bpATG起始密码子，3484bpTAA终止密码子编码1137个通读的蛋白质氨基酸序列该基因被命名为SR1GenBank注册号：AY1938921MAATTRSLASIYDVFLSFTGQDTRHGFTGYLYKALDDRGIYTFIDDQELPRGDEIKPALS61DAIQGSRIAITVLSQNYAFSTFCLDELVTILHCKSEGLLVIPVFYKVDPSHVRHQKGSYG121EAMAKHQKRFKANKEKLQKWRMALQQVADLSGYHFKDGDAYEYKFIQSIVEQVSREINRA181PLHVADYPVGLGSQVIEVRKLLDVGSDDVVHIIGIHGMGGLGKTTLAVAVYNLIAPHFDE241SCFLQNVREESNLKHLQSSLLSKLLGEKDITLTSWQEGASMIQHRLRRKKVLLILDDVDK301REQLKAIVGKPDWFGPGSRVIITTRDKHLLKYHEVERTYEVKVLNHNAALHLLTWNAFKR361EKIDPIYDDVLNRVVTYASGLPLALEVIGSNLYGKTVAEWESALETYKRIPSNEILKILQ421VSFDALEEEQQNVFLDIACCFKGHEWTEVDDIFRALYGNGKKYHIGVLVEKSLIKYNRNN481RGTVQMHNLIQDMGREIERQRSPEEPGKRKRLWSPKDIIQVLKHNTGTSKIEIICLDSSI541SDKEETVEWNENAFMKMENLKILIIRNGKFSIGPNYIPEGLRVLEWHRYPSNCLPSNFDP601INLVICKLPDSSITSFEFHGSSKKLGHLTVLNFDKCKFLTQIPDVSDLPNLKELSFRKCE661SLVAVDDSVGFLNKLKKLSAYGCRKLTSFPPLNLTSLRRLQISGCSSLEYFPEILGEMVK721IRVLELHDLPIKELPFSFQNLIGLSRLYLRRCRIVQLRCSLAMMSKLSVFRIENCNKWHW781VESEEGEETVGALWWRPEFSAKNCNLCDDFFLTGFKRFAHVGYLNLSGNNFTILPEFFKE841LKFLRTLDVSDCEHLQKIRGLPPNLKDFRAINCASLTSSSKSMLLNQELYEAGGTKFMFP901GTRIPEWFNQQSSGHSSSFWFRNKFPAKLLCLLIAPVSVPLYSLFPPKVSFGHHVPYPKV