CN101175849B - 使用变异型乙酰乳酸合成酶基因的转化方法 - Google Patents
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Abstract
使用对PC类除草剂的特异性优异的变异ALS基因，有效地选择转化细胞。包括以下步骤，即使用含有目标基因和编码相当于来自水稻的野生型乙酰乳酸合成酶的氨基酸序列中的第95位的甘氨酸变异为丙氨酸的变异型乙酰乳酸合成酶的基因的重组载体，转化宿主细胞的步骤；和在嘧啶基羧基类除草剂的存在下培养上述步骤中所得的转化细胞的步骤，使用编码所述变异型乙酰乳酸合成酶的基因作为选择标记物。
Description
技术领域
本发明涉及使用在野生型乙酰乳酸合成酶的规定位置上导入变异的变异型乙酰乳酸合成酶的转化方法及植物培育方法。
背景技术
已知乙酰乳酸合成酶(以下称为“ALS”)是酪氨酸、缬氨酸和异亮氨酸等支链氨基酸生物合成途径中的控速酶，是植物生长所必须的酶。众所周知ALS广泛存在于高等植物整体中，也在各种微生物例如酵母菌(Saccharomyces cerevisiae)、大肠杆菌(Escherichia coli)、鼠伤寒沙门氏菌(Salmonella typhimurium)等中发现了上述酶的存在。
已知大肠杆菌及鼠伤寒沙门氏菌中存在3种ALS的同工酶。上述各种同工酶是由承担酶的催化活性的分子量大的催化亚单位和通过与支链氨基酸结合发挥反馈抑制剂功能的分子量小的控制亚单位形成的杂低聚物(Chipman et al.，Biochim.Biophys.Acta.1385，401-419，1998[非专利文献1])。催化亚单位分别位于Ilv IH、Ilv GM、Ilv BN操纵子。另一方面，酵母菌中，ALS虽然为单一的酶，但与细菌一样，由催化亚单位和控制亚单位形成(Panget al.，Biochemistry，38，5222-5231，1999[非专利文献2])，催化蛋白质亚单位位于ILV2位点。
也已知植物中的ALS与上述微生物相同，由催化亚单位和控制亚单位形成(Hershey et al.，Plant Molecular Biology.40，795-806，1999)[非专利文献3])。例如，作为双子叶植物的烟草中，ALS的催化亚单位被SuRA及SuRB 2个基因位点编码(Lee et al.，EMBO J.7，1241-1248，1988[非专利文献4])，为玉米时，ALS的催化亚单位被als1及als2 2个基因位点编码(Burr et al.，Trendsin Genetics 7，55-61，1991[非专利文献5]；Lawrence et al.，Plant Mol.Biol.18，1185-1187， 1992[非专利文献6])。关于编码催化亚单位的基因，为双子叶植物时，不仅烟草，拟南芥(Arabidopsis)、油菜、棉、苍耳(Xanthium)、反枝苋(Amaranthus)以及地肤(kochia)等均已完全确定了碱基序列(参见Chipman et al.，Biochim.Biophys.Acta.1385，401-419，1998[非专利文献1]及国际公开公报WO97/08327[专利文献1])。单子叶植物中，玉米或水稻也完全确定了碱基序列。
已知磺酰脲类除草剂、咪唑啉酮类除草剂、三唑嘧啶类除草剂以及嘧啶基羧基类除草剂(pyrimidinylcarboxy herbicide)(以下，称为“PC类除草剂”。)等除草剂通过抑制该ALS来控制植物的生长(Ray，Plant Physiol.75，827-831，1984[非专利文献7]；Shaner et al.，PlantPhysiol.76，545-546，1984[非专利文献8]；Subramanian et al.，PlantPhysiol.96，310-313，1991[非专利文献9]；Shimizu et al.，J.Pestic.Sci.19，59-67，1994[非专利文献10])。
作为对上述除草剂具有抗性的植物，在编码ALS的基因中具有引起不分种类而存在的保守区域中的1个或2个氨基酸置换的1个或2个碱基置换的植物是已知的。例如可以举出，编码对磺酰脲类除草剂具有强抗性的ALS的基因(参见Kathleen et al.，EMBO J.7，1241-1248，1988〔非专利文献11〕；Mourad et al.，Planta，188，491-497，1992[非专利文献12]；Guttieri et al.，Weed Sci.43，175-178，1995[非专利文献13]；Bernasconi et al.，J.Biol.Chem.270，17381-17385，1995[非专利文献14]及特开昭63-71184号公报[专利文献2])，编码对咪唑啉酮类除草剂具有强抗性的ALS的基因(参见Mourad et al.，Planta，188，491-497，1992[非专利文献12]；Lee et al.，FEBS Lett.452，341-345，1999[非专利文献15]及特开平5-227964号公报[专利文献3])，编码对PC除草剂具有强抗性的ALS的基因(参见WO02/443 85A1[专利文献4]及WO03/083118A1[专利文献5])或编码对磺酰脲类除草剂、咪唑啉酮类除草剂、PC除草剂具有抗性的ALS的基因(参见Kathleen et al.，EMBO J.7，1241-1248，1988[非专利文献11]；Bernasconi et al.，J.Biol.Chem.270，17381-17385，1995 [非专利文献14]；Hattori et al.， Mol.Gen.Genet.246，419-425，1995[非专利文献16]；Alison et al.，Plant Physiol.111，1353，1996[非专利文献17]；Rajasekarauet al.，PlantSci.119，115-124，1996[非专利文献18]、特开昭63-71184号公报[专利文献2]、特开平4-311392号公报[专利文献6]、Bernasconi et al.，US Patent 5633437，1997[专利文献7]、WO02/44385A1[专利文献4]及WO03/083118A1[专利文献5])。
另外，人们尝试了使具有对磺酰脲类除草剂显示特异抗性的ALS的个体与具有对咪唑啉酮类除草剂显示特异抗性的ALS的个体杂交，制作对两者显示抗性的植物(Mourad et al.，Mol.Gen.Genet，243，178-184，1994[非专利文献19])。并且，也人为地将编码ALS的基因转化为除草剂抗性基因(参见Ott et al.，J.Mol.Biol.263，359-368，1996[非专利文献20]、特开昭63-71184号公报[专利文献2]、特开平5-227964号公报[专利文献3]、特表平11-504213号公报[专利文献8])，明确了缺失1个氨基酸对磺酰脲类除草剂和咪唑啉酮类除草剂两者均显示抗性(参见特开平5-227964号公报[专利文献3])。
如上所述地潜心研究了对除草剂具有抗性的ALS及编码ALS的基因，但至今尚无以PC类除草剂抗性为指标，仅对PC类除草剂具有特异的抗性的变异ALS基因的报告。如果能得到对特定的除草剂具有特异的抗性的变异ALS基因，则可以在各种用途中使用该变异ALS基因，但至今没有关于在对PC类除草剂的特异性方面有用的变异ALS基因的报告。
非专利文献1 Chipman et al.，Biochim.Biophys.Acta.1385，401-419，1998
非专利文献2 Panget al.，Biochemistry，38，5222-5231，1999
非专利文献3 Hershey et al.，Plant Molecular Biology.40，795-806，1999
非专利文献4 Lee et al.，EMBO J.7，1241-1248，1988
非专利文献5 Burr et al.，Trendsin Genetics 7，55-61，1991
非专利文献6 Lawrence et al.，Plant Mol.Biol.18，1185-1187， 1992
非专利文献7 Ray，Plant Physiol.75，827-831，1984
非专利文献8 Shaner et al.，Plant Physiol.76，545-546，1984
非专利文献9 Subramanian et al.，Plant Physiol.96，310-313，1991
非专利文献10 Shimizu et al.，J.Pestic.Sci.19，59-67，1994
非专利文献11 Kathleen et al.，EMBO J.7，1241-1248，1988
非专利文献12 Mourad et al.，Planta，188，491-497，1992
非专利文献13 Guttieri et al.，Weed Sci.43，175-178，1995
非专利文献14 Bernasconi et al.，J.Biol.Chem，270，17381-17385，1995
非专利文献15 Lee et al.，FEBS Lett.452，341-345，1999
非专利文献16 Hattori et al.，Mol.Gen.Genet.246，419-425，1995
非专利文献17 Alison et al.，Plant Physiol.111，1353，1996
非专利文献18 Rajasekarauet al.，Plant Sci.119，115-124，1996
非专利文献19 Mourad et al.，Mol.Gen.Genet，243，178-184，1994
非专利文献20 Ott et al.，J.Mol.Biol.263，359-368，1996
专利文献1 国际公开公报WO97/08327号
专利文献2 特开昭63-71184号公报
专利文献3 特开平5-227964号公报
专利文献4 国际公开公报WO02/44385号
专利文献5 国际公开公报WO03/083118号
专利文献6 特开平4-311392号公报
专利文献7 Bernasconi et al.，美国专利号USP 5,633,437号
专利文献8 特表平11-504213号公报
发明内容
鉴于上述实际情况，本发明的目的在于提供可以使用对PC类除 草剂的特异性优异的变异ALS基因，有效地选择转化细胞的方法。
为了达到上述目的，本发明人进行了深入的研究，结果明确了具有特定变异的ALS对PC类除草剂显示极高的抗性，并且发现可以使用编码具有该变异的ALS的基因作为选择标记物，从而完成了本发明。
即，本发明包含以下内容。
(1)一种转化方法，所述方法包括以下步骤：
使用含有目标基因和编码变异型乙酰乳酸合成酶的基因的重组载体，转化宿主细胞的步骤，上述变异型乙酰乳酸合成酶是相当于来自水稻的野生型乙酰乳酸合成酶的氨基酸序列中的第95位的甘氨酸变异为丙氨酸的变异型乙酰乳酸合成酶；和
在嘧啶基羧基类除草剂的存在下培养上述步骤中所得的转化细胞的步骤，
其特征在于，使用编码上述变异型乙酰乳酸合成酶的基因作为选择标记物。
(2)如(1)所述的转化方法，其特征在于，编码上述变异型乙酰乳酸合成酶的基因为编码以下的(a)或(b)的蛋白质的基因。
(a)由序列号2中记载的氨基酸序列形成的蛋白质。
(b)由序列号2中记载的氨基酸序列的第95位的丙氨酸之外的至少1个以上氨基酸被置换、缺失或加成的氨基酸序列形成，且对嘧啶基羧基类除草剂具有抗性，具有乙酰乳酸合成酶活性的蛋白质。
(3)如(1)所述的转化方法，其特征在于，上述宿主细胞是植物细胞。
(4)一种植物培育方法，所述方法包括以下步骤：
使用含有目标基因和编码变异型乙酰乳酸合成酶的基因的重组载体，转化植物细胞的步骤，上述变异型乙酰乳酸合成酶是相当于来自水稻的野生型乙酰乳酸合成酶的氨基酸序列中的第95位的甘氨酸变异为丙氨酸的变异型乙酰乳酸合成酶；和
在嘧啶基羧基类除草剂的存在下培养上述步骤中所得的转化植 物的步骤，
其特征在于，使用编码上述变异型乙酰乳酸合成酶的基因作为选择标记物。
(5)如(4)所述的植物培育方法，其特征在于，编码上述变异型乙酰乳酸合成酶的基因为编码以下的(a)或(b)的蛋白质的基因。
(a)由序列号2中记载的氨基酸序列形成的蛋白质。
(b)由序列号2中记载的氨基酸序列的第95位的丙氨酸之外的至少1个以上氨基酸被置换、缺失或加成的氨基酸序列形成，且对嘧啶基羧基类除草剂具有抗性，具有乙酰乳酸合成酶活性的蛋白质。
本说明书包含作为本申请优先权基础的日本专利申请2005-136186号说明书及/或附图中所记载的内容。
附图说明
图1表示比较来自水稻的变异型ALS蛋白质的氨基酸序列和来自水稻的野生型ALS蛋白质的氨基酸序列得到的结果。
图2-1表示比较来自水稻的变异型ALS基因的碱基序列和编码来自水稻的野生型ALS蛋白质的基因的碱基序列得到的结果。
图2-2表示比较来自水稻的变异型ALS基因的碱基序列和编码来自水稻的野生型ALS蛋白质的基因的碱基序列得到的结果。
图2-3表示比较来自水稻的变异型ALS基因的碱基序列和编码来自水稻的野生型ALS蛋白质的基因的碱基序列得到的结果。
图3是表示观察来自含有双草醚钠(bispyribac-sodium)的生根培养基中的G95A-1系统及G95A-2系统的克隆个体中的生根情况得到的结果的照片。
图4是说明G95A变异型ALS表达载体的制作方法的模式图。
图5是表示以对野生型ALS的抑制达到50％的浓度为基准的G95A变异型ALS的药物抗性比(RS比)的特性图。
具体实施方式
下面详细说明本发明。
本发明的乙酰乳酸合成酶蛋白质(以下称为“变异型ALS蛋白质”)可以通过使野生型ALS蛋白质中的规定部位变异而得到。在来自水稻的野生型ALS蛋白质中，从N末端的甲硫氨酸开始数的第95位氨基酸是甘氨酸。本发明的变异型ALS蛋白质中，该第95位甘氨酸被置换为丙氨酸。即，来自水稻的本发明的变异型ALS蛋白质具有第95位甘氨酸被置换为丙氨酸(标记为G95A)的氨基酸序列。编码来自水稻的变异型ALS蛋白质的基因(以下称为“变异型ALS基因”)的碱基序列及变异型ALS蛋白质的氨基酸序列分别示于序列号1及序列号2。
图1表示比较来自水稻的变异型ALS蛋白质的氨基酸序列和来自水稻的野生型ALS蛋白质的氨基酸序列的结果。需要说明的是，图1中，第1列氨基酸序列表示野生型ALS蛋白质，第2列氨基酸序列表示变异型ALS蛋白质。
来自水稻的变异型ALS基因(序列号1)与编码来自水稻的野生型ALS蛋白质的基因相比较，编码野生型ALS蛋白质中的第95位的甘氨酸的密码子被置换为编码丙氨酸的密码子。图2-1～图2-3表示比较来自水稻的变异型ALS基因的碱基序列和编码来自水稻的野生型ALS蛋白质的基因的碱基序列得到的结果。需要说明的是，图2-1～图2-3中，第一列碱基序列表示野生型ALS基因，第二列碱基序列表示编码变异型ALS蛋白质的基因。
该变异型ALS基因可以通过在编码存在于日本型水稻品种台中65号的基因组DNA中的野生型ALS蛋白质的基因中导入上述变异而得到。作为导入变异的方法，可以使用现有的公知方法，例如可以使用部位特异性变异导入法。部位特异性变异导入法可以使用市售的试剂盒、例如Mutan-K(宝酒造株式会社制)、GeneEditor(普洛麦格(Promega)社制)、ExSite(stratagene社制)等加以实施。需要说明的是，编码变异ALS蛋白质的基因可以由变异株培养细胞中得到，所述变异株培养细胞是在PC类除草剂存在下培养PC类除草剂敏感性的野生株培养细胞后表现出对PC类除草剂的抗性的细胞。
本发明的变异型ALS基因不限定于序列号1所示的来自水稻的基因，可以广泛从来自植物的ALS基因中得到。例如，可以通过在来自玉米、小麦、大麦、大豆、棉、油菜、糖萝卜、黑麦草(Loliummultiflorum)、烟草及拟南芥(Thale Cress)等的ALS基因中导入相同的变异，得到本发明的变异型ALS基因。此处，所谓相同的变异是指将相当于来自水稻的野生型ALS蛋白质的第95位甘氨酸(因植物的不同，有时不是第95位)的甘氨酸变异为丙氨酸的变异。
来自玉米的2种变异型ALS蛋白质的氨基酸序列分别示于序列号3和4。来自小麦的2种变异型ALS蛋白质的部分氨基酸序列分别示于序列号5和6。来自棉的2种变异型ALS蛋白质的氨基酸序列分别示于序列号7和8。来自油菜的2种变异型ALS蛋白质的氨基酸序列分别示于序列号9和10。来自烟草的2种变异型ALS蛋白质的氨基酸序列分别示于序列号11和12。来自黑麦草的变异型ALS蛋白质的氨基酸序列示于序列号13。来自拟南芥的变异型ALS蛋白质的氨基酸序列示于序列号14。
变异型ALS蛋白质无论来自何种植物，只要相当于来自水稻的野生型ALS蛋白质的第95位甘氨酸的甘氨酸被置换为丙氨酸，就对PC类除草剂特异性地显示抗性。
变异型ALS蛋白质与野生型ALS蛋白质相比较，对PC类除草剂具有优异的抗性。其可以通过将编码变异型ALS蛋白质的基因组合入大肠杆菌等的表达载体，确定由被该表达载体转化的大肠杆菌得到的变异型ALS的除草剂敏感性来判断。
此处，作为PC类除草剂，如化学式1所示，可以举出双草醚钠、嘧硫草醚钠(pyrithiobac-sodium)、嘧草醚(pyriminobac)。
化学式1
嘧硫草醚钠 嘧草醚
对PC类除草剂特异性地显示抗性是指对除PC类除草剂之外的磺酰脲类除草剂、咪唑啉酮类除草剂的抗性显著低于对PC类除草剂的抗性。需要说明的是，作为磺酰脲类除草剂，如化学式2所示，可以举出氯磺隆(chlorsulfuron)、苄嘧磺隆(bensulfuron-methyl)、吡嘧磺隆(pyrazosulfuron-ethyl)、唑吡嘧磺隆(imazosulfuron)。
化学式2
氯磺隆 苄嘧磺隆
吡嘧磺隆 唑吡嘧磺隆
作为咪唑啉酮类除草剂，如化学式3所示，可以举出咪唑喹啉酸(imazaquin)、咪唑烟酸(imazapyr)。
化学式3
咪唑喹啉酸 咪唑烟酸
本发明中，可以构筑通过利用变异型ALS基因，能有效地转化目标基因的转化方法。即，变异型ALS基因可以用作植物转化试验中的选择性标记物。例如，使用目标基因转化植物细胞时，将具有变异型ALS基因与目标基因的重组载体导入植物细胞内后，在PC类除草剂的存在下培养该植物细胞。结果判定目标基因与变异型ALS基因一起导入在PC类除草剂的存在下生存的植物细胞中。另外，目标基因以及编码变异型ALS蛋白质的基因是否被导入植物细胞的染色体中可以在观察该植物的表现型后，通过基因组杂交(genomic southern hybridization)或PCR调查上述基因是否存在于基因组上而确认。
作为转化植物的方法，可以使用现有的公知方法。例如，可以举出使用根癌农杆菌(Agrobacterium tumefaciens)将外来基因导入目标植物细胞的方法。
具体而言，在含有根癌农杆菌的Ti质粒的T-DNA序列的双载体中插入变异型ALS基因以及目标基因。将该Ti质粒转化至大肠杆菌等中。然后，将在大肠杆菌等中增殖的保持变异型ALS基因以及目标基因的双载体转化至含有辅助质粒(helper plasmid)的根癌农杆菌中。接下来，使该根癌农杆菌转染作为目标的植物后，鉴定转化植物。被鉴定的转化植物为培养细胞时，可以通过现有的公知方法使该植物细胞再生为完整的植物。
另外，将具有变异型ALS基因以及目标基因的重组载体转化至目标植物时，可以使用现有的公知方法直接导入植物体中。并且，作为转化具有变异型ALS基因以及目标基因的重组载体的方法，可以使用聚乙二醇法、电穿孔(ElectroPoration)法、基因枪(particle gun)法等。
另一方面，变异型ALS基因以及目标基因可以转化到单子叶植物和 双子叶植物等所有种类的植物中。作为转化编码变异型ALS蛋白质的基因的对象作物，例如可以举出水稻、玉米、小麦、大麦、大豆、棉、油菜、黑麦草以及烟草等。并且，通过导入变异型基因和目标基因，可以转化草坪草或树木等。
任一情况下，都可以通过将变异型ALS基因转化到植物中而赋予该植物对PC类除草剂的特异抗性。特别是由于PC类除草剂与磺酰脲类除草剂或咪唑啉酮类除草剂不同，为水溶性，故容易处理，可以排除有机溶剂对宿主细胞的影响，所以，优选用作转化中的药物。另外，PC类除草剂显示比咪唑啉酮类除草剂强约100倍的ALS抑制活性，故用微量的PC除草剂即可筛选转化体。
下面，使用实施例更加详细地说明本发明。但本发明的技术范围并不限定于下述实施例。
[实施例1]诱导来自花药培养的愈伤组织(callus)
在水稻品种“台中65号”孕穗期，选取能够最多地获得单核期花药的叶耳间距6～8cm的幼穗。此时，茎在旗叶前的叶的节下进行在水中剪枝，将直接包裹幼穗的旗叶和旗叶前的叶的2片叶子以外的叶子除去。
用含水的纸巾包裹茎的基部，用聚乙烯袋被覆，在黑暗中·10℃下进行5～10天的低温处理。然后，用干净的钳子取出幼穗，用70％乙醇灭菌10分钟，在灭菌后的纸巾(Crecia，东京)上吸干水分。用灭菌后的镊子打开含有单核期的花药的半透明的颖花，仅取出花药，放置于愈伤组织诱导培养基(N6CI培养基，表1)中。将其在持续明亮的环境中·30℃下进行培养，每隔三周在新的培养基上进行传代培养。
表1
·愈伤组织诱导培养基(N6CI)pH5.8
| N6无机盐 N6维生素 蔗糖 2.4-D L(-)-脯氨酸脱乙酰吉兰糖胶(gelrite)酪蛋白氨基酸(Casamino acids) | 30g/l 2mg/l 2.878g/l 3g/l 0.3g/l |
[实施例2]用双草醚钠筛选花药培养愈伤组织
将愈伤组织诱导后第5周的来自花药培养的愈伤组织在含有0.25μM双草醚钠的愈伤组织诱导培养基中培养4周。然后，将增殖的愈伤组织用含有0.5μM双草醚钠的再分化培养基(表2)培养4周，得到白化的再分化植物。传代培养均隔2周进行。
表2
·再分化培养基(pH5.8)
| MS无机盐N6维生素蔗糖山梨醇2.4-DNAABAP酪蛋白氨基酸L(-)-脯氨酸脱乙酰吉兰糖胶 | 30g/l 50g/l 2mg/l 1mg/l 2mg/l 2g/l 2.878g/l 4g/l |
使总量为1升，放入高压釜中，加入双草醚钠
[实施例3]双草醚钠抗性鉴定
以上述方法筛选的个体的2个系统为G95A-1系统和G95A-2系统，由于为白化个体，故用MS培养基培养，分株进行增殖。为了鉴定双草醚钠抗性的程度，将从G95A-1系统中分出的克隆个体移植在含有0、1、5、10、20μM的双草醚钠的生根培养基(表3)中(图3A：培养皿的左侧，由于白化而呈白色)。克隆个体少的G95A-2系统仅用5μM进行鉴定。作为对照区，使用播种后第2周的野生型台中65号(图3各培养皿右侧)，观察移植后第1周(图3B)、第2周(图3C)的植物体。结果为G95A-1及G95A-2两个系统均在所有浓度的双草醚钠的培养基中可见新的生根，显示了抗性，对照区的野生型在含有双草醚钠的培养基中均枯死。
表3
·生根鉴定培养基(pH5.8)
| MS无机盐N6维生素蔗糖琼脂 | 30g/l 8g/l |
使总量为1升，放入高压釜中，加入双草醚钠
[实施例4]双草醚钠抗性的白化系统的ALS基因序列解析
将上述2个系统的叶(约0.5×1cm)放入1.5ml管中，在50℃下使其干燥2小时以上。在管内放入4粒直径为3mm的玻璃珠BZ-3(井内盛荣堂)，用混合机MM300(Retsch)粉碎叶后，加入300μl的提取缓冲液(200mMTris-HCl(pH7.5)、250mM NaCl、25mM EDTA、0.5％SDS)，混悬。将该混悬液以14,000rpm的转速离心5分钟，将200μl上清液转移至新管中，加入200μl异丙醇。将其以14,000rpm的转速离心5分钟，除去上清液，将所得的沉淀真空干燥3分钟。在该沉淀中加入50μl的1/5×TE后，再以14,000rpm的转速离心1分钟，以其为基因组DNA溶液。
以制得的基因组DNA为模板，使用以下所示的引物，通过PCR-定向测序(direct sequencing)解析ALS基因的整个区域的序列。PCR中，使用ExTaq(宝日医生物技术(takara-bio)株式会社)。反应在94℃下初期变性1分钟后，将94℃30秒、58℃30秒、72℃40秒的循环进行40次。组合ALSF2和ALS2R的引物时，加入PCRx增强子(英杰(invitrogen)社)，在94℃下初期变性1分钟后，将94℃1分钟、50℃1分钟、72℃1分钟的循环进行40次。对各个PCR产物实施琼脂糖凝胶电泳，使用Mini Elute GelExtraction kit(QIAGEN社)进行精制。
以该PCR产物为模板，使用ABI Sequencing kit，由下述引物进行测序反应。使用ALSF2、ALS2R的引物时，加入测序Rx增强子溶液A(英杰社)。测序反应条件为将96℃10秒、50℃5秒、60℃4分钟的循环进行35次。测序反应后，用ABI PRISM 310 Genetic Analyzer(AppliedBiosystems，U.S.A.)确定碱基序列。
ALSF2(5’-CCACCACCCACCATGGCTACG-3’，正义引物，与ALS基因的第-12-9位对应；序列号15)
ALS2R(5’-GAAGAGGTGGTTGGTGATGA-3’，反义引物，与ALS基因的第326-345位对应；序列号16)
ALS 12(5’-GCAACCAACCTCGTGTCCGC-3，正义引物，与ALS基因的第436-455位相对应；序列号17)
ALS22(5’-GAAGGCTTCCTGTATGACGC-3’，反义引物，与ALS基因的第620-639位相对应；序列号18)
ALS13(5’-GAATTGCGCTGGTTTGTTGA-3’，正义引物，与ALS基因的第868-887位相对应；序列号19)
ALS23(5’-CTCAATTTTCCCTGTCACACG-3’，反义引物，与ALS基因的第1051-1071位相对应；序列号20)
ALS24F(5’-GGTAGCTTCCTCATGAACAT-3’，正义引物，与ALS基因的第1537-1556位相对应；序列号21)
ALS24R(5’-AATGTTCATGAGGAAGCTAC-3’，反义引物，与ALS基因的第1538-1557位相对应；序列号22)
ALS25(5’-CATTCAGGTCAAACATAGGCC-3’，反义引物，与ALS基因的第1919-1989位相对应；序列号23)
由此确定上述2系统的ALS基因序列，结果两者的ALS的第95位氨基酸均通过1个碱基置换从甘氨酸(GGC)置换为丙氨酸(GCC)。
[实施例5]制作表达GST融合G95A变异型ALS的载体
导入pUC18载体中的来自水稻的W548L/S627I2点变异型ALS(参见WO02/44385A1)为模板，将用缩聚了第95位的甘氨酸部分的密码子得到的正义引物ALS-M5(5’-TACCCGGGCNNNGCGTCCATGGAGATCCA-3’：与氨基酸序列中的第92-101位相对应；序列号24)和与第191-196位氨基酸序列对应的反义引物ALS-RspA(5’-TGTGCTTGG TGATGGA-3’；序列号25)扩增的PCR生成物克隆至pT7Blue-T载体，用该载体通过常规方法转化大肠杆菌(HB-101 株)。
由相同的引物组合进行菌落PCR，进行序列解析，得到第95位的甘氨酸(GGC)部分变异为丝氨酸(AGC)、半胱氨酸(TGC)、酪氨酸(TAT)、丙氨酸(GCA)、缬氨酸(GTG)、亮氨酸(CTG)、异亮氨酸(ATA)、甲硫氨酸(ATG)、色氨酸(TGG)、苯丙氨酸(TTT)、天冬氨酸(GAT)、谷氨酸(GAG)、精氨酸(CGG)的菌落。对于丙氨酸变异体，液体培养大肠杆菌后，提取质粒，用Sma I切断。电泳后，由琼脂糖凝胶精制变异型ALS基因片段，用Sma I消化后，进行BAP处理将其精制，将得到的来自水稻的W548L/S627I2点变异型ALS基因连接到pUC18载体上。由得到的带有G95A/W548L/S627I 3点变异型ALS基因的pUC18载体上切下含有G95A部分的NcoI片段，NcoI处理后，与BAP处理后的导入了野生型ALS基因的大肠杆菌蛋白质表达载体(pGEX-2T)进行连接，得到具有G95A1点变异型ALS基因的pGEX-2T表达载体(图4)。
[实施例6]确认导入了G95A变异型ALS基因的pGEX-2T载体的碱基序列
将由该载体转化得到的大肠杆菌(JM109株)在2ml*10根中37℃下培养12小时，用质粒提取装置(TOMY DP-480)提取(500μl)质粒后，通过离心浓缩，浓缩至200μl左右。用GFX PCR和GelPurification kit(Amersham Bioscience)进行脱盐，最终用200μl灭菌水洗脱。使用BigDye Terminator ver.1.1 cycle sequencing kit(AppliedBiosystems)，对该质粒进行测序反应。[总容量20μl(模板DNA13μl、质粒(3.2pmol/μl)1μl、pre-mix4μl、稀释缓冲液2μl)，反应条件：初期变性96℃(5分钟)、将变性96℃(5秒)-退火50℃(5秒)-延伸60℃(4分钟)的循环进行40次，最终循环的延伸为60℃(9分钟)]测序反应后，使用AutoSeq G-50 column(Amersham Bioscience)，用凝胶过滤除去反应液中的荧光碱基。将反应样品用ABI PRIZM 310genetic analyser进行测定，确认序列。作为测序用的引物，使用以下 序列的引物。
PGEX-5(5’-GGGCTGGCAAGCCACGTTTGGTG-3’，正义引物，ALS基因的上游；序列号26)
ALS-RspC(5’-CAGCGACGTGTTCGCCTA-3’，正义引物，与ALS基因的第25 8-275位对应；序列号27)
ALS-M1(5’-CCCCAGCCGCATGATCGGCACCGACGCCTT-3’，正义引物，与ALS基因的第510-539位对应；序列号28)
ALS-Rsp3(5’-CTGGGACACCTCGATGAAT-3’，正义引物，与ALS基因的第720-738位对应；序列号29)
ALS-RSp7(5’-AACTGGGATACCAGTCAGCTC-3’，反义引物，与ALS基因的第886-906位对应；序列号30)
ALS-Rsp1(5’-GCTCTGCTACAACAGAGCACA-3’，正义引物，与ALS基因的第1192-1212位对应；序列号31)
3-1-3(5’-GATTGCCTCACCTTTCG-3’，反义引物，与ALS基因的第1346-1362位对应；序列号32)
4-83-10(5’-CAGCCCAAATCCCATTG-3’，反义引物，与ALS基因的第1457-173位对应；序列号33)
3-1-4(5’-AGGTGTCACAGTTGTTG-3’，正义引物，与ALS基因的第1506-1522位对应；序列号34)
ALS-RspB(5’-TCAAGGACATGATCCTGGATGG-3’，正义引物，与ALS基因的第1892-1913位对应；序列号35)
ALS-Rsp2(5’-AGTCCTGCCATCACCATCCAG-3’，反义引物，与ALS基因的第1906-1926位对应；序列号36)
pGEX-3(5’-CCGGGAGCTGCATGTGTCAGAGG-3’，反义引物，ALS基因的下游；序列号37)
[实施例7]G95A变异型ALS的表达与ALS的配制
将用实施例6制得的具有G95A变异型ALS基因的pGEX-2T以及具有野生型ALS基因的pGEX-2T(参见WO02/44385A1)转化的大肠杆菌 在27℃下分别在含有2ml氨苄西林的LB液体培养基中振荡培养(前培养)。使用1ml该前培养液，在含有250ml氨苄西林的LB液体培养基中分别进行主培养。培养一夜后，加入1mM IPTG，进一步培养3小时～4小时，由此进行GSP融合蛋白质的表达诱导。需要说明的是，菌体用ALS提取缓冲液(含有30％甘油和0.5mM MgCl2的磷酸钾缓冲液pH7.5)洗涤后在-80℃下保存。
按照下述方法由大肠杆菌配制和精制ALS。首先，将预先保存在-80℃的大肠杆菌的颗粒混悬在ALS提取缓冲液中(相对于由50ml培养液中得到的颗粒，添加2.5ml ALS提取缓冲液)。对该混悬液进行超声波处理(Heat Systems-Ultrasonics社制，Sonicator W-225R，微型芯片，输出控制8，间隔约1秒，40秒2次)后，在4℃、15000×g下离心20分钟，以该上清液作为粗酶溶液。由此，配制GST融合G95A变异型ALS蛋白质以及GST融合野生型ALS蛋白质的粗酶溶液。
[实施例8]测定表达ALS的活性
用于活性测定反应的反应溶液是在由20mM的丙酮酸钠、0.5mM的硫胺素焦磷酸、0.5mM MgCl2、10μM黄素腺嘌呤二核苷酸、10mM缬氨酸(为了抑制来自大肠杆菌的ALS活性而添加)以及20mM磷酸钾缓冲液(pH7.5)形成的溶液中，混合作为活性测定对象的GST融合ALS得到的溶液。使用0.5ml该反应溶液。测定反应在添加作为测定对象的GST融合ALS后，在37℃下进行30分钟。另外，测定反应通过添加0.05ml6N硫酸使其停止。然后，将反应结束后的反应溶液在37℃下培养60分钟。由此，使反应溶液中所含的乙酰乳酸转化为3-羟基丁酮。接下来，为了定量反应溶液中所含的3-羟基丁酮，添加0.05ml0.5％(w/v)的肌酸和0.05ml溶解在2.5N氢氧化钠中的5％(w/v)的α-萘酚，在37℃下培养10分钟。然后，通过比色反应溶液在525nm下的吸光度，对3-羟基丁酮进行定量，评价ALS活性。需要说明的是，以反应0小时作为对照区。测定药物的抑制活性时，将双草醚钠和嘧硫草醚钠制成100倍浓度的水溶液，添加至反应液中。水溶性低的嘧 草醚、氯磺隆、苄嘧磺隆、咪唑喹啉酸以及咪唑烟酸制成100倍浓度的丙酮溶液，添加至反应液中。
[实施例9]G95A变异型ALS的药物感受性
测定各种ALS抑制剂对表达的G95A变异型ALS的抑制活性的结果为双草醚钠、嘧硫草醚钠以及嘧草醚的抑制活性极弱(以100μM实现50％以下的抑制活性)，氯磺隆的抑制活性强，另外，苄嘧磺隆、咪唑喹啉酸以及咪唑烟酸也显示抑制活性(表4)。
表4
G95A变异型ALS的药物感受性
BS：双草醚钠；PS：嘧硫草醚钠，PM：嘧草醚
CS：氯磺隆；BM：苄嘧磺隆
IQ：咪唑喹啉酸；IP：咪唑烟酸
需要说明的是，表4中没有单位的数值均为抑制50％的浓度，单位为μM，单位为％的数值表示在100μM时的抑制百分比。SE为标准误差。
比较各药物将G95A变异型ALS抑制50％的浓度与将野生型ALS(GST融合野生型ALS)抑制50％的浓度，算出以将野生型ALS抑制50％的浓度为基准的G95A变异型ALS的药物抗性比(RS比)，双草醚钠、嘧硫草醚钠以及嘧草醚的RS比分别为16,000倍以上、9,100倍以上、13,000倍以上，但氯磺隆、苄嘧磺隆、咪唑喹啉酸以及咪唑烟酸的RS比为0.19、40、0.82、4.9，G95A变异型ALS对PC除草剂特异性地显示强抗性(表5及图5)。需要说明的是，图5中，BS为双草醚钠，PS为 嘧硫草醚钠，PM为嘧草醚，CS为氯磺隆，BM为苄嘧磺隆，IQ为咪唑喹啉酸，IP为咪唑烟酸。
表5
G95A变异型ALS的药物抗性比
| 药物 | 抑制50％的浓度(μm) | 抗性比(RS比) | |
| 野生型 | G95A变异型 | ||
| 双草醚钠 | 0.0063 | ＞100 | ＞16000 |
| 嘧硫草醚钠 | 0.011 | ＞100 | ＞9100 |
| 嘧草醚 | 0.0080 | ＞100 | ＞13000 |
| 氯磺隆 | 0.013 | 0.0025 | 0.19 |
| 苄嘧磺隆 | 0.0070 | 0.28 | 40 |
| 咪唑喹啉酸 | 2.2 | 1.8 | 0.82 |
| 咪唑烟酸 | 9.6 | 47 | 4.9 |
[总结]
由以上的实施例可知，在来自水稻的野生型ALS蛋白质中导入了G95A变异的变异型ALS蛋白质对嘧啶基羧基类除草剂特异性地显示抗性。通过利用上述显示优异的特异性的变异型ALS蛋白质的特性，能确实有效地在PC类除草剂的存在下筛选表达变异型ALS蛋白质的细胞和不表达的细胞。
需要说明的是，上述实施例中，使用来自水稻的ALS基因，但本发明的技术范围不限定于使用来自水稻的变异型ALS基因的转化方法。通常，已知ALS基因在不同的植物间显示高同源性。另外，ALS基因中的特定变异对多种植物赋予相同的影响。由此，通过本实施例可知，例如来自玉米、小麦、大麦、大豆、棉、油菜、黑麦草以及烟草的具有与G95A变异相同的变异的变异型ALS蛋白质也同样对嘧啶基羧基类除草剂特异性地显示抗性。
产业上的可利用性
如以上详细说明所述，根据本发明，利用对PC类除草剂显示极高的抗性的变异型乙酰乳酸合成酶作为选择标记物，可以提供高效的转化方 法。
本说明书中引用的所有刊物、专利和专利申请直接作为参考引入本说明书中。
序列表
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Leu Ala Ser Ser Gly Arg Pro Gly Pro Val Leu Val Asp Ile Pro Lys
210 215 220
Asp Ile Gln Gln Gln Met Ala Val Pro Val Trp Asp Lys Pro Met Ser
225 230 235 240
Leu Pro Gly Tyr Ile Ala Arg Leu Pro Lys Pro Pro Ala Thr Glu Leu
245 250 255
Leu Glu Gln Val Leu Arg Leu Val Gly Glu Ser Arg Arg Pro Val Leu
260 265 270
Tyr Val Gly Gly Gly Cys Ala Ala Ser Gly Glu Glu Leu Arg Arg Phe
275 280 285
Val Glu Leu Thr Gly Ile Pro Val Thr Thr Thr Leu Met Gly Leu Gly
290 295 300
Asn Phe Pro Ser Asp Asp Pro Leu Ser Leu Arg Met Leu Gly Met His
305 310 315 320
Gly Thr Val Tyr Ala Asn Tyr Ala Val Asp Lys Ala Asp Leu Leu Leu
325 330 335
Ala Leu Gly Val Arg Phe Asp Asp Arg Val Thr Gly Lys Ile Glu Ala
340 345 350
Phe Ala Ser Arg Ala Lys Ile Val His Val Asp Ile Asp Pro Ala Glu
355 360 365
Ile Gly Lys Asn Lys Gln Pro His Val Ser Ile Cys Ala Asp Val Lys
370 375 380
Leu Ala Leu Gln Gly Met Asn Ala Leu Leu Glu Gly Ser Thr Ser Lys
385 390 395 400
Lys Ser Phe Asp Phe Gly Ser Trp Asn Asp Glu Leu Asp Gln Gln Lys
405 410 415
Arg Glu Phe Pro Leu Gly Tyr Lys Thr Ser Asn Glu Glu Ile Gln Pro
420 425 430
Gln Tyr Ala Ile Gln Val Leu Asp Glu Leu Thr Lys Gly Glu Ala Ile
435 440 445
Ile Gly Thr Gly Val Gly Gln His Gln Met Trp Ala Ala Gln Tyr Tyr
450 455 460
Thr Tyr Lys Arg Pro Arg Gln Trp Leu Ser Ser Ala Gly Leu Gly Ala
465 470 475 480
Met Gly Phe Gly Leu Pro Ala Ala Ala Gly Ala Ser Val Ala Asn Pro
485 490 495
Gly Val Thr Val Val Asp Ile Asp Gly Asp Gly Ser Phe Leu Met Asn
500 505 510
Val Gln Glu Leu Ala Met Ile Arg Ile Glu Asn Leu Pro Val Lys Val
515 520 525
Phe Val Leu Asn Asn Gln His Leu Gly Met Val Val Gln Trp Glu Asp
530 535 540
Arg Phe Tyr Lys Ala Asn Arg Ala His Thr Tyr Leu Gly Asn Pro Glu
545 550 555 560
Asn Glu Ser Glu Ile Tyr Pro Asp Phe Val Thr Ile Ala Lys Gly Phe
565 570 575
Asn Ile Pro Ala Val Arg Val Thr Lys Lys Asn Glu Val Arg Ala Ala
580 585 590
Ile Lys Lys Met Leu Glu Thr Pro Gly Pro Tyr Leu Leu Asp Ile Ile
595 600 605
Val Pro His Gln Glu His Val Leu Pro Met Ile Pro Ser Gly Gly Ala
610 615 620
Phe Lys Asp Met Ile Leu Asp Gly Asp Gly Arg Thr Val Tyr
625 630 635
<210>5
<211>598
<212>PRT
<213>小麦
<400>5
Ala Ala Ser Pro Ala Ala Thr Ser Ala Ala Pro Pro Ala Thr Ala Leu
1 5 10 15
Arg Pro Trp Gly Pro Ser Glu Pro Arg Lys Gly Ala Asp Ile Leu Val
20 25 30
Glu Ala Leu Glu Arg Cys Gly Ile Val Asp Val Phe Ala Tyr Pro Gly
35 40 45
Ala Ala Ser Met Glu Ile His Gln Ala Leu Thr Arg Ser Pro Val Ile
50 55 60
Thr Asn His Leu Phe Arg His Glu Gln Gly Glu Ala Phe Ala Ala Ser
65 70 75 80
Gly Tyr Ala Arg Ala Ser Gly Arg Val Gly Val Cys Val Ala Thr Ser
85 90 95
Gly Pro Gly Ala Thr Asn Leu Val Ser Ala Leu Ala Asp Ala Leu Leu
100 105 110
Asp Ser Ile Pro Met Val Ala Ile Thr Gly Gln Val Pro Arg Arg Met
115 120 125
Ile Gly Thr Asp Ala Phe Gln Glu Thr Pro Ile Val Glu Val Thr Arg
130 135 140
Ser Ile Thr Lys His Asn Tyr Leu Val Leu Asp Val Glu Asp Ile Pro
145 150 155 160
Arg Val Ile Gln Glu Ala Phe Phe Leu Ala Ser Ser Gly Arg Pro Gly
165 170 175
Pro Val Leu Val Asp Ile Pro Lys Asp Ile Gln Gln Gln Met Ala Val
180 185 190
Pro Val Trp Asp Thr Pro Met Ser Leu Pro Gly Tyr Ile Ala Arg Leu
195 200 205
Pro Lys Pro Pro Ser Thr Glu Ser Leu Glu Gln Val Leu Arg Leu Val
210 215 220
Gly Glu Ser Arg Arg Pro Ile Leu Tyr Val Gly Gly Gly Cys Ala Ala
225 230 235 240
Ser Gly Glu Glu Leu Arg Arg Phe Val Glu Leu Thr Gly Ile Pro Val
245 250 255
Thr Thr Thr Leu Met Gly Leu Gly Asn Phe Pro Ser Asp Asp Pro Leu
260 265 270
Ser Leu Arg Met Leu Gly Met His Gly Thr Val Tyr Ala Asn Tyr Ala
275 280 285
Val Asp Lys Ala Asp Leu Leu Leu Ala Phe Gly Val Arg Phe Asp Asp
290 295 300
Arg Val Thr Gly Lys Ile Glu Ala Phe Ala Ser Arg Ser Lys Ile Val
305 310 315 320
His Ile Asp Ile Asp Pro Ala Glu Ile Gly Lys Asn Lys Gln Pro His
325 330 335
Val Ser Ile Cys Ala Asp Val Lys Leu Ala Leu Gln Gly Leu Asn Asp
340 345 350
Leu Leu Asn Gly Ser Lys Ala Gln Gln Gly Leu Asp Phe Gly Pro Trp
355 360 365
His Lys Glu Leu Asp Gln Gln Lys Arg Glu Phe Pro Leu Gly Phe Lys
370 375 380
Thr Phe Gly Glu Ala Ile Pro Pro Gln Tyr Ala Ile Gln Val Leu Asp
385 390 395 400
Glu Leu Thr Lys Gly Glu Ala Ile Ile Ala Thr Gly Val Gly Gln His
405 410 415
Gln Met Trp Ala Ala Gln Tyr Tyr Thr Tyr Lys Arg Pro Arg Gln Trp
420 425 430
Leu Ser Ser Ser Gly Leu Gly Ala Met Gly Phe Gly Leu Pro Ala Ala
435 440 445
Ala Gly Ala Ala Val Ala Asn Pro Gly Val Thr Val Val Asp Ile Asp
450 455 460
Gly Asp Gly Ser Phe Leu Met Asn Ile Gln Glu Leu Ala Leu Ile Arg
465 470 475 480
Ile Glu Asn Leu Pro Val Lys Val Met Ile Leu Asn Asn Gln His Leu
485 490 495
Gly Met Val Val Gln Trp Glu Asp Arg Phe Tyr Lys Ala Asn Arg Ala
500 505 510
His Thr Tyr Leu Gly Asn Pro Glu Asn Glu Ser Glu Ile Tyr Pro Asp
515 520 525
Phe Val Thr Ile Ala Lys Gly Phe Asn Val Pro Ala Val Arg Val Thr
530 535 540
Lys Lys Ser Glu Val Thr Ala Ala Ile Lys Lys Met Leu Glu Thr Pro
545 550 555 560
Gly Pro Tyr Leu Leu Asp Ile Ile Val Pro His Gln Glu His Val Leu
565 570 575
Pro Met Ile Pro Ser Gly Gly Ala Phe Lys Asp Met Ile Met Glu Gly
580 585 590
Asp Gly Arg Thr Ser Tyr
595
<210>6
<211>598
<212>PRT
<213>小麦
<400>6
Ala Ala Ser Pro Ala Ala Thr Ser Val Ala Pro Pro Ala Thr Ala Leu
1 5 10 15
Arg Pro Trp Gly Pro Ser Glu Pro Arg Lys Gly Ala Asp Ile Leu Val
20 25 30
Glu Ala Leu Glu Arg Cys Gly Ile Val Asp Val Phe Ala Tyr Pro Gly
35 40 45
Ala Ala Ser Met Glu Ile His Gln Ala Leu Thr Arg Ser Pro Val Ile
50 55 60
Thr Asn His Leu Phe Arg His Glu Gln Gly Glu Ala Phe Ala Ala Ser
65 70 75 80
Gly Tyr Ala Arg Ala Ser Gly Arg Val Gly Val Cys Val Ala Thr Ser
85 90 95
Gly Pro Gly Ala Thr Asn Leu Val Ser Ala Leu Ala Asp Ala Leu Leu
100 105 110
Asp Ser Ile Pro Met Val Ala Ile Thr Gly Gln Val Pro Arg Arg Met
115 120 125
Ile Gly Thr Asp Ala Phe Gln Glu Thr Pro Ile Val Glu Val Thr Arg
130 135 140
Ser Ile Thr Lys His Asn Tyr Leu Val Leu Asp Val Glu Asp Ile Pro
145 150 155 160
Arg Val Ile Gln Glu Ala Phe Phe Leu Ala Ser Ser Gly Arg Pro Gly
165 170 175
Pro Val Leu Val Asp Ile Pro Lys Asp Ile Gln Gln Gln Met Ala Val
180 185 190
Pro Val Trp Asp Thr Pro Met Ser Leu Pro Gly Tyr Ile Ala Arg Leu
195 200 205
Pro Lys Pro Pro Ser Thr Glu Ser Leu Glu Gln Val Leu Arg Leu Val
210 215 220
Gly Glu Ser Arg Arg Pro Ile Leu Tyr Val Gly Gly Gly Cys Ala Ala
225 230 235 240
Ser Gly Glu Glu Leu Arg Arg Phe Val Glu Leu Thr Gly Ile Pro Val
245 250 255
Thr Thr Thr Leu Met Gly Leu Gly Asn Phe Pro Ser Asp Asp Pro Leu
260 265 270
Ser Leu Arg Met Leu Gly Met His Gly Thr Val Tyr Ala Asn Tyr Ala
275 280 285
Val Asp Lys Ala Asp Leu Leu Leu Ala Phe Gly Val Arg Phe Asp Asp
290 295 300
Arg Val Thr Gly Lys Ile Glu Ala Phe Ala Ser Arg Ser Lys Ile Val
305 310 315 320
His Ile Asp Ile Asp Pro Ala Glu Ile Gly Lys Asn Lys Gln Pro His
325 330 335
Val Ser Ile Cys Ala Asp Val Lys Leu Ala Leu Gln Gly Leu Asn Ala
340 345 350
Leu Leu Asn Gly Ser Lys Ala Gln Gln Gly Leu Asp Phe Gly Pro Trp
355 360 365
His Lys Glu Leu Asp Gln Gln Lys Arg Glu Phe Pro Leu Gly Phe Lys
370 375 380
Thr Phe Gly Glu Ala Ile Pro Pro Gln Tyr Ala Ile Gln Val Leu Asp
385 390 395 400
Glu Leu Thr Lys Gly Glu Ala Ile Ile Ala Thr Gly Val Gly Gln His
405 410 415
Gln Met Trp Ala Ala Gln Tyr Tyr Thr Tyr Lys Arg Pro Arg Gln Trp
420 425 430
Leu Ser Ser Ser Gly Leu Gly Ala Met Gly Phe Gly Leu Pro Ala Ala
435 440 445
Ala Gly Ala Ala Val Ala Asn Pro Gly Val Thr Val Val Asp Ile Asp
450 455 460
Gly Asp Gly Ser Phe Leu Met Asn Ile Gln Glu Leu Ala Leu Ile Arg
465 470 475 480
Ile Glu Asn Leu Pro Val Lys Val Met Ile Leu Asn Asn Gln His Leu
485 490 495
Gly Met Val Val Gln Trp Glu Asp Arg Phe Tyr Lys Ala Asn Arg Ala
500 505 510
His Thr Tyr Leu Gly Asn Pro Glu Asn Glu Ser Glu Ile Tyr Pro Asp
515 520 525
Phe Val Thr Ile Ala Lys Gly Phe Asn Val Pro Ala Val Arg Val Thr
530 535 540
Lys Lys Ser Glu Val Thr Ala Ala Ile Lys Lys Met Leu Glu Thr Pro
545 550 555 560
Gly Pro Tyr Leu Leu Asp Ile Ile Val Pro His Gln Glu His Val Leu
565 570 575
Pro Met Ile Pro Ser Gly Gly Ala Phe Lys Asp Met Ile Met Glu Gly
580 585 590
Asp Gly Arg Thr Ser Tyr
595
<210>7
<211>659
<212>PRT
<213>棉
<400>7
Met Ala Ala Ala Thr Ser Asn Ser Ala Leu Pro Lys Leu Ser Thr Leu
1 5 10 15
Thr Ser Ser Phe Lys Ser Ser Ile Pro Ile Ser Lys Ser Ser Leu Pro
20 25 30
Phe Ser Thr Thr Pro Gln Lys Pro Thr Pro Tyr Arg Ser Phe Asp Val
35 40 45
Ser Cys Ser Leu Ser His Ala Ser Ser Asn Pro Arg Ser Ala Ala Ala
50 55 60
Ser Val Thr Gln Lys Thr Ala Pro Pro His Tyr Phe Ile Ser Arg Tyr
65 70 75 80
Ala Asp Asp Glu Pro Arg Lys Gly Ala Asp Ile Leu Val Glu Ala Leu
85 90 95
Glu Arg Glu Gly Val Lys Asp Val Phe Ala Tyr Pro Gly Ala Ala Ser
100 105 110
Met Glu Ile His Gln Ala Leu Thr Arg Ser Lys Ile Ile Arg Asn Val
115 120 125
Leu Pro Arg His Glu Gln Gly Gly yal Phe Ala Ala Glu Gly Tyr Ala
130 135 140
Arg Ser Ser Gly Ile Ser Gly Val Cys Ile Ala Thr Ser Gly Pro Gly
145 150 155 160
Ala Thr Asn Leu Val Ser Gly Leu Ala Asp Ala Met Leu Asp Ser Ile
165 170 175
Pro Leu Val Ala Ile Thr Gly Gln Val Pro Arg Arg Met Ile Gly Thr
180 185 190
Asp Ala Phe Gln Glu Thr Pro Ile Val Glu Val Thr Arg Ser Ile Thr
195 200 205
Lys His Asn Tyr Leu Val Leu Asp Val Asp Asp Ile Pro Arg Ile Val
210 215 220
Ser Glu Ala Phe Phe Leu Ala Ser Ser Gly Arg Pro Gly Pro Val Leu
225 230 235 240
Ile Asp Val Pro Lys Asp Ile Gln Gln Gln Leu Ala Val Pro Lys Trp
245 250 255
Asn His Ser Leu Arg Leu Pro Gly Tyr Leu Ser Arg Leu Pro Lys Ala
260 265 270
Pro Ala Glu Ala His Leu Glu Gln Ile Val Arg Leu Val Ser Glu Ser
275 280 285
Lys Lys Pro Val Leu Tyr Val Gly Gly Gly Cys Leu Asn Ser Ser Glu
290 295 300
Glu Leu Lys Arg Phe Val Glu Leu Thr Gly Ile Pro Val Ala Ser Thr
305 310 315 320
Leu Met Gly Leu Gly Ala Phe Pro Ile Ser Asp Glu Leu Ser Leu Gln
325 330 335
Met Leu Gly Met His Gly Thr Val Tyr Ala Asn Tyr Ala Val Asp Lys
340 345 350
Ser Asp Leu Leu Leu Ala Phe Gly Val Arg Phe Asp Asp Arg Val Thr
355 360 365
Gly Lys Leu Glu Ala Phe Ala Ser Arg Ala Lys Ile Val His Ile Asp
370 375 380
Ile Asp Ser Ala Glu Ile Gly Lys Asn Lys Gln Pro His Met Ser Val
385 390 395 400
Cys Ser Asp Val Lys Leu Ala Leu Gln Gly Ile Asn Lys Ile Leu Glu
405 410 415
Thr Thr Gly Ala Lys Leu Asn Leu Asp Tyr Ser Glu Trp Arg Gln Glu
420 425 430
Leu Asn Glu Gln Lys Leu Lys Phe Pro Leu Ser Tyr Lys Thr Phe Gly
435 440 445
Glu Ala Ile Pro Pro Gln Tyr Ala Ile Gln Val Leu Asp Glu Leu Thr
450 455 460
Gly Gly Asn Ala Ile Ile Ser Thr Gly Val Gly Gln His Gln Met Trp
465 470 475 480
Ala Ala Gln Phe Tyr Lys Tyr Lys Lys Pro Arg Gln Trp Leu Thr Ser
485 490 495
Gly Gly Leu Gly Ala Met Gly Phe Gly Leu Pro Ala Ala Ile Gly Ala
500 505 510
Ala Val Ala Asn Pro Glu Ala Val Val Val Asp Ile Asp Gly Asp Gly
515 520 525
Ser Phe Ile Met Asn Val Gln Glu Leu Ala Thr Met Arg Val Glu Asn
530 535 540
Leu Pro Val Lys Ile Leu Leu Leu Asn Asn Gln His Leu Gly Met Val
545 550 555 560
Val Gln Trp Glu Asp Arg Phe Tyr Lys Ala Asn Arg Ala His Thr Tyr
565 570 575
Leu Gly Asp Pro Ser Asn Glu Ser Glu Ile Phe Pro Asn Met Leu Lys
580 585 590
Phe Ala Glu Ala Cys Gly Ile Pro Ala Ala Arg Val Thr Lys Lys Glu
595 600 605
Asp Leu Lys Ala Ala Ile Gln Lys Met Leu Asp Thr Pro Gly Pro Tyr
610 615 620
Leu Leu Asp Val Ile Val Pro His Gln Glu His Val Leu Pro Met Ile
625 630 635 640
Pro Ser Gly Gly Ala Phe Lys Asp Val Ile Thr Glu Gly Asp Gly Arg
645 650 655
Thr Gln Tyr
<210>8
<211>659
<212>PRT
<213>棉
<400>8
Met Ala Ala Ala Thr Ala Asn Ser Ala Leu Pro Lys Leu Ser Thr Leu
1 5 10 15
Thr Ser Ser Phe Lys Ser Ser Ile Pro Ile Ser Lys Ser Ser Leu Pro
20 25 30
Phe Ser Thr Thr Pro Gln Lys Pro Thr Pro Tyr Arg Ser Phe Asp Val
35 40 45
Ser Cys Ser Leu Ser His Ala Ser Ser Asn Pro Arg Ser Ala Ala Thr
50 55 60
Ser Val Thr Pro Lys Asn Ala Pro Pro His Asp Phe Ile Ser Arg Tyr
65 70 75 80
Ala Asp Asp Glu Pro Arg Lys Gly Ala Asp Ile Leu Val Glu Ala Leu
85 90 95
Val Arg Glu Gly Val Lys Asp Val Phe Ala Tyr Pro Gly Ala Ala Ser
100 105 110
Met Glu Ile His Gln Ala Leu Thr Arg Ser Lys Ile Ile Arg Asn Val
115 120 125
Leu Pro Arg His Glu Gln Gly Gly Val Phe Ala Ala Glu Gly Tyr Ala
130 135 140
Arg Ser Ser Gly Ile Pro Gly Val Cys Ile Ala Thr Ser Gly Pro Gly
145 150 155 160
Ala Thr Asn Leu Val Ser Gly Leu Ala Asp Ala Met Leu Asp Ser Ile
165 170 175
Pro Leu Val Ala Ile Thr Gly Gln Val Pro Arg Arg Met Ile Gly Thr
180 185 190
Asp Ala Phe Gln Glu Thr Pro Ile Val Glu Val Thr Arg Ser Ile Thr
195 200 205
Lys His Asn Tyr Leu Val Leu Asp Val Asp Asp Ile Pro Arg Ile Val
210 215 220
Ser Glu Ala Phe Phe Leu Ala Ser Ser Gly Arg Pro Gly Pro Val Leu
225 230 235 240
Ile Asp Val Pro Lys Asp Ile Gln Gln Gln Leu Ala Val Pro Lys Trp
245 250 255
Asn His Ser Leu Arg Leu Pro Gly Tyr Leu Ser Arg Leu Pro Lys Ala
260 265 270
Pro Gly Glu Ala His Leu Glu Gln Ile Val Arg Leu Val Ser Glu Ser
275 280 285
Lys Lys Pro Val Leu Tyr Val Gly Gly Gly Cys Leu Asn Ser Ser Glu
290 295 300
Glu Leu Lys Arg Phe Val Glu Leu Thr Gly Ile Pro Val Ala Ser Thr
305 310 315 320
Leu Met Gly Leu Gly Ala Phe Pro Ile Ser Asp Asp Leu Ser Leu Gln
325 330 335
Met Leu Gly Met His Gly Thr Val Tyr Ala Asn Tyr Ala Val Asp Lys
340 345 350
Ser Asp Leu Leu Leu Ala Phe Gly Val Arg Phe Asp Asp Arg Val Thr
355 360 365
Gly Lys Leu Glu Ala Phe Ala Ser Arg Ala Lys Ile Val His Ile Asp
370 375 380
Ile Asp Ser Ala Glu Ile Gly Lys Asn Lys Gln Pro His Val Ser Val
385 390 395 400
Cys Ser Asp Val Lys Leu Ala Leu Gln Gly Ile Asn Lys Ile Leu Glu
405 410 415
Thr Lys Val Ala Lys Leu Asn Leu Asp Tyr Ser Glu Trp Arg Gln Glu
420 425 430
Leu Asn Glu Gln Lys Leu Lys Phe Pro Leu Ser Tyr Lys Thr Phe Gly
435 440 445
Glu Ala Ile Pro Pro Gln Tyr Ala Ile Gln Val Leu Asp Glu Leu Thr
450 455 460
Gly Gly Asn Ala Ile Ile Ser Thr Gly Val Gly Gln His Gln Met Trp
465 470 475 480
Ala Ala Gln Phe Tyr Lys Tyr Lys Lys Pro Arg Gln Trp Leu Thr Ser
485 490 495
Gly Gly Leu Gly Ala Met Gly Phe Gly Leu Pro Ala Ala Ile Gly Ala
500 505 510
Ala Val Ala Asn Pro Glu Ala Val Val Val Asp Ile Asp Gly Asp Gly
515 520 525
Ser Phe Ile Met Asn Val Gln Glu Leu Ala Thr Ile Arg Val Glu Asn
530 535 540
Leu Pro Val Lys Ile Leu Leu Leu Asn Asn Gln His Leu Gly Met Val
545 550 555 560
Val Gln Trp Glu Asp Arg Phe Tyr Lys Ala Asn Arg Ala His Thr Tyr
565 570 575
Leu Gly Asp Pro Ser Asn Glu Ser Glu Ile Phe Pro Asn Met Leu Lys
580 585 590
Phe Ala Glu Ala Cys Gly Ile Pro Ala Ala Arg Val Thr Lys Lys Glu
595 600 605
Asp Leu Lys Ala Ala Met Gln Lys Met Leu Asp Thr Pro Gly Pro Tyr
610 615 620
Leu Leu Asp Val Ile Val Pro His Gln Glu His Val Leu Pro Met Ile
625 630 635 640
Pro Ser Gly Gly Ala Phe Lys Asp Val Ile Thr Glu Gly Asp Gly Arg
645 650 655
Thr Gln Tyr
<210>9
<211>637
<212>PRT
<213>油菜
<400>9
Met Ala Ser Phe Ser Phe Phe Gly Thr Ile Pro Ser Ser Pro Thr Lys
1 5 10 15
Ala Ser Val Phe Ser Leu Pro Val Ser Val Thr Thr Leu Pro Ser Phe
20 25 30
Pro Arg Arg Arg Ala Thr Arg Val Ser Val Ser Ala Asn Ser Lys Lys
35 40 45
Asp Gln Asp Arg Thr Ala Ser Arg Arg Glu Asn Pro Ser Thr Phe Ser
50 55 60
Ser Lys Tyr Ala Pro Asn Val Pro Arg Ser Gly Ala Asp Ile Leu Val
65 70 75 80
Glu Ala Leu Glu Arg Gln Gly Val Asp Val Val Phe Ala Tyr Pro Gly
85 90 95
Ala Ala Ser Met Glu Ile His Gln Ala Leu Thr Arg Ser Asn Thr Ile
100 105 110
Arg Asn Val Leu Pro Arg His Glu Gln Gly Gly Ile Phe Ala Ala Glu
115 120 125
Gly Tyr Ala Arg Ser Ser Gly Lys Pro Gly Ile Cys Ile Ala Thr Ser
130 135 140
Gly Pro Gly Ala Met Asn Leu Val Ser Gly Leu Ala Asp Ala Leu Phe
145 150 155 160
Asp Ser Val Pro Leu Ile Ala Ile Thr Gly Gln Val Pro Arg Arg Met
165 170 175
Ile Gly Thr Met Ala Phe Gln Glu Thr Pro Val Val Glu Val Thr Arg
180 185 190
Thr Ile Thr Lys His Asn Tyr Leu Val Met Glu Val Asp Asp Ile Pro
195 200 205
Arg Ile Val Arg Glu Ala Phe Phe Leu Ala Thr Ser Val Arg Pro Gly
210 215 220
Pro Val Leu Ile Asp Val Pro Lys Asp Val Gln Gln Gln Phe Ala Ile
225 230 235 240
Pro Asn Trp Glu Gln Pro Met Arg Leu Pro Leu Tyr Met Ser Thr Met
245 250 255
Pro Lys Pro Pro Lys Val Ser His Leu Glu Gln Ile Leu Arg Leu Val
260 265 270
Ser Glu Ser Lys Arg Pro Val Leu Tyr Val Gly Gly Gly Cys Leu Asn
275 280 285
Ser Ser Glu Glu Leu Arg Arg Phe Val Glu Leu Thr Gly Ile Pro Val
290 295 300
Ala Ser Thr Phe Met Gly Leu Gly Ser Tyr Pro Cys Asp Asp Glu Glu
305 310 315 320
Phe Ser Leu Gln Met Leu Gly Met His Gly Thr Val Tyr Ala Asn Tyr
325 330 335
Ala Val Glu Tyr Ser Asp Leu Leu Leu Ala Phe Gly Val Arg Phe Asp
340 345 350
Asp Arg Val Thr Gly Lys Leu Glu Ala Phe Ala Ser Arg Ala Lys Ile
355 360 365
Val His Ile Asp Ile Asp Ser Thr Glu Ile Gly Lys Asn Lys Thr Pro
370 375 380
His Val Ser Val Cys Cys Asp Val Gln Leu Ala Leu Gln Gly Met Asn
385 390 395 400
Glu Val Leu Glu Asn Arg Arg Asp Val Leu Asp Phe Gly Glu Trp Arg
405 410 415
Cys Glu Leu Asn Glu Gln Arg Leu Lys Phe Pro Leu Arg Tyr Lys Thr
420 425 430
Phe Gly Glu Glu Ile Pro Pro Gln Tyr Ala Ile Gln Leu Leu Asp Glu
435 440 445
Leu Thr Asp Gly Lys Ala Ile Ile Thr Thr Gly Val Gly Gln His Gln
450 455 460
Met Trp Ala Ala Gln Phe Tyr Arg Phe Lys Lys Pro Arg Gln Trp Leu
465 470 475 480
Ser Ser Gly Gly Leu Gly Ala Met Gly Phe Gly Leu Pro Ala Ala Met
485 490 495
Gly Ala Ala Ile Ala Asn Pro Gly Ala Val Val Val Asp Ile Asp Gly
500 505 510
Asp Gly Ser Phe Ile Met Asn Ile Gln Glu Leu Ala Thr Ile Arg Val
515 520 525
Glu Asn Leu Pro Val Lys Val Leu Leu Ile Asn Asn Gln His Leu Gly
530 535 540
Met Val Leu Gln Trp Glu Asp His Phe Tyr Ala Ala Asn Arg Ala Asp
545 550 555 560
Ser Phe Leu Gly Asp Pro Ala Asn Pro Glu Ala Val Phe Pro Asp Met
565 570 575
Leu Leu Phe Ala Ala Ser Cys Gly Ile Pro Ala Ala Arg Val Thr Arg
580 585 590
Arg Glu Asp Leu Arg Glu Ala Ile Gln Thr Met Leu Asp Thr Pro Gly
595 600 605
Pro Phe Leu Leu Asp Val Val Cys Pro His Gln Asp His Val Leu Pro
610 615 620
Leu Ile Pro Ser Gly Gly Thr Phe Lys Asp Ile Ile Val
625 630 635
<210>10
<211>599
<212>PRT
<213>油菜
<400>10
Met Ser His Leu Leu Pro Leu Lys Lys Pro Thr Arg Thr Arg Leu Ser
1 5 10 15
Ser Pro Ala Thr Leu Pro Asp Glu Pro Arg Lys Gly Ala Asp Ile Leu
20 25 30
Val Glu Ala Leu Glu Arg Gln Gly Val Glu Thr Val Phe Ala Tyr Pro
35 40 45
Gly Ala Ala Ser Met Glu Ile His Gln Ala Leu Thr Arg Ser Ser Thr
50 55 60
Ile Arg Asn Val Leu Pro Arg His Glu Gln Gly Gly Val Phe Ala Ala
65 70 75 80
Glu Gly Tyr Ala Arg Ser Ser Gly Lys Pro Gly Ile Cys Ile Ala Thr
85 90 95
Ser Gly Pro Gly Ala Thr Asn Leu Val Ser Gly Leu Ala Asp Ala Met
100 105 110
Leu Asp Ser Val Pro Leu Val Ala Ile Thr Gly Gln Val Pro Arg Arg
115 120 125
Met Ile Gly Thr Asp Ala Phe Gln Glu Thr Pro Ile Val Glu Val Thr
130 135 140
Arg Ser Ile Thr Lys His Asn Tyr Leu Val Met Asp Val Asp Asp Ile
145 150 155 160
Pro Arg Ile Val Gln Glu Ala Phe Phe Leu Ala Thr Ser Gly Arg Pro
165 170 175
Gly Pro Val Leu Val Asp Val Pro Lys Asp Ile Gln Gln Gln Leu Ala
180 185 190
Ile Pro Asn Trp Asp Gln Pro Met Arg Leu Pro Gly Tyr Met Ser Arg
195 200 205
Leu Pro Gln Pro Pro Glu Val Ser Gln Leu Gly Gln Ile Val Arg Leu
210 215 220
Ile Ser Glu Ser Lys Arg Pro Val Leu Tyr Val Gly Gly Gly Ser Leu
225 230 235 240
Asn Ser Ser Glu Glu Leu Gly Arg Phe Val Glu Leu Thr Gly Ile Pro
245 250 255
Val Ala Ser Thr Leu Met Gly Leu Gly Ser Tyr Pro Cys Asn Asp Glu
260 265 270
Leu Ser Leu Gln Met Leu Gly Met His Gly Thr Val Tyr Ala Asn Tyr
275 280 285
Ala Val Glu His Ser Asp Leu Leu Leu Ala Phe Gly Val Arg Phe Asp
290 295 300
Asp Arg Val Thr Gly Lys Leu Glu Ala Phe Ala Ser Arg Ala Lys Ile
305 310 315 320
Val His Ile Asp Ile Asp Ser Ala Glu Ile Gly Lys Asn Lys Thr Pro
325 330 335
His Val Ser Val Cys Gly Asp Val Lys Leu Ala Leu Gln Gly Met Asn
340 345 350
Lys Val Leu Glu Asn Arg Ala Glu Glu Leu Lys Leu Asp Phe Gly Val
355 360 365
Trp Arg Ser Glu Leu Ser Glu Gln Lys Gln Lys Phe Pro Leu Ser Phe
370 375 380
Lys Thr Phe Gly Glu Ala Ile Pro Pro Gln Tyr Ala Ile Gln Ile Leu
385 390 395 400
Asp Glu Leu Thr Glu Gly Lys Ala Ile Ile Ser Thr Gly Val Gly Gln
405 410 415
His Gln Met Trp Ala Ala Gln Phe Tyr Lys Tyr Arg Lys Pro Arg Gln
420 425 430
Trp Leu Ser Ser Ser Gly Leu Gly Ala Met Gly Phe Gly Leu Pro Ala
435 440 445
Ala Ile Gly Ala Ser Val Ala Asn Pro Asp Ala Ile Val Val Asp Ile
450 455 460
Asp Gly Asp Gly Ser Phe Ile Met Asn Val Gln Glu Leu Ala Thr Ile
465 470 475 480
Arg Val Glu Asn Leu Pro Val Lys Ile Leu Leu Leu Asn Asn Gln His
485 490 495
Leu Gly Met Val Met Gln Trp Glu Asp Arg Phe Tyr Lys Ala Asn Arg
500 505 510
Ala His Thr Tyr Leu Gly Asp Pro Ala Arg Glu Asn Glu Ile Phe Pro
515 520 525
Asn Met Leu Gln Phe Ala Gly Ala Cys Gly Ile Pro Ala Ala Arg Val
530 535 540
Thr Lys Lys Glu Glu Leu Arg Glu Ala Ile Gln Thr Met Leu Asp Thr
545 550 555 560
Pro Gly Pro Tyr Leu Leu Asp Val Ile Cys Pro His Gln Glu His Val
565 570 575
Leu Pro Met Ile Pro Ser Gly Gly Thr Phe Lys Asp Val Ile Thr Glu
580 585 590
Gly Asp Gly Arg Thr Lys Tyr
595
<210>11
<211>667
<212>PRT
<213>烟草
<400>11
Met Ala Ala Ala Ala Pro Ser Pro Ser Ser Ser Ala Phe Ser Lys Thr
1 5 10 15
Leu Ser Pro Ser Ser Ser Thr Ser Ser Thr Leu Leu Pro Arg Ser Thr
20 25 30
Phe Pro Phe Pro His His Pro His Lys Thr Thr Pro Pro Pro Leu His
35 40 45
Leu Thr His Thr His Ile His Ile His Ser Gln Arg Arg Arg Phe Thr
50 55 60
Ile Ser Asn Val Ile Ser Thr Asn Gln Lys Val Ser Gln Thr Glu Lys
65 70 75 80
Thr Glu Thr Phe Val Ser Arg Phe Ala Pro Asp Glu Pro Arg Lys Gly
85 90 95
Ser Asp Val Leu Val Glu Ala Leu Glu Arg Glu Gly Val Thr Asp Val
100 105 110
Phe Ala Tyr Pro Gly Ala Ala Ser Met Glu Ile His Gln Ala Leu Thr
115 120 125
Arg Ser Ser Ile Ile Arg Asn Val Leu Pro Arg His Glu Gln Gly Gly
130 135 140
Val Phe Ala Ala Glu Gly Tyr Ala Arg Ala Thr Gly Phe Pro Gly Val
145 150 155 160
Cys Ile Ala Thr Ser Gly Pro Gly Ala Thr Asn Leu Val Ser Gly Leu
165 170 175
Ala Asp Ala Leu Leu Asp Ser Val Pro Ile Val Ala Ile Thr Gly Gln
180 185 190
Val Pro Arg Arg Met Ile Gly Thr Asp Ala Phe Gln Glu Thr Pro Ile
195 200 205
Val Glu Val Thr Arg Ser Ile Thr Lys His Asn Tyr Leu Val Met Asp
210 215 220
Val Glu Asp Ile Pro Arg Val Val Arg Glu Ala Phe Phe Leu Ala Arg
225 230 235 240
Ser Gly Arg Pro Gly Pro Ile Leu Ile Asp Val Pro Lys Asp Ile Gln
245 250 255
Gln Gln Leu Val Ile Pro Asp Trp Asp Gln Pro Met Arg Leu Pro Gly
260 265 270
Tyr Met Ser Arg Leu Pro Lys Leu Pro Asn Glu Met Leu Leu Glu Gln
275 280 285
Ile Val Arg Leu Ile Ser Glu Ser Lys Lys Pro Val Leu Tyr Val Gly
290 295 300
Gly Gly Cys Ser Gln Ser Ser Glu Asp Leu Arg Arg Phe Val Glu Leu
305 310 315 320
Thr Gly Ile Pro Val Ala Ser Thr Leu Met Gly Leu Gly Ala Phe Pro
325 330 335
Thr Gly Asp Glu Leu Ser Leu Ser Met Leu Gly Met His Gly Thr Val
340 345 350
Tyr Ala Asn Tyr Ala Val Asp Ser Ser Asp Leu Leu Leu Ala Phe Gly
355 360 365
Val Arg Phe Asp Asp Arg Val Thr Gly Lys Leu Glu Ala Phe Ala Ser
370 375 380
Arg Ala Lys Ile Val His Ile Asp Ile Asp Ser Ala Glu Ile Gly Lys
385 390 395 400
Asn Lys Gln Pro His Val Ser Ile Cys Ala Asp Ile Lys Leu Ala Leu
405 410 415
Gln Gly Leu Asn Ser Ile Leu Glu Ser Lys Glu Gly Lys Leu Lys Leu
420 425 430
Asp Phe Ser Ala Trp Arg Gln Glu Leu Thr Glu Gln Lys Val Lys His
435 440 445
Pro Leu Asn Phe Lys Thr Phe Gly Asp Ala Ile Pro Pro Gln Tyr Ala
450 455 460
Ile Gln Val Leu Asp Glu Leu Thr Asn Gly Asn Ala Ile Ile Ser Thr
465 470 475 480
Gly Val Gly Gln His Gln Met Trp Ala Ala Gln Tyr Tyr Lys Tyr Arg
485 490 495
Lys Pro Arg Gln Trp Leu Thr Ser Gly Gly Leu Gly Ala Met Gly Phe
500 505 510
Gly Leu Pro Ala Ala Ile Gly Ala Ala Val Gly Arg Pro Asp Glu Val
515 520 525
Val Val Asp Ile Asp Gly Asp Gly Ser Phe Ile Met Asn Val Gln Glu
530 535 540
Leu Ala Thr Ile Lys Val Glu Asn Leu Pro Val Lys Ile Met Leu Leu
545 550 555 560
Asn Asn Gln His Leu Gly Met Val Val Gln Trp Glu Asp Arg Phe Tyr
565 570 575
Lys Ala Asn Arg Ala His Thr Tyr Leu Gly Asn Pro Ser Asn Glu Ala
580 585 590
Glu Ile Phe Pro Asn Met Leu Lys Phe Ala Glu Ala Cys Gly Val Pro
595 600 605
Ala Ala Arg Val Thr His Arg Asp Asp Leu Arg Ala Ala Ile Gln Lys
610 615 620
Met Leu Asp Thr Pro Gly Pro Tyr Leu Leu Asp Val Ile Val Pro His
625 630 635 640
Gln Glu His Val Leu Pro Met Ile Pro Ser Gly Gly Ala Phe Lys Asp
645 650 655
Val Ile Thr Glu Gly Asp Gly Arg Ser Ser Tyr
660 665
<210>12
<211>664
<212>PRT
<213>烟草
<400>12
Met Ala Ala Ala Ala Ala Ala Pro Ser Pro Ser Phe Ser Lys Thr Leu
1 5 10 15
Ser Ser Ser Ser Ser Lys Ser Ser Thr Leu Leu Pro Arg Ser Thr Phe
20 25 30
Pro Phe Pro His His Pro His Lys Thr Thr Pro Pro Pro Leu His Leu
35 40 45
Thr Pro Thr His Ile His Ser Gln Arg Arg Arg Phe Thr Ile Ser Asn
50 55 60
Val Ile Ser Thr Thr Gln Lys Val Ser Glu Thr Gln Lys Ala Glu Thr
65 70 75 80
Phe Val Ser Arg Phe Ala Pro Asp Glu Pro Arg Lys Gly Ser Asp Val
85 90 95
Leu Val Glu Ala Leu Glu Arg Glu Gly Val Thr Asp Val Phe Ala Tyr
100 105 110
Pro Gly Ala Ala Ser Met Glu Ile His Gln Ala Leu Thr Arg Ser Ser
115 120 125
Ile Ile Arg Asn Val Leu Pro Arg His Glu Gln Gly Gly Val Phe Ala
130 135 140
Ala Glu Gly Tyr Ala Arg Ala Thr Gly Phe Pro Gly Val Cys Ile Ala
145 150 155 160
Thr Ser Gly Pro Gly Ala Thr Asn Leu Val Ser Gly Leu Ala Asp Ala
165 170 175
Leu Leu Asp Ser Val Pro Ile Val Ala Ile Thr Gly Gln Val Pro Arg
180 185 190
Arg Met Ile Gly Thr Asp Ala Phe Gln Glu Thr Pro Ile Val Glu Val
195 200 205
Thr Arg Ser Ile Thr Lys His Asn Tyr Leu Val Met Asp Val Glu Asp
210 215 220
Ile Pro Arg Val Val Arg Glu Ala Phe Phe Leu Ala Arg Ser Gly Arg
225 230 235 240
Pro Gly Pro Val Leu Ile Asp Val Pro Lys Asp Ile Gln Gln Gln Leu
245 250 255
Val Ile Pro Asp Trp Asp Gln Pro Met Arg Leu Pro Gly Tyr Met Ser
260 265 270
Arg Leu Pro Lys Leu Pro Asn Glu Met Leu Leu Glu Gln Ile Val Arg
275 280 285
Leu Ile Ser Glu Ser Lys Lys Pro Val Leu Tyr Val Gly Gly Gly Cys
290 295 300
Ser Gln Ser Ser Glu Glu Leu Arg Arg Phe Val Glu Leu Thr Gly Ile
305 310 315 320
Pro Val Ala Ser Thr Leu Met Gly Leu Gly Ala Phe Pro Thr Gly Asp
325 330 335
Glu Leu Ser Leu Ser Met Leu Gly Met His Gly Thr Val Tyr Ala Asn
340 345 350
Tyr Ala Val Asp Ser Ser Asp Leu Leu Leu Ala Phe Gly Val Arg Phe
355 360 365
Asp Asp Arg Val Thr Gly Lys Leu Glu Ala Phe Ala Ser Arg Ala Lys
370 375 380
Ile Val His Ile Asp Ile Asp Ser Ala Glu Ile Gly Lys Asn Lys Gln
385 390 395 400
Pro His Val Ser Ile Cys Ala Asp Ile Lys Leu Ala Leu Gln Gly Leu
405 410 415
Asn Ser Ile Leu Glu Ser Lys Glu Gly Lys Leu Lys Leu Asp Phe Ser
420 425 430
Ala Trp Arg Gln Glu Leu Thr Val Gln Lys Val Lys Tyr Pro Leu Asn
435 440 445
Phe Lys Thr Phe Gly Asp Ala Ile Pro Pro Gln Tyr Ala Ile Gln Val
450 455 460
Leu Asp Glu Leu Thr Asn Gly Ser Ala Ile Ile Ser Thr Gly Val Gly
465 470 475 480
Gln His Gln Met Trp Ala Ala Gln Tyr Tyr Lys Tyr Arg Lys Pro Arg
485 490 495
Gln Trp Leu Thr Ser Gly Gly Leu Gly Ala Met Gly Phe Gly Leu Pro
500 505 510
Ala Ala Ile Gly Ala Ala Val Gly Arg Pro Asp Glu Val Val Val Asp
515 520 525
Ile Asp Gly Asp Gly Ser Phe Ile Met Asn Val Gln Glu Leu Ala Thr
530 535 540
Ile Lys Val Glu Asn Leu Pro Val Lys Ile Met Leu Leu Asn Asn Gln
545 550 555 560
His Leu Gly Met Val Val Gln Trp Glu Asp Arg Phe Tyr Lys Ala Asn
565 570 575
Arg Ala His Thr Tyr Leu Gly Asn Pro Ser Asn Glu Ala Glu Ile Phe
580 585 590
Pro Asn Met Leu Lys Phe Ala Glu Ala Cys Gly Val Pro Ala Ala Arg
595 600 605
Val Thr His Arg Asp Asp Leu Arg Ala Ala Ile Gln Lys Met Leu Asp
610 615 620
Thr Pro Gly Pro Tyr Leu Leu Asp Val Ile Val Pro His Gln Glu His
625 630 635 640
Val Leu Pro Met Ile Pro Ser Gly Gly Ala Phe Lys Asp Val Ile Thr
645 650 655
Glu Gly Asp Gly Arg Ser Ser Tyr
660
<210>13
<211>640
<212>PRT
<213>黑麦草
<400>13
Met Ala Thr Ala Thr Ser Thr Ala Val Ala Phe Ser Gly Ala Thr Ala
1 5 10 15
Thr Leu Pro Lys Pro Arg Thr Leu Pro Arg His Leu Leu Pro Ser Ser
20 25 30
Arg Arg Ala Leu Ala Ala Pro Ile Arg Cys Ser Ala Val Ser Pro Ser
35 40 45
Pro Ser Pro Ala Pro Pro Ala Thr Ala Leu Arg Pro Trp Gly Pro Ser
50 55 60
Glu Pro Arg Lys Gly Ala Asp Ile Leu Val Glu Ala Leu Glu Arg Cys
65 70 75 80
Gly Ile Ser Asp Val Phe Ala Tyr Pro Gly Ala Ala Ser Met Glu Ile
85 90 95
His Gln Ala Leu Thr Ser Ser Pro Leu Ile Thr Asn His Leu Phe Arg
100 105 110
His Glu Gln Gly Glu Ala Phe Ala Ala Ser Gly Tyr Ala Arg Ala Ser
115 120 125
Gly Arg Val Gly Val Cys Val Ala Thr Ser Gly Pro Gly Ala Thr Asn
130 135 140
Leu Val Ser Ala Leu Ala Asp Ala Leu Leu Asp Ser Ile Pro Met Val
145 150 155 160
Ala Ile Thr Gly Gln Val Pro Arg Arg Met Ile Gly Thr Asp Ala Phe
165 170 175
Gln Glu Thr Pro Ile Val Glu Val Thr Arg Ser Ile Thr Lys His Asn
180 185 190
Tyr Leu Val Leu Asp Val Glu Asp Ile Pro Arg Val Ile Gln Glu Ala
195 200 205
Phe Phe Leu Ala Ser Ser Gly Arg Pro Gly Pro Val Leu Val Asp Ile
210 215 220
Pro Lys Asp Ile Gln Gln Gln Met Ala Val Pro Val Trp Asp Ala Pro
225 230 235 240
Met Ser Leu Pro Gly Tyr Ile Ala Arg Leu Pro Lys Pro Pro Ala Thr
245 250 255
Glu Leu Leu Glu Gln Val Leu Arg Leu Val Gly Glu Glu Arg Arg Pro
260 265 270
Ile Leu Tyr Val Gly Gly Gly Cys Ser Ala Ser Gly Glu Asp Val Arg
275 280 285
Arg Phe Val Glu Leu Thr Gly Ile Pro Val Thr Thr Thr Leu Met Gly
290 295 300
Leu Gly Asn Phe Pro Ser Asp Asp Pro Leu Ser Leu Arg Met Leu Gly
305 310 315 320
Met His Gly Thr Val Tyr Ala Asn Tyr Ala Val Asp Lys Ala Asp Leu
325 330 335
Leu Leu Ala Phe Gly Val Arg Phe Asp Asp Arg Val Thr Gly Lys Ile
340 345 350
Glu Ala Phe Ala Ser Arg Ser Lys Ile Val His Ile Asp Ile Asp Pro
355 360 365
Ala Glu Ile Gly Lys Asn Lys Gln Pro His Val Ser Ile Cys Ala Asp
370 375 380
Val Lys Leu Ala Leu Gln Gly Leu Asn Ala Val Leu Thr Gly Ser Lys
385 390 395 400
Cys Asp Lys Ser Phe Asp Phe Ala Ser Trp His Asp Glu Leu Glu Gln
405 410 415
Gln Lys Arg Glu Phe Pro Leu Gly Tyr Lys Thr Phe Gly Glu Ala Ile
420 425 430
Pro Pro Gln Tyr Ala Ile Gln Val Leu Asp Glu Leu Thr Lys Gly Glu
435 440 445
Ala Ile Ile Ala Thr Gly Val Gly Gln His Gln Met Trp Ala Ala Gln
450 455 460
Tyr Tyr Thr Tyr Lys Arg Pro Arg Gln Val Leu Ser Ser Ala Gly Leu
465 470 475 480
Gly Ala Met Gly Phe Gly Leu Pro Ala Ala Ala Gly Thr Ala Val Ala
485 490 495
Asn Pro Gly Val Thr Val Val Asp Ile Asp Gly Asp Gly Ser Phe Leu
500 505 510
Met Asn Ile Gln Glu Leu Ala Leu Ile Arg Ile Glu Asn Leu Pro Val
515 520 525
Lys Val Met Ile Leu Asn Asn Gln His Leu Gly Met Val Val Gln Trp
530 535 540
Glu Asp Arg Phe Tyr Lys Ala Asn Arg Ala His Thr Tyr Leu Gly Asn
545 550 555 560
Pro Glu Asn Glu Ser Glu Ile Tyr Pro Asp Phe Val Thr Ile Ala Lys
565 570 575
Gly Phe Asn Val Pro Ala Val Arg Val Thr Lys Arg Ser Glu Val Arg
580 585 590
Ala Ala Ile Lys Lys Met Leu Glu Thr Pro Gly Pro Tyr Leu Leu Asp
595 600 605
Ile Ile Val Pro His Gln Glu His Val Leu Pro Met Ile Pro Ser Gly
610 615 620
Gly Ala Phe Lys Asp Ile Ile Met Glu Gly Asp Gly Arg Ile Ser Tyr
625 630 635 640
<210>14
<211>670
<212>PRT
<213>拟南芥
<400>14
Met Ala Ala Ala Thr Thr Thr Thr Thr Thr Ser Ser Ser Ile Ser Phe
1 5 10 15
Ser Thr Lys Pro Ser Pro Ser Ser Ser Lys Ser Pro Leu Pro Ile Ser
20 25 30
Arg Phe Ser Leu Pro Phe Ser Leu Asn Pro Asn Lys Ser Ser Ser Ser
35 40 45
Ser Arg Arg Arg Gly Ile Lys Ser Ser Ser Pro Ser Ser Ile Ser Ala
50 55 60
Val Leu Asn Thr Thr Thr Asn Val Thr Thr Thr Pro Ser Pro Thr Lys
65 70 75 80
Pro Thr Lys Pro Glu Thr Phe Ile Ser Arg Phe Ala Pro Asp Gln Pro
85 90 95
Arg Lys Gly Ala Asp Ile Leu Val Glu Ala Leu Glu Arg Gln Gly Val
100 105 110
Glu Thr Val Phe Ala Tyr Pro Gly Ala Ala Ser Met Glu Ile His Gln
115 120 125
Ala Leu Thr Arg Ser Ser Ser Ile Arg Asn Val Leu Pro Arg His Glu
130 135 140
Gln Gly Gly Val Phe Ala Ala Glu Gly Tyr Ala Arg Ser Ser Gly Lys
145 150 155 160
Pro Gly Ile Cys Ile Ala Thr Ser Gly Pro Gly Ala Thr Asn Leu Val
165 170 175
Ser Gly Leu Ala Asp Ala Leu Leu Asp Ser Val Pro Leu Val Ala Ile
180 185 190
Thr Gly Gln Val Pro Arg Arg Met Ile Gly Thr Asp Ala Phe Gln Glu
195 200 205
Thr Pro Ile Val Glu Val Thr Arg Ser Ile Thr Lys His Asn Tyr Leu
210 215 220
Val Met Asp Val Glu Asp Ile Pro Arg Ile Ile Glu Glu Ala Phe Phe
225 230 235 240
Leu Ala Thr Ser Gly Arg Pro Gly Pro Val Leu Val Asp Val Pro Lys
245 250 255
Asp Ile Gln Gln Gln Leu Ala Ile Pro Asn Trp Glu Gln Ala Met Arg
260 265 270
Leu Pro Gly Tyr Met Ser Arg Met Pro Lys Pro Pro Glu Asp Ser His
275 280 285
Leu Glu Gln Ile Val Arg Leu Ile Ser Glu Ser Lys Lys Pro Val Leu
290 295 300
Tyr Val Gly Gly Gly Cys Leu Asn Ser Ser Asp Glu Leu Gly Arg Phe
305 310 315 320
Val Glu Leu Thr Gly Ile Pro Val Ala Ser Thr Leu Met Gly Leu Gly
325 330 335
Ser Tyr Pro Cys Asp Asp Glu Leu Ser Leu His Met Leu Gly Met His
340 345 350
Gly Thr Val Tyr Ala Asn Tyr Ala Val Glu His Ser Asp Leu Leu Leu
355 360 365
Ala Phe Gly Val Arg Phe Asp Asp Arg Val Thr Gly Lys Leu Glu Ala
370 375 380
Phe Ala Ser Arg Ala Lys Ile Val His Ile Asp Ile Asp Ser Ala Glu
385 390 395 400
Ile Gly Lys Asn Lys Thr Pro His Val Ser Val Cys Gly Asp Val Lys
405 410 415
Leu Ala Leu Gln Gly Met Asn Lys Val Leu Glu Asn Arg Ala Glu Glu
420 425 430
Leu Lys Leu Asp Phe Gly Val Trp Arg Asn Glu Leu Asn Val Gln Lys
435 440 445
Gln Lys Phe Pro Leu Ser Phe Lys Thr Phe Gly Glu Ala Ile Pro Pro
450 455 460
Gln Tyr Ala Ile Lys Val Leu Asp Glu Leu Thr Asp Gly Lys Ala Ile
465 470 475 480
Ile Ser Thr Gly Val Gly Gln His Gln Met Trp Ala Ala Gln Phe Tyr
485 490 495
Asn Tyr Lys Lys Pro Arg Gln Trp Leu Ser Ser Gly Gly Leu Gly Ala
500 505 510
Met Gly Phe Gly Leu Pro Ala Ala Ile Gly Ala Ser Val Ala Asn Pro
515 520 525
Asp Ala Ile Val Val Asp Ile Asp Gly Asp Gly Ser Phe Ile Met Asn
530 535 540
Val Gln Glu Leu Ala Thr Ile Arg Val Glu Asn Leu Pro Val Lys Val
545 550 555 560
Leu Leu Leu Asn Asn Gln His Leu Gly Met Val Met Gln Trp Glu Asp
565 570 575
Arg Phe Tyr Lys Ala Asn Arg Ala His Thr Phe Leu Gly Asp Pro Ala
580 585 590
Gln Glu Asp Glu Ile Phe Pro Asn Met Leu Leu Phe Ala Ala Ala Cys
595 600 605
Gly Ile Pro Ala Ala Arg Val Thr Lys Lys Ala Asp Leu Arg Glu Ala
610 615 620
Ile Gln Thr Met Leu Asp Thr Pro Gly Pro Tyr Leu Leu Asp Val Ile
625 630 635 640
Cys Pro His Gln Glu His Val Leu Pro Met Ile Pro Ser Gly Gly Thr
645 650 655
Phe Asn Asp Val Ile Thr Glu Gly Asp Gly Arg Ile Lys Tyr
660 665 670
<210>15
<211>21
<212>DNA
<213>人工序列
<220>
<223>人工序列说明：合成DNA
<400>15
ccaccaccca ccatggctac g 21
<210>16
<211>20
<212>DNA
<213>人工序列
<220>
<223>人工序列说明：合成DNA
<400>16
gaagaggtgg ttggtgatga 20
<210>17
<211>20
<212>DNA
<213>人工序列
<220>
<223>人工序列说明：合成DNA
<400>17
gcaaccaacc tcgtgtccgc 20
<210>18
<211>20
<212>DNA
<213>人工序列
<220>
<223>人工序列说明：合成DNA
<400>18
gaaggcttcc tgtatgacgc 20
<210>19
<211>20
<212>DNA
<213>人工序列
<220>
<223>人工序列说明：合成DNA
<400>19
gaattgcgct ggtttgttga 20
<210>20
<211>21
<212>DNA
<213>人工序列
<220>
<223>人工序列说明：合成DNA
<400>20
ctcaattttc cctgtcacac g 21
<210>21
<211>20
<212>DNA
<213>人工序列
<220>
<223>人工序列说明：合成DNA
<400>21
ggtagcttcc tcatgaacat 20
<210>22
<211>20
<212>DNA
<213>人工序列
<220>
<223>人工序列说明：合成DNA
<400>22
aatgttcatg aggaagctac 20
<210>23
<211>21
<212>DNA
<213>人工序列
<220>
<223>人工序列说明：合成DNA
<400>23
cattcaggtc aaacataggc c 21
<210>24
<211>29
<212>DNA
<213>人工序列
<220>
<223>人工序列说明：合成DNA
<400>24
tacccgggcn nngcgtccat ggagatcca 29
<210>25
<211>16
<212>DNA
<213>人工序列
<220>
<223>人工序列说明：合成DNA
<400>25
tgtgcttggt gatgga 16
<210>26
<211>23
<212>DNA
<213>人工序列
<220>
<223>人工序列说明：合成DNA
<400>26
gggctggcaa gccacgtttg gtg 23
<210>27
<211>18
<212>DNA
<213>人工序列
<220>
<223>人工序列说明：合成DNA
<400>27
cagcgacgtg ttcgccta 18
<210>28
<211>30
<212>DNA
<213>人工序列
<220>
<223>人工序列说明：合成DNA
<400>28
ccccagccgc atgatcggca ccgacgcctt 30
<210>29
<211>19
<212>DNA
<213>人工序列
<220>
<223>人工序列说明：合成DNA
<400>29
ctgggacacc tcgatgaat 19
<210>30
<211>21
<212>DNA
<213>人工序列
<220>
<223>人工序列说明：合成DNA
<400>30
aactgggata ccagtcagct c 21
<210>31
<211>21
<212>DNA
<213>人工序列
<220>
<223>人工序列说明：合成DNA
<400>31
gctctgctac aacagagcac a 21
<210>32
<211>17
<212>DNA
<213>人工序列
<220>
<223>人工序列说明：合成DNA
<400>32
gattgcctca cctttcg 17
<210>33
<211>17
<212>DNA
<213>人工序列
<220>
<223>人工序列说明：合成DNA
<400>33
cagcccaaat cccattg 17
<210>34
<211>17
<212>DNA
<213>人工序列
<220>
<223>人工序列说明：合成DNA
<400>34
aggtgtcaca gttgttg 17
<210>35
<211>22
<212>DNA
<213>人工序列
<220>
<223>人工序列说明：合成DNA
<400>35
tcaaggacat gatcctggat gg 22
<210>36
<211>21
<212>DNA
<213>人工序列
<220>
<223>人工序列说明：合成DNA
<400>36
agtcctgcca tcaccatcca g 21
<210>37
<211>23
<212>DNA
<213>人工序列
<220>
<223>人工序列说明：合成DNA
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ccgggagctg catgtgtcag agg 23
Claims (2)
1.一种转化方法，所述方法包括以下步骤：
使用含有目标基因和编码变异型乙酰乳酸合成酶的基因的重组载体，转化宿主细胞的步骤，和在嘧啶基羧基类除草剂的存在下培养上述步骤中所得的转化细胞的步骤，
其特征在于，使用编码所述变异型乙酰乳酸合成酶的基因作为选择标记物，
所述变异型乙酰乳酸合成酶是由序列号2中记载的氨基酸序列形成的变异型乙酰乳酸合成酶，所述嘧啶基羧基类除草剂是选自双草醚钠、嘧硫草醚钠、嘧草醚中的至少一种化合物。
2.如权利要求1所述的转化方法，其特征在于，所述宿主细胞是植物细胞。
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| Application Number | Priority Date | Filing Date | Title |
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| JP2005136186 | 2005-05-09 | ||
| JP136186/2005 | 2005-05-09 | ||
| PCT/JP2006/309622 WO2006121178A1 (ja) | 2005-05-09 | 2006-05-09 | 変異型アセト乳酸シンターゼ遺伝子を用いた形質転換方法 |
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| Publication Number | Publication Date |
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| CN101175849A CN101175849A (zh) | 2008-05-07 |
| CN101175849B true CN101175849B (zh) | 2011-04-06 |
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| Application Number | Title | Priority Date | Filing Date |
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| CN2006800160362A Expired - Fee Related CN101175849B (zh) | 2005-05-09 | 2006-05-09 | 使用变异型乙酰乳酸合成酶基因的转化方法 |
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| US (1) | US8017400B2 (zh) |
| EP (1) | EP1889902B1 (zh) |
| JP (1) | JP4968685B2 (zh) |
| KR (1) | KR101323617B1 (zh) |
| CN (1) | CN101175849B (zh) |
| CA (1) | CA2607645C (zh) |
| ES (1) | ES2372971T3 (zh) |
| PT (1) | PT1889902E (zh) |
| WO (1) | WO2006121178A1 (zh) |
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- 2006-05-09 EP EP06746369A patent/EP1889902B1/en active Active
- 2006-05-09 CN CN2006800160362A patent/CN101175849B/zh not_active Expired - Fee Related
- 2006-05-09 KR KR1020077028724A patent/KR101323617B1/ko active IP Right Grant
- 2006-05-09 US US11/920,161 patent/US8017400B2/en active Active
- 2006-05-09 WO PCT/JP2006/309622 patent/WO2006121178A1/ja active Application Filing
- 2006-05-09 ES ES06746369T patent/ES2372971T3/es active Active
- 2006-05-09 CA CA2607645A patent/CA2607645C/en not_active Expired - Fee Related
- 2006-05-09 PT PT06746369T patent/PT1889902E/pt unknown
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Also Published As
| Publication number | Publication date |
|---|---|
| EP1889902B1 (en) | 2011-10-12 |
| PT1889902E (pt) | 2011-12-16 |
| CA2607645A1 (en) | 2006-11-16 |
| ES2372971T3 (es) | 2012-01-30 |
| US20090217420A1 (en) | 2009-08-27 |
| CN101175849A (zh) | 2008-05-07 |
| EP1889902A1 (en) | 2008-02-20 |
| US8017400B2 (en) | 2011-09-13 |
| KR20080033165A (ko) | 2008-04-16 |
| JPWO2006121178A1 (ja) | 2008-12-18 |
| WO2006121178A1 (ja) | 2006-11-16 |
| EP1889902A4 (en) | 2008-10-08 |
| CA2607645C (en) | 2014-02-04 |
| KR101323617B1 (ko) | 2013-11-06 |
| JP4968685B2 (ja) | 2012-07-04 |
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