CN103436547B - 一种具有草甘膦耐性的基因及其应用 - Google Patents

一种具有草甘膦耐性的基因及其应用 Download PDF

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CN103436547B
CN103436547B CN201310396762.3A CN201310396762A CN103436547B CN 103436547 B CN103436547 B CN 103436547B CN 201310396762 A CN201310396762 A CN 201310396762A CN 103436547 B CN103436547 B CN 103436547B
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强胜
毛婵娟
陈世国
戴伟民
宋小玲
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Abstract

本发明公开了一种具有草甘膦耐性的基因及其应用。该基因的核苷酸序列为SEQIDNO：1、SEQIDNO：3或SEQIDNO：5及其保持抗草甘膦活性的突变基因，以及编码的蛋白质氨基酸序列分别为SEQIDNO：2、SEQIDNO：4或SEQIDNO：6及其在第280到294位的保守区1、第416到433位的保守区2中氨基酸突变，利用该来自于植物的具有草甘膦耐性的基因及其突变制备抗/耐草甘膦植株，可以明显的显示出抗/耐草甘膦效果，这为培育抗/耐草甘膦除草剂作物提供了新的选择，增加抗/耐草甘膦除草剂转基因技术的多样性，从而降低了抗/耐除草剂转基因技术的生态风险。

Description

一种具有草甘膦耐性的基因及其应用
技术领域
本发明属于植物分子生物学与植物遗传工程领域，具体地说，本发明涉及一种植物源抗/耐草甘膦基因以及该基因编码的蛋白质，并且对其进行人工改造，提供一种人工突变获得高抗草甘膦基因突变体的方法以及基因突变后获得的植物源高抗/耐草甘膦基因。通过遗传转化的方法使该基因在植物中表达而使植物获得对草甘膦的抗性能力，从而可以利用草甘膦于作物田间选择性地防除杂草。本发明也可以运用在作物育种、植物细胞培养的筛选。
背景技术
自从1946年开始使用2,4-D，化学除草剂已走过60多年的历程，为全球粮食生产和农业现代化做出了巨大贡献（PowelsandYu,2010）。其中草甘膦（N-（膦羧基甲基）甘氨酸）是迄今为止最为重要、应用最为广泛和最优秀的除草剂（DukeandPowles,2008）。自1974年美国孟山都公司开发草甘膦以来，由于其具有广谱、低毒、安全、无土壤残留的特点，迅速占据了世界除草剂的主导地位。草甘膦毒性作用机理主要是竟争性抑制EPSP合酶即5-烯醇式丙酮酸-3-磷酸莽草酸合酶(EPSPS)，该酶广泛存在于真菌、细菌、藻类、高等植物体内，但不存在于动物体内。草甘膦是磷酸烯醇式丙酮酸(PEP)的类似物，二者的分子式极为相似，它能与PEP竞争性结合EPSP合酶，形成EPSP合酶·3-磷酸莽草酸(S3P)·草甘膦的复合物，阻断EPSP的合成，从而阻断芳香族氨基酸的合成，导致植物死亡。而草甘膦无选择性地杀死作物和杂草，限制了它在农业生产上的使用，同时也给农业生产带来了巨大的损失。自1996开始，抗草甘膦转基因作物（如大豆、玉米、棉花和油菜）研制和商业化，才使得草甘膦被广泛应用于农作物田杂草的防除（Powles,2008）。目前，草甘膦已成为生产量最大的农药品种，占全球农药市场的近20％，年销售超过20亿美元。许多研究者通过多种方法来选育抗草甘膦的作物新品种。
目前通过基因工程培育抗草甘膦植物是最有效的获取抗性作物的手段。基因工程方法多采用细菌来源的抗性EPSP合成酶基因，该基因的产物与草甘膦结合活性下降而不能被其竞争抑制，保证了植物芳香族氨基酸的正常合成。常用的抗性基因来源有鼠沙门氏菌、根癌农杆菌、大肠杆菌、假单胞菌等。许多研究者利用这些抗性基因，采用基因工程的方法获得了表达细菌EPSP合成酶的转基因植物表现对草甘膦的抗性（SostandAmrherin,1990;BlackburnandBoutin,2003;Maskell,1998;GalloandIrvine,1996;Zhouetal.,1995;Mannerlofetal.,1997;Penalozaetal.,1995;Zboinskaetal.,1992），其中转基因抗草甘膦大豆已在美国、巴西等国家大面积栽种。但这些来源于细菌的抗性基因作物作为食品或食品原料，其安全性一直是争论的焦点。
在植物中也发现了由于EPSP合酶单个氨基酸位点突变造成其对草甘膦具有抗药性。牛筋草（Eleusineindica）对草甘膦的抗性提高了8～12倍，在于其EPSPS第106位脯氨酸变成了丝氨酸或苏氨酸（Pro106Ser/Thr）（Baersonetal..,2002;Ngetal.,2003)；抗草甘膦的瑞士黑麦草（Loliumrigidum）不同种群的EPSPS的106位脯氨酸有2种不同突变，被苏氨酸或丙氨酸取代（Pro106Thr/Ala）（WakelinandPreson,2006;Yuetal.,2007）；EPSP合酶第182位氨基酸由脯氨酸（P）变成了丝氨酸（S）是多花黑麦草（Loliummultiflorum）种群的草甘膦抗性机制之一（González-Torralva,2012）。通过这些有效位点的基因突变，降低了EPSPS对草甘膦的亲和性，从而增强了杂草对草甘膦的抗性。
同时，对抗草甘膦杂草EPSPS基因研究证实，杂草对草甘膦的抗药性大多是由氨基酸突变造成的极性变化所致。抗草甘膦牛筋草和瑞士黑麦草EPSPS编码基因106位非极性脯氨酸分别突变为极性丝氨酸和苏氨酸(Baerson,2002;WakelinandPreston,2006)；抗草甘膦瑞士黑麦草和田旋花EPSPS基因的101位分别由非极性脯氨酸和非极性苯丙氨酸突变为极性丝氨酸(SimarmataandPenner,2008;Zhangetal.,2011)。氨基酸极性的改变，可能会影响EPSP合酶与草甘膦的亲和性。
孟山都公司已经对抗草甘膦牛筋草的EPSPS基因申请了专利保护。但是为了提高转基因农作物的抗性水平和增加抗性基因的多样性，生产应用中仍然需要新的抗/耐草甘膦基因和以此为基础的抗/耐草甘膦植物。相对于细菌等来源的抗草甘膦基因，植物来源的进化抗性或天然抗性基因，在培育抗/耐草甘膦除草剂作物后的生态环境和食品安全风险要低于其它生物类别的异源基因，提高民众心理的认可。
发明内容
本发明的目的是为了解决现有技术中存在的缺陷，提供一种基于自然界的植物天然耐性为基础，经过前期对大量植物进行的草甘膦耐性筛选得到的新的抗/耐草甘膦基因，并且对其进行改良得到一系列突变基因，利用这些基因可以用来产生转基因抗草甘膦植物，也可作为植物细胞培育中的筛选标记。
为了达到上述目的，本发明提供了三种具有草甘膦耐性的基因（LS-EPSPS）LsEPSPS1、LsEPSPS2和LsEPSPS3，核苷酸序列分别为SEQIDNO：1、SEQIDNO：3和SEQIDNO：5。
本发明还提供了一种抗/耐草甘膦蛋白质多肽，该抗/耐草甘膦蛋白质多肽的氨基酸序列为SEQIDNO：2、SEQIDNO：4、SEQIDNO：6或与上述氨基酸序列相比具有不小84%相同性的氨基酸序列。
SEQIDNO：2、SEQIDNO：4和SEQIDNO：6分别表示为SEQIDNO：1、SEQIDNO：3和SEQIDNO：5编码的氨基酸序列。这三种氨基酸序列均包含四个特异性位点，麦冬中为144m，184i，232a以及273m，土麦冬和阔叶土麦冬中为146m，186i，234a以及275m，相当于大肠杆菌EPSP合酶氨基酸序列中70、107、153以及192位。
氨基酸序列SEQIDNO：2、SEQIDNO：4和SEQIDNO：6表示的蛋白质多肽包括信号肽和成熟蛋白两部分。
本发明还提供一种通过突变获得保持有抗草甘膦能力的突变基因的方法，该方法包括以下步骤：
（1）获得突变基因，所述突变基因编码的蛋白质氨基酸序列SEQIDNO：7（此序列为SEQIDNO:4去除信号肽后的氨基酸序列）的第280到294位（保守区1）和第416到433位（保守区2）氨基酸序列至少各发生一个氨基酸突变；
（2）然后通过草甘膦筛选突变基因，从而选择高抗草甘膦的突变基因。
其中突变基因采用易错倾向PCR、定点突变、人工合成技术或者其他基因突变技术获得。
通过上述方法，本发明提供了一种具有草甘膦耐性的基因，基因编码的蛋白质的氨基酸序列在SEQIDNO：7的第280到294位的保守区1、第416到433位的保守区2中至少各发生一个氨基酸突变。特别在保守区1和保守区2个至少引入1个或者1个以上如下位点的特变:保守区1：1)第284位赖氨酸；2)第285位苯丙氨酸；3)第289位亮氨酸；保守区2：1)第416位缬氨酸；2)第421位精氨酸；3)第422位天冬氨酸；4)第424位甘氨酸；5)第425位半胱氨酸。
本发明同时也提供一种抗草甘膦的突变基因，其编码的蛋白质在以下两个保守区域至少各发生一个氨基酸突变。保守区1：1)第284位氨基酸由赖氨酸变为精氨酸；2)第285位氨基酸由苯丙氨酸变为酪氨酸；3)第289位氨基酸由亮氨酸变为组氨酸；保守区2：1)第416位氨基酸由缬氨酸变为谷氨酸；2)第421位氨基酸由精氨酸变为甘氨酸；3)第422位氨基酸由天冬氨酸变为缬氨酸；4)第424位氨基酸由甘氨酸变为半胱氨酸；5)第425位氨基酸由半胱氨酸变为酪氨酸。
本发明进一步提供一种具有草甘膦耐性的蛋白质，其第280到294位的氨基酸序列为SEQIDNO:8-SEQIDNO:14中的任意一种，第280到294位的氨基酸序列为SEQIDNO:15-SEQIDNO:28中的任意一种。
本发明还提供了一种具有草甘膦耐性的蛋白质，该蛋白质的氨基酸序列为SEQIDNO：29-SEQIDNO：52中的任意一种。
本发明还提供了核苷酸序列为编码上述蛋白质多肽的基因。
利用上述具有草甘膦耐性基因的核酸序列，可以用来人工引入一个或多个变异，然后通过草甘膦筛选变异分子，而获得保持有抗/耐草甘膦能力的变异分子。例如利用低保真PCR的方法可以产生很多LSEPSPS的变异分子，而这些变异分子可以通过草甘膦筛选获得继续保持或增强有抗/耐草甘膦能力的变异分子。上述具有抗/耐草甘膦的LS-EPSPS基因LsEPSPS1、LsEPSPS2和LsEPSPS3及其获得的保持有草甘膦耐性的突变基因LSE-EPSPS统称为抗/耐草甘膦基因（LSEPSPS）。
本发明还提供了能够与上述蛋白质多肽结合的抗体。
利用本发明的基因编码的氨基酸序列，可设计和人工添加信号肽序列以有利于在植物中的表达。
利用本发明的基因编码的氨基酸序列，可以设计和人工合成密码子优化的有利于在植物中表达的核酸序列。例如Campbell和Gowri（1990）的报道（PlantPhysiology,92:1-11）。
本发明还提供了一种包含上述抗/耐草甘膦基因的表达载体，所述表达载体为原核表达载体或植物转化质粒。
构建包含表达构件的原核表达载体时，表达构件包括启动子和抗/耐草甘膦基因，载体质粒可以是PET系列、pMAL-p2X或pRSET-A等。在原核生物表达系统中，启动子可以是T7噬菌体启动子或者lac(乳糖启动子)、trp(色氨酸启动子)、PL和PR(λ噬菌体的左向和右向启动子)以及tac（乳糖和色氨酸的杂合启动子)等强启动子。
利用上述抗/耐草甘膦基因进一步构建包含表达构件的植物转化质粒时，表达构件包括启动子，带有叶绿体信号肽的抗/耐草甘膦基因和终止子。在转化单子叶植物时启动子可以是玉米Ubiqutin启动子，或者水稻Actin启动子，或者其他启动子。而终止子可以是根据根癌农杆菌终止子或者其他终止子。抗/耐草甘膦的LSE-EPSPS基因在5’端连接一个在同一个表达阅读框内表达的编码信号肽的DNA片段，这个信号肽可以引导LSEPSPS蛋白质进入叶绿体。这个信号肽可以是Rubisco的亚基的信号肽（decastroSilvaFilho1996PlantMol.Biol.30:767一780)，或者植物EPSPS信号肽（Archer1990J.Bioenerg．Biomemb.22(b):789一810），或者其他能使EPSPS发挥功能的信号肽，或者无信号肽。这个表达构件可以通过农杆菌（如Agrobacterium菌株LAB4404）转入整合到植物的基因组中，稳定遗传和表达。
本发明还提供了包含上述抗/耐草甘膦基因的植物细胞，以及包含该植物细胞的抗/耐草甘膦植株。
本发明还提供了上述抗/耐草甘膦基因在制备抗/耐草甘膦植株方面的用途，以及上述抗/耐草甘膦基因作为植物转基因筛选标记的用途。
利用抗/耐草甘膦基因制备抗/耐草甘膦植株，可通过含有LSEPSPS表达构件的植物转化质粒获得。植物转化的方法和步骤各种植物有所不同，同种植物不同的品种也可能有所不同。但是植物转化的技术和方法是已知和成熟的。通常通过农杆菌或基因枪导入植物的未成熟胚、成熟胚、未分化的愈伤组织或原生质体。然后用不同浓度的草甘膦培养基进行筛选培养。再进行分化而获得转化芽，经过生根培养基培养就可以获得可以种植的种苗。也可以通过花粉介导法，通过农杆菌导入植物的花粉从而获得转基因的种子。更进一步，草甘膦可以喷施在转化苗及其后代筛选除去未转化植株和后代分离失去LSEPSPS基因的植株。具体可通过以下步骤制备：
（1）利用DNA重组技术，构建含有所述抗/耐草甘膦基因的植物转化质粒；
（2）将步骤（1）中构建的植物转化质粒通过基因枪或农杆菌浸染方法或花粉介导法导入植物组织，利用草甘膦培养基筛选含有抗/耐草甘膦基因的植物细胞；
（3）将步骤（2）中筛选的植物细胞进行分化获得转化芽，经过生根培养获得植物种苗，从而得到所述抗/耐草甘膦植株。
植物可选取水稻、玉米、棉花、小麦、大豆、马铃薯、甘薯、谷子、高粱、大麦、油菜、甘蔗、烟草、草坪草（狗牙根、结缕草、黑麦草、早熟禾、暖地大叶草、剪股颖、海滨雀稗）或牧草（紫苜蓿、红三叶草、雀稗）、蔬菜（青菜、白菜、黄瓜、芹菜、辣椒、茄子）、花卉（月季、康乃馨、菊花）。
抗/耐草甘膦基因作为植物转基因筛选标记时，通过已知的分子克隆技术可以使LSEPSPS在植物细胞中表达，通常构建一个在植物中表达的表达构件。这个表达构件包括启动子、带有叶绿体信号肽的LSEPSPS和终止子。这个表达构件可作为筛选标志被克隆到植物转化质粒。植物转化质粒通常还含有目的基因和其他DNA序列。植物转化质粒可以通过基因枪或农杆菌浸染方法或花粉介导法导入植物组织，而含有合适浓度的草甘膦培养基则可以选择性地杀死没有导入质粒DNA的植物细胞，从而选择含有LSEPSPS目的基因的植物细胞。
本发明适用于所有植物，包括双子叶和单子叶植物。
本发明相比现有技术具有以下优点：本发明基于自然界的植物天然耐性为基础，经过前期大量植物对草甘膦的耐性筛选，代替依赖于在长期草甘膦环境中生存的植物或微生物的抗性生物型筛选，发现了百合科麦冬草的草甘膦耐药性，并利用易错倾向PCR技术进行基因改造使其获得更高的草甘膦抗性，为培育抗/耐草甘膦除草剂作物提供了新的选择，增加抗/耐草甘膦除草剂转基因技术的多样性，从而降低了抗/耐除草剂转基因技术的生态风险；再则，由于本发明的基因是植物本身来源，在培育抗/耐草甘膦除草剂作物后的生态环境和食品安全风险要低于不同生物类别的异源基因，提高民众认可。
具体实施方式
下面结合具体实施例对本发明进行详细说明。
实施例1.麦冬、土麦冬和阔叶土麦冬的草甘膦抗性实验
选取长势均匀的麦冬、土麦冬和阔叶土麦冬，进行喷雾处理。草甘膦浓度设置为0、375、750、1500、3000、6000、12000、24000ai.g/ha，试验按照设计用量处理供试植物材料，每一处理重复四次。喷药后观察植株受损伤程度，每周调查并记录药害等级，绘制抑制率曲线并进行Logistic回归分析，计算ED50值。重复三次。结果发现，麦冬、土麦冬和阔叶土麦冬的草甘膦抗性分别是一般植物的4.73、5.06和5.8倍。
实施例2.EPSPS基因的克隆
以麦冬、土麦冬和阔叶土麦冬总RNA反转录产物为模板，利用EPSPS基因保守序列设计引物，进行RT-PCR反应，将扩增得到的片段克隆至pMD19-T载体中，进行测序及BLAST。
得到保守区的序列后，再通过RACE（rapid-amplificationofcDNAends）技术，进行EPSP合成酶基因的全长克隆。以接头引物AP进行麦冬、土麦冬和阔叶土麦冬RNA的反转录，利用已经得到的基因保守区序列设计基因特异性引物，并与3’端的特异引物进行RT-PCR反应。
根据已经得到的目的基因片段，利用TdT末端转移酶法进行5’端的克隆。设计基因特异性引物反转录后进行单引物的扩增，然后纯化扩增产物，用dCTP和TdT对纯化的扩增产物加尾，使用锚定引物进行PCR扩增带有dC尾的产物，最后使用巢式重新扩增初步PCR产物。将扩增得到的序列进行克隆测序。
最后，将保守区、3’端序列，5’端序列进行拼接，获得麦冬草EPSPS基因的cDNA的全长序列。麦冬基因的核苷酸序列为SEQIDNO:1，并命名为LsEPSPS1，其编码的氨基酸为SEQIDNO:2；土麦冬EPSPS基因的核苷酸序列为SEQIDNO:3，并命名为LsEPSPS2，其编码的氨基酸序列为SEQIDNO:4；阔叶土麦冬EPSPS基因的核苷酸序列为SEQIDNO:5，并命名为LsEPSPS3，其编码的氨基酸序列为SEQIDNO:6。
实施例3.麦冬、土麦冬和阔叶土麦冬EPSPS基因编码氨基酸序列的分析比对
通过MAGA软件分析比较了与麦冬、土麦冬和阔叶土麦冬EPSP合酶一致性较高的约260条EPSP合酶氨基酸序列，发现麦冬、土麦冬与阔叶土麦冬的EPSP合酶的氨基酸序列与其他植物相比存在四处特异的氨基酸，分别是144m，184i，232a以及273m（序列编号根据麦冬EPSP合酶氨基酸序列SEQIDNO:2，相当于大肠杆菌EPSP合酶的70、107、153及192位，土麦冬和阔叶土麦冬中为146m，186i，234a以及275m），这些位点的氨基酸在麦冬、土麦冬与阔叶土麦冬中均保守，但在其他植物的相同位点均不存在。正是由于这些氨基酸的存在导致了麦冬、土麦冬与阔叶土麦冬对草甘膦的耐药性。
144和273位的Met中含有甲硫基—SCH3，其他氨基酸中均不含有，可能由于甲硫基的存在导致EPSP合酶结构发生改变。
184位的Ile与其他植物中的Val和Thr相比，R基团多一个—CH2—，可能由于—CH2—的增加致使同一位点所需的空间体积增大，使得与草甘膦的结合的空间位阻增大。
232位的Ala与其他植物中的Val相比，少了两个—CH3，使得R基团长度和空间位阻都减少，可能造成EPSP合酶结构发生改变。
将麦冬的EPSP合酶的氨基酸序列（共516个氨基酸）进行二级结构预测，并与已知的EPSP合酶主链图进行比较，发现整个氨基酸部位总共有12个螺旋（除信号肽），第144位甲硫氨酸位于第2和第3个螺旋的转角处，大约处于第3区；第184位异亮氨酸位于第3个螺旋上；第232位丙氨酸位于第4和第5螺旋的第2个折叠处；第273位甲硫氨酸位于第5和第6个螺旋的折叠处。
144Met与草甘膦结合位点22Lys以及S3P结合位点23Ser、27Arg较为接近；184Ile与草甘膦结合位点96Gly、94Asn同处于第三个螺旋上，其增加的主链长度可能会使草甘膦结合的空间位阻增大从而降低草甘膦与EPSP合酶的亲和性；232Ala与124Arg很接近；273Met与其空间构像可能会影响169Ser、170Ser、171Gln以及197Ser都处于第5个结构域。这四个位点均处于底物与酶的结合有关的区域，此四个氨基酸的变化，可能导致EPSP合酶结构的变化，使其与底物的结合程度发生变化，从而导致草甘膦的耐药性。
实施例4.错误倾向PCR突变扩增
根据已知的土麦冬的EPSPS基因设计引物，并以此为模板,进行错误倾向PCR致突变反应,体系总体积100ul:10×致突变PCR缓冲液10Ll,10×dNTP混合物10ul,引物各10ul,MnCl210ul,模板10ul,TaqDNA聚合酶1ul,超纯水39ul.将混合好的反应体系以10ul分装入eppendorf管中进行PCR循环反应,反应条件为:94℃变性5min,94℃变性1min,50℃退火1min,72℃延伸1min,72℃延伸10min.30个循环后,110%琼脂糖凝胶电泳,溴化乙锭(EB)染色后对其进行紫外分析检测.用Promega公司DNA纯化试剂盒回收琼脂糖上PCR产物.扩增得到约1300bp的目的片段。
实施例5.重组质粒的构建及阳性克隆菌株的筛选及鉴定
用SphI和HindIII限制性酶分别切割pQE80L质粒载体和PCR突变产物,得到具有粘性末端的基因片段(大小分别约为4.7kb和1.3kb),将酶切后的基因片段用T4连接酶连接,再进行感受态细胞E.coli转化.挑取将能在含草甘膦100mmol/L培养基上生长的菌落，进行质粒的提取，并再次转化E.coli，使得原本在100mmol/L草甘膦培养基上不能生长的大肠杆菌获得草甘膦抗性。经测序得到该核苷酸序列为SEQIDNO:29-SEQIDNO:52。
实施例6.高抗草甘膦LSE-EPSPS氨基酸序列突变位点的分析
在100mmol/L草甘膦上能够正常生长的克隆可能包含一个抗草甘膦性能比原始基因LS-EPSPS更加高的突变体。因此测定了实施例2获得的抗草甘膦克隆的突变区域的核苷酸序列，结果发现这此基因在保守区1(从第280到294号氨基酸)和保守区2(从第416到433号氨基酸)的突变结果如下:
表1EPSP合酶氨基酸序列保守区突变情况
保守区域 序列 突变情况
280-294 280GGDVKFAEVLEKMG294A K284R,F285Y,L289H
416-433 416VPVTIRDPGCTRKTFPD433Y V416E,R421G,D422V,G424C,C425Y
实施例7.保守区氨基酸突变功能验证
为了验证保守区280-294以及416-433区域的突变位点是否导致草甘膦抗性，本发明通过定点突变的方法获得一系列的突变基因，并将其导入大肠杆菌，在含有100mmol/L草甘膦培养基上37℃培养48h，观察生长情况，对能够正常生长的突变基因进行统计，结果发现，草甘膦抗性蛋自质的第280到294位的氨基酸序列为SEQIDNO:8-SEQIDNO:14中的任意一种，第280到294位的氨基酸序列为SEQIDNO:15-SEQIDNO:28中的任意一种。符合上述条件的蛋白质具有草甘膦抗性。
实施例8.LS-EPSPS和LSE-EPSPS基因在大肠杆菌中的表达
LS-EPSPS基因克隆在载体PET-28a中并转入大肠杆菌BL21(DE3)获得转基因菌株。转基因菌株的阅读框在PET系统的T7启动子的控制下而获得表达。
转LS-EPSPS-PET菌株和转PET-28a菌株（空载体，对照）在LB中培养，当OD600达到0.6时，分别将转基因菌株和PET-28a在37℃含Kan的LB液体培养基中生长至OD值0.4左右，1%的接种量接入草甘膦浓度分别为0、1500、3000、4500、6000、7500、9000、10500以及12000mg/L的含Kan的LB液体培养基中，同时加IPTG至终浓度为1mmol/L，12～16h后测OD值，从而得到转基因菌株和PET-28a在不同草甘膦浓度下的生长曲线并进行Logistic回归分析，实验结果发现在7500和9000mg/L浓度下，转LS-EPSPS-PET菌株的OD%（OD值占对照的百分比）值约为70%和60%，而转PET-28a菌株的OD%仅为对照的20%和1%，说明在7500和9000mg/L浓度下，转LS-EPSPS基因菌株对草甘膦的抗性大大高于空载菌株。根据Logistic方程，转LS-EPSPS基因菌株的ED50值也大大高于空载体PET-28a。这些说明LS-EPSPS基因具有抗/耐草甘膦能力。
用EcoRI和HindIII限制性酶分别切割pET-28a质粒载体和PCR突变产物LSE-EPSPS基因,得到具有粘性末端的基因片段(大小分别约为5.8kb和1.3kb),将酶切后的基因片段用T4连接酶连接,再进行感受态细胞BL21(DE3)转化,获得重组菌株，重组菌株的阅读框在PET系统的T7启动子的控制下而获得表达。分别用5m1含有50ug/mlKan的LB液体培养基培养转化子BL21(LSE-EPSPS)，BL21(LS-EPSPS)，以200rpm,37℃空气浴的条件至细胞浓度达lO8/ml,以1%的接种量，转接到新鲜的含有50ug/mlKan的LB液体培养基中，200rpm、37℃继续培养至OD600=0.75时，加入IPTG至1mmol/L以诱导重组蛋白的合成。在前述条件下，继续培养3小时。离心收取菌体，采用Laemmli所述的方法，先用SDS-PAGE样品缓冲液重悬菌体，SDS-PAGE凝胶电泳分离蛋白质。电泳完毕，用20%的考马氏亮蓝染色。
实施例9.EPSP合成酶粗提物的制备
分别用5m1含有50ug/mlKan的LB液体培养基培养转化BL21(LSE-EPSPS)，BL21(LS-EPSPS)，以200rpm,37℃空气浴的条件至细胞浓度达lO8/ml,以1%的接种量，转接到新鲜的含有50ug/mlKan的LB液体培养基中，200rpm、37℃继续培养至OD600=0.75时，加入IPTG至1mM以诱导重组蛋白的合成。在前述条件下，继续培养3小时。离心收取菌体。用缓冲液A(50mMTris-C1,0.1mMDTT,pH7.2)重悬菌体。细胞悬液置-70℃冻结，再在室温中重融。然后用Fluko公司的乳化机处理细胞悬液使其破壁。以12000rpm离心30分钟，弃去细胞碎片。上清液即为EPSP合成酶粗提物，可用于有关该酶的各项指标的测定。
实施例10.离体EPSP合酶活性测定
称取麦冬、土麦冬、阔叶土麦冬以及野生拟南芥幼苗0.5g，加150ml提取缓冲液A[100mmol/L的TrispH7.5、1mmol/L的EDTA、10%(V/V)的甘油、1mg/L的BSA、10mmol/L的维生素C(Vc)、1mmol/L的苯脒(benzamidine)、5mmol/L的DTT、20mg/mlPVPP]，研磨匀浆，用6层纱布过滤，12000rpm、4℃下离心20min，上清液为EPSP合酶的粗酶液。40μl酶促反应体系(50mmol/L的HEPESpH7.5、1mmol/L的(NH4)6MO7O24、1mmol/L的PEP、2mmol/L的S3P、1mg/L的BSA，草甘膦浓度设置为0、100、200、500、800、1000以及2000umol/L)于25℃下预热5min，加入10μl酶液，25℃下酶促反应15min，迅速放入沸水中停止酶促反应，冷却至室温，再参照Lanzetta和Alvarez(1979)的方法加800μl孔雀石绿盐酸盐显色反应30min，后再加100μl34%的柠檬酸钠溶液，30min后于660nm的分光光度计上测定OD660值。结果发现，野生型拟南芥EPSP合酶活性在500umol/L时出现明显的下降，麦冬EPSP合酶活性在800umol/L时出现明显的下降，土麦冬和阔叶土麦冬EPSP合酶活性在1000umol/L时才出现明显的下降。进行Logistic回归分析并计算ED50值，拟南芥、麦冬、土麦冬和阔叶土麦冬EPSP合酶的ED50值分别为560、860、1090以及1070umol/L，由此可见，麦冬、土麦冬和阔叶土麦冬EPSP合酶的活性明显高于野生型拟南芥。
实施例11.真核表达载体的构建
一般地含有35S启动子的植物表达载体比较适合外源基因向双子叶植物的转移，启动效率很强，容易在转基因后代中转录和表达，但35S启动子不适合在单子叶植物中发挥作用；单子叶转基因植物主要使用actin、emu、ubquitinpromoter三种启动子，它们来源于单子叶植物，比较适合外源基因向单子叶植物的转移。终止子较常用的是胭脂碱合成酶基因终止子NOS，标记基因可以是新霉素磷酸转移酶(nptⅡ)、氯霉素乙酰转移酶基因（cat）、荧光素酶基因（luc）、绿色荧光蛋白基因(gfp)或者β-葡萄糖苷酸酶基因（gus）等等。以植物表达载体pBI121-LSEPSPS的构建为例：利用DNA重组技术，将LSEPSPS基因克隆至植物表达载体pBI121中，通过酶切、电泳鉴定LSEPSPS基因已成功构建到植物表达载体pBI121中；然后将重组质粒pBI121-LSEPSPS通过冻融法导入根癌农杆菌EHA105中，Kan平板筛选阳性克隆，生长的单菌落进行PCR鉴定，证明重组质粒已转入根癌农杆菌EHA105中。对于不同的植物可选择不同的表达载体，也可对其进行重组和改造，例如替换或增加启动子、终止子或标记基因。
实施例12.转基因拟南芥的抗性鉴定
用花序浸染法转化野生型拟南芥，将LS-EPSPS基因和人工合成LSE-EPSPS相似序列以及由于低保真PCR扩增得到的变异的LSE-EPSPS基因构建好PBI121-LSEPSPS的植物表达载体并用冻融法转入农杆菌EHA105，制备好已转化了相应质粒的农杆菌菌液10ml，在转化前1d，转入大瓶培养过夜，第2d取出使用时菌液的OD600应当在1.2到1.6之间。室温下5000r/m离心15min，弃上清液，将农杆菌沉淀悬浮于相应体积的渗透培养基中，使OD600在0.8左右。将上述农杆菌悬浮液注人喷雾器中，对准植物的地上部分进行喷洒，直到植株上有水珠滴下时为止。用保鲜膜将这些植物(T0代植物)罩起来以保持湿度，置入培养室中培养，2到3d后可去掉保鲜膜，转化后lw左右方可浇水。继续培养至植物成熟，收种子(Tl代)放在干燥环境中lw左右，进行转化子的筛选。将成功转化的植株进行抗性鉴定，其具备草甘膦抗性，可以作为转基因筛选标记。
将筛选获得转基因拟南芥植株，以非转基因和转LS-EPSP基因的拟南芥组培苗的茎作为外植体在进行愈伤组织的诱导，培养基为6-BA2.0mg/L和NAA0.1mg/L的MS培养基。在愈伤生长2～3周后，进行草甘膦处理。将通过Kan抗性筛选的转基因抗性愈伤组织和野生愈伤组织分别接种到草甘膦的浓度为0mg/L、50mg/L、100mg/L、200mg/L、400mg/L、600mg/L、800mg/L和1000mg/LMS培养基中，然后于25℃，16h/8h进行光、暗交替培养，两周后统计结果。对照拟南芥愈伤组织在草甘膦浓度为50mg/L时，已变黄并逐步坏死。而转基因拟南芥愈伤组织在600mg/L时仍可以正常生长，当浓度达800mg/L时生长才受到抑制，褐化变黄。
实施例13.转变基因抗/耐草甘膦油菜的获得
目前常用的转基因方法主要有农杆菌介导转化法、激光微束穿刺法、PEG法、电击法、微注射法和花粉介导法。以花粉介导法为例介绍转基因抗/耐草甘膦油菜的获得。
进入油菜盛花期后，选取主茎或一次分枝上10～15个1～2d内将要开放的花蕾徒手去雄，并摘除所选茎或分枝上其他花蕾，然后套袋。同时在同一品种中选取第2天将开放的花序进行套袋,以备第2天取粉用。翌日上午当天开放的套袋植株的花粉约0.4g，悬浮在25ml7.5%的蔗糖溶液中，进行第1次超声波处理，然后在溶液中加入20μg含EPSPS基因的质粒DNA进行第2次超声波处理。第2次处理后，在溶液中加入10μL1/10000(W/V)硼酸，然后用处理过的花粉授在前一天去雄的油菜柱头上，套袋并挂牌，同时标注授粉花蕾数。授粉后5～6d去袋，使处理的授粉株充分发育。采收种子。
实施例14.转基因抗/耐草甘膦玉米的获得
在温室对受体材料以7d为周期分期播种，玉米试材按试验需要进行控制授粉，授粉后10～13d，取玉米幼胚作为转化受体。用次氯酸钠消毒玉米棒后，剥出幼胚(剥幼胚时要谨慎，不要创伤胚)，用侵染液(加AS)清洗幼胚4遍，然后加入一定浓度的农杆菌菌液，放置10～30min，取出后用灭菌滤纸吸干，放到共培养培养基上，在25℃(黑暗)共培养2～5d，然后将幼胚转移到静息培养基，在28℃条件下暗培养7d。侵染和共培养环节菌液浓度OD500=0．3～0．5，侵染时间10min)、共培养时间3d。从静息培养基上把幼胚移入含有草甘膦的抗性筛选培养基上，暗培养。先在含10mg/L除草剂低选择压下培养14d，再经过80mg/L除草剂高压选择，第3次筛选浓度加大至160mg/L。每次继代注意淘汰呈褐色和水渍化的愈伤组织，并把生长正常的愈伤组织用镊子夹碎，分开选择培养。在继代筛选过程中注意经常观察发现污染或农杆菌复出的立即移出，未污染材料继续培养。另外，经常把出现褐化污染的组织块剔出或转移好的未被污染的组织块到新的相同培养基上。组织块膨大后，经常把大块组织剥小，连续继代筛选3次后，将选择到的抗性愈伤组织转到再生Ⅰ培养基上，恢复培养15d或更长时间(暗培养)。再将抗性愈伤组织转到再生Ⅱ培养基上发芽，培养条件为28℃，每日3000lx光强下，光照12h。待再生的玉米植株长到3片叶时，可将幼苗分移植到含生根培养基的瓶中，在室内培养。当幼苗长出较粗的根后，将幼苗从罐头瓶中取出，用水冲净培养基，移栽于混有营养土和蛭石(1∶3)的小花盆中，当玉米又长出2～3片新叶时，可将其移入大田或大花盆中，待长出三四叶后提取叶片DNA进行PCR检测。确定含有转入的LSEPSPS基因。
实施例15.转基因抗/耐草甘膦棉花的获得
大量提取植物表达载体质粒DNA，选择次日将开放的花蕾进行自交。由于棉花是常异交植物，天然异交率在10%左右，因而外来花粉往往会造成品种间的混杂。在开花前一天，可见花冠快速伸长，黄色或乳白色的花冠呈指状突出于花蕾，次日即开放成为花朵。选择这样的花蕾，于指状花冠的前端用细线扎紧，并将细线的另一端系于铃柄，作为收获时的标记；在开花后20～24h左右即次日，选择果枝和花位较好的幼子房作为转化对象。一般选择每个果枝的第一和第二个果节位的花朵进行转基因操作。这些果节上的棉铃一般成铃率较高，有利于收获较多的种子；用50田微量进样器作为微注射的工具。每次使用前和使用后，应以淡洗涤剂清洗，再用蒸馏水漂清；注射时，摘除或剥去花瓣，抹平花柱。在剥除花瓣时，注意不能损伤幼子房的表皮层，以免增加脱落率；用右手持微量注射器，左手轻扶摘除花瓣后的幼子房，从抹平花柱处沿子房的纵轴方向进针至子房长度的约三分之二处，并后退至约三分之一处。
花粉管通道法转化得到的棉花种子(T0代)收获后于在温室盆栽幼苗，在2～3叶期，以草甘膦胺盐水剂喷洒棉花叶片(草甘膦施用量1000克/公顷)。一周后观察植株生长状况，拔除叶片表面有退绿斑点的植株。为获得高抗草甘膦的转基因棉株可连续进行3次筛选，时间间隔为10～14天。生长正常的被认为是获得抗性基因的植株。共获得3株。
实施例16.转基因抗/耐草甘膦烟草的获得
农杆菌活化；共培养：将侵染过的叶块摆放在铺有2层滤纸的烟草芽分化培养基(MS+IAA0.5mg/L+6-BA2mg/L)上，25oC暗培养4天；抗性芽的筛选：将经过共培养的烟草外植体转移到抗性芽筛选培养基(MS+IAA0.5mg/L+6-BA2mg/L+Kan100mg/L+Carb500mg/L)上，2～3周后即可生芽。生根：待抗性芽长到1cm左右时，将其转移到生根培养基(MS+Kan100mg/L+Carb500mg/L)上，1～2周后即有不定根形成。将烟草无菌苗移温室栽，长有6～8片真叶时，分别涂摸1‰、2‰、3‰、4‰、5‰、6‰浓度的草甘膦除草剂(商品名为农达Roundup)。继续培养两周观察烟草的草甘膦抗性，敏感的植株叶片出现黄花，而抗性植株生长正常，共获得了5株。
实施例17.转基因抗/耐草甘膦黄瓜的获得
菌株培养：从平板上挑取含构建好的植物表达载体的根癌农杆菌单菌落接种于5mlLB+50mg/L卡那霉素（Kan）的培养液中，于28℃，200r/min条件下培养16h后，转接到新鲜的90mlLB+50mg/L卡那霉素的培养液中，在同样的条件下继续培养至对数生长期时(4～5h，OD≌0.8～1.2)，用液体MS洗涤3次后，重悬于MS液体培养基中，使其OD值为0.2～0.3即可用于转化。愈伤组织诱导和植株再生：将黄瓜种子浸入75%的乙醇中处理30s，再用次氯酸钙溶液处理15min，无菌水冲洗5次，播种在发芽培养基上，25℃黑暗萌发。取萌发2～3天的黄瓜子叶，切成5mm方块，与处于对数生长期经MS培养基适当稀释的农杆菌（含有LSEPSPS基因）共浸染一定时间，将浸染后的外植体置于再生培养基上，24～25℃暗培养2天，再转至含羧苄青霉素和一定浓度草甘膦的诱芽培养基，24～25℃暗培养2～3周，进行愈伤组织分化。由子叶产生的愈伤组织获得的抗性芽，转至1/2MS培养基(含羧苄青霉素和一定浓度草甘膦)获得转化植株。
实施例18.转基因抗/耐草甘膦月季的获得
月季成熟小叶外植体叶背朝下接种到添加了2，4-D0.5mg/L和pCAP3.0mg/L的愈伤组织诱导培养基上，光照条件下培养20d。将继代20～30d的新鲜愈伤组织置于农杆菌菌液（OD600=0.8）中侵染20min,，然后倒出愈伤组织，在无菌滤纸上适当晾干后接入含100uMAS、改良1/2MS盐类的共培养培养基中，25℃黑暗条件下共培养，用EHA105浸染的培养物共培养3～4d，以愈伤组织周围农杆菌长至肉眼可见菌落时为好。共培养结束后，愈伤组织用无菌水清洗1～2遍，在无菌滤纸上晾干后接入含70mg/L潮霉素和300mg/L头孢霉素的选择培养基，25℃黑暗条件下选择培养，2～3周继代一次，进行抗性愈伤的选择和增殖。对选择3个月的抗性愈伤组织进行GUS反应检测，一团抗性愈伤组织上的不同部分呈现深蓝色，浅蓝色或无蓝色反应，表现为明显的嵌合体。PCR检测，抗性愈伤组织中能扩增出目的片段。对抗性愈伤组织进行扩繁，然后进行再生植株的诱导和检测。
实施例19.转基因抗/耐草甘膦狗牙根的获得
狗牙根茎节的分生能力强，选用含茎节长约0.5～1.0cm的茎段为外植体。用自来水洗净表面，70%酒精浸泡30s，无菌水冲洗3次，0.1%氯化汞消毒10min，无菌水冲洗3次。取灭菌过的滤纸，吸干表面水分，接种于诱导培养基。愈伤组织的诱导以含2，4-D2mg/L+NAA3mg/L+6-BA0.2mg/L的MS培养基。继代培养2周，然后转移至含6-BA3mg/L+KT4.0mg/LMS分化培养基。置于28℃、16h光照、光10～30uEm-2s-1的人工气候室分化,每2周以相同培养基继代1次。将分化的小植株接于含NAA0.3mg/L的MS生根培养基，经2周生根培养后移至不含激素的1/2MS培养基壮苗，尔后移栽。取OD600≈0.5的农杆菌菌液，25℃浸泡10min，用灭过菌的滤纸吸去多余菌液，放入MS诱导培养基，暗条件共培养2d，然后用含Carb500mg/L的无菌水清洗愈伤。50mg/L潮霉素用作筛选物质，经3轮筛选，再生的植株苗长约15cm时转移至温室炼苗2周，成活后进行GUS组织化学染色和PCR鉴定，获得了阳性植株，移栽在网室自然生长。
实施例20.抗LSEPSPS单克隆抗体的制备
将含有LSEPSPS基因的重组单克隆表达菌落置于LB培养液（含有Kan50mg/L）中,37℃,300rpm培养,至细菌生长达到对数生长晚期后，加入终浓度为0.025mmol/L的IPTG，22℃低温诱导表达8h，离心10000rpm，10min，收集菌体，加入蛋白上样缓冲液，煮沸5min，进行12%SDS－PAGE鉴定，使之在大肠杆菌BL21（DE3）中获得高效表达。将制备的菌体经过超声破碎之后，以4℃，12000rpm离心20分钟，收集上清,进行亲合层析纯化。采用纯化的重组EPSPS抗原免疫Balb/c小鼠，取小鼠脾脏B淋巴细胞小鼠骨髓瘤细胞SP2/O在聚乙二醇（PEG）作用下进行融合，通过ELISA筛选稳定分泌抗LSEPSPS的单克隆杂交瘤细胞株。将杂交瘤细胞株注入小鼠腹腔，生的腹水中含有高效价的单克隆抗体。用饱和硫酸铵法纯化腹水IgG。
实施例21.变异来源和蛋白表达来源LSEPSPS分子的草甘膦抗性鉴定
LSEPSPS可以用来人工引入单个或多个变异而获得保持有抗草甘膦能力的LSEPSPS变异分子。例如利用低保真PCR的方法可以产生很多LSEPSPS变异分子，而这些变异分子可以通过草甘膦筛选获得继续保持有抗草甘膦能力的LSEPSPS变异分子。密码子具有简并性：除了甲硫氨酸和色氨酸外，每一个氨基酸都至少有两个密码子。这样可以在一定程度内，使氨基酸序列不会因为某一个碱基被意外替换而导致氨基酸错误。LSEPSPS基因翻译得到的氨基酸序列推导其编码的碱基序列，会得到多条与LSEPSPS相似的序列，此序列亦具有草甘膦抗性。将人工合成LSEPSPS相似序列以及由于低保真PCR扩增得到的变异的LSEPSPS基因构建好PBI121-LSEPSPS的植物表达载体并用冻融法转入农杆菌EHA105，制备好已转化了相应质粒的农杆菌菌液10ml，在转化前1d，转入大瓶培养过夜，第2d取出使用时菌液的OD600应当在1.2到1.6之间。室温下5000r/m离心15min，弃上清液，将农杆菌沉淀悬浮于相应体积的渗透培养基中，使OD600在0.8左右。将上述农杆菌悬浮液注人喷雾器中，对准植物的地上部分进行喷洒，直到植株上有水珠滴下时为止。用保鲜膜将这些植物(T0代植物)罩起来以保持湿度，置入培养室中培养，2到3d后可去掉保鲜膜，转化后lw左右方可浇水。继续培养至植物成熟，收种子(Tl代)放在干燥环境中lw左右，进行转化子的筛选。将成功转化的植株进行抗性鉴定，其具备草甘膦抗性，可以作为转基因筛选标记。
显然，本发明不限于以上实施例，还可以有许多变形。本领域的普通技术人员能从本发明公开的内容直接导出或联想到的所有变形，均应认为是本发明的保护范围。
<110>南京农业大学
<120>一种具有草甘膦耐性的基因及其应用
<160>52
<170>PatentInVersion3.3
<210>1
<211>1551
<212>DNA
<213>麦冬种（Ophiopogonjaponicus）
<220>
<400>SEQIDNO:1
atggagcaagcgatcatggctcagggcgtcgccaccaacctcggcctctctacttcctcc60
cttcggaatccgaagctcatcgcggcctcttctccttcttcttcgattcggatcggatcg120
gggctgaaactagggttttcggttggcttgaagggggaggttgggaggaggagggcggtt180
agggtttcggcgtcggtggcggcggcggagaagccgtcgacggtgccggagatcgtgctg240
cagccgatcaaggagatctccgggacgatcaagctcccgggatccaagtcgttgtccaat300
cggattctgcttcttgccgcgctcgctgagggaactactgttgtggacaatttgttggac360
agtgatgacattagctatatgcttgctgcacttaaaacacttgggctctctgtagaagac420
gatagtgtcatgaaacgtgcaactgttgtgggatctggaggtcaatttcctgttgggaaa480
gattccaaagaggtccaactttttctaggaaatgcagggactgcaatgcgtcctctgaca540
gctgctgttattgctgctggtggaaatgcaagttacatacttgatggggtaccacgaatg600
agggagagacctataggggacttggttgttggtttgaagcagttgggtgcagatgttgat660
tgcattttgggaactgactgtccacctgttcgtgccaatgcaaatggaggtcttccaggg720
ggaaaggttaaactctctggatcaattagcagtcagtatttgactgcgttgcttatggct780
gcgcccttagctcttggagatgtggagattgagatcatggataaacttatttcagtccca840
tatgttgaaatgacgcttaaactgatggaacgttttggagtcagtgtggagcattccagt900
agctgggataggttcttcatcaagggcggccaaaagtacaagtcccctggaaatgcttat960
gtggaaggtgatgcttcgagcgctagttattttctcgcaggcgcagcagtcaccggcggc1020
acggtaactgttgagggttgtggcacaagcagtttgcagggtgatgtaaaattcgctgaa1080
gttcttgagaaaatgggggcaaaggttacatggacagagaatagcgtcacagtaacaggc1140
ccgccgcaagatccttccaagaagaagcgattgcaagccattgatgtcaatatgaataaa1200
atgcctgatgttgccatgactctagctgttgttgctctttatgctgatggtccgactgcc1260
attagagacgttgcttcatggcgagtaaaggagactgaacgaatgattgctatctgtaca1320
gagcttagaaagctgggagcaacggtggaagaaggacccgattactgcgtgatcaccccg1380
cccgagaagttgaacgtggcagcaatcgacacctacgacgatcacaggatggccatggcg1440
ttctctcttgcagcctgtgcggatgcacccgtcacgatcagagaccccggctgcacccgc1500
aaaactttcccggactattttgaggtattgcagaggtttgcgaagcactaa1551
<210>2
<211>516
<212>PRT
<213>麦冬种（Ophiopogonjaponicus）
<220>
<221>ACT_SITE
<222>(144,184,232,273)
<223>Met,Ile,Ala,Met
<220>
<221>SIGNAL
<222>(1)...(63)
<220>
<221>CHAIN
<222>(64)...(516)
<400>SEQIDNO:2
MetGluGlnAlaIleMetAlaGlnGlyValAlaThrAsnLeuGlyLeu
151015
SerThrSerSerLeuArgAsnProLysLeuIleAlaAlaSerSerPro
202530
SerSerSerIleArgIleGlySerGlyLeuLysLeuGlyPheSerVal
354045
GlyLeuLysGlyGluValGlyArgArgArgAlaValArgValSerAla
505560
SerValAlaAlaAlaGluLysProSerThrValProGluIleValLeu
65707580
GlnProIleLysGluIleSerGlyThrIleLysLeuProGlySerLys
859095
SerLeuSerAsnArgIleLeuLeuLeuAlaAlaLeuAlaGluGlyThr
100105110
ThrValValAspAsnLeuLeuAspSerAspAspIleSerTyrMetLeu
115120125
AlaAlaLeuLysThrLeuGlyLeuSerValGluAspAspSerValMet
130135140
LysArgAlaThrValValGlySerGlyGlyGlnPheProValGlyLys
145150155160
AspSerLysGluValGlnLeuPheLeuGlyAsnAlaGlyThrAlaMet
165170175
ArgProLeuThrAlaAlaValIleAlaAlaGlyGlyAsnAlaSerTyr
180185190
IleLeuAspGlyValProArgMetArgGluArgProIleGlyAspLeu
195200205
ValValGlyLeuLysGlnLeuGlyAlaAspValAspCysIleLeuGly
210215220
ThrAspCysProProValArgAlaAsnAlaAsnGlyGlyLeuProGly
225230235240
GlyLysValLysLeuSerGlySerIleSerSerGlnTyrLeuThrAla
245250255
LeuLeuMetAlaAlaProLeuAlaLeuGlyAspValGluIleGluIle
260265270
MetAspLysLeuIleSerValProTyrValGluMetThrLeuLysLeu
275280285
MetGluArgPheGlyValSerValGluHisSerSerSerTrpAspArg
290295300
PhePheIleLysGlyGlyGlnLysTyrLysSerProGlyAsnAlaTyr
305310315320
ValGluGlyAspAlaSerSerAlaSerTyrPheLeuAlaGlyAlaAla
325330335
ValThrGlyGlyThrValThrValGluGlyCysGlyThrSerSerLeu
340345350
GlnGlyAspValLysPheAlaGluValLeuGluLysMetGlyAlaLys
355360365
ValThrTrpThrGluAsnSerValThrValThrGlyProProGlnAsp
370375380
ProSerLysLysLysArgLeuGlnAlaIleAspValAsnMetAsnLys
385390395400
MetProAspValAlaMetThrLeuAlaValValAlaLeuTyrAlaAsp
405410415
GlyProThrAlaIleArgAspValAlaSerTrpArgValLysGluThr
420425430
GluArgMetIleAlaIleCysThrGluLeuArgLysLeuGlyAlaThr
435440445
ValGluGluGlyProAspTyrCysValIleThrProProGluLysLeu
450455460
AsnValAlaAlaIleAspThrTyrAspAspHisArgMetAlaMetAla
465470475480
PheSerLeuAlaAlaCysAlaAspAlaProValThrIleArgAspPro
485490495
GlyCysThrArgLysThrPheProAspTyrPheGluValLeuGlnArg
500505510
PheAlaLysHis
515
<210>3
<211>1557
<212>DNA
<213>土麦冬种（LiriopesSpicatae）
<220>
<400>SEQIDNO:3
atggagcaagcgatcatggctaagggcgtcgccaccaacctcggcctctccacttcctcc60
cttgggaatccgaagctcatcgtggcttcttcttctcctcctccttcttcgattcggatc120
ggatcggggctgaaactagggttttcggttggtttgaaggggggagttgggatgaggagg180
gcggttacggtttcggcgtcggtggcggcggcggagaaaccgtcgacggtgccggagatc240
gtgctgcagccgatcaaggagatctcggggacgatcaagctcccgggatccaagtcgttg300
tccaatcggattctgcttcttgccgcgctcgctgagggaactactgttgtggacaattta360
ttggacagtgatgacattcgctatatgcttgctgcacttaaaacgcttgggctcactgta420
gaagacgatagtgtcatgaaacgtgcaactgttgtgggatctggtggtcaatttcctgtt480
gggaaagattccaaagaggttcaactttttctaggaaatgcagggactgcaatgcgtcct540
ctgacagctgctgttattgctgctggtggaaatgcaagttacatacttgatggggtacca600
cgaatgagggagaggcctataggggacttggttgttggcttgaagcagttaggtgcagat660
gttgattgcattttgggaactgactgtcctcctgttcgtgccaatgcacatggaggtctt720
ccaggaggaaaggtgaaactctctggatcaattagcagtcagtatttgactgcgttgctt780
atggcagcgcccttagctcttggagacgtggagattgagatcatggataaacttatttca840
gtcccatatgttgaaatgacgcttaaactgatggaacgttttggggttagtgtggagcat900
tccagtagctgggataggttcttcatcaagggtggtcaaaagtacaagtcccctggaaat960
gcttatgtggaaggtgatgcttcgagcgctagttattttctcgcaggtgcagcggtcact1020
ggcggcacggtaactgttgagggttgtggcacgagcagtttgcagggagatgtaaaattc1080
gctgaagttcttgagaaaatgggggcaaaggttacatggacagagaatagcgtcacagta1140
acaggcccaccgcaagatccttccaagaagaagcgattgcgagccgttgatgtcaatatg1200
aataaaatgcctgatgttgccatgactctagctgttgttgctctttatgctgatggtccg1260
actgccattagagacgttgcttcttggcgagtaaaggagactgaacgaatgattgctatt1320
tgtacagaacttagaaagctgggagcaacagtggaggaaggacccgattactgcgtgatc1380
accccaccggagaagctgaacgcgaatgcaatcgacacctacgacgatcacaggatggct1440
atggcgttctctctcgcagcctgtgcggatgtacccgtcacgatcagagaccccggttgc1500
acccgcaaaactttccccgactattttgaggtattgcagaggttcacaacgcactaa1557
<210>4
<211>518
<212>PRT
<213>土麦冬种（LiriopesSpicatae）
<220>
<221>ACT_SITE
<222>(146,186,234,275)
<223>Met,Ile,Ala,Met
<220>
<221>SIGNAL
<222>(1)...(65)
<220>
<221>CHAIN
<222>(66)...(518)
<400>SEQIDNO:4
MetGluGlnAlaIleMetAlaLysGlyValAlaThrAsnLeuGlyLeu
151015
SerThrSerSerLeuGlyAsnProLysLeuIleValAlaSerSerSer
202530
ProProProSerSerIleArgIleGlySerGlyLeuLysLeuGlyPhe
354045
SerValGlyLeuLysGlyGlyValGlyMetArgArgAlaValThrVal
505560
SerAlaSerValAlaAlaAlaGluLysProSerThrValProGluIle
65707580
ValLeuGlnProIleLysGluIleSerGlyThrIleLysLeuProGly
859095
SerLysSerLeuSerAsnArgIleLeuLeuLeuAlaAlaLeuAlaGlu
100105110
GlyThrThrValValAspAsnLeuLeuAspSerAspAspIleArgTyr
115120125
MetLeuAlaAlaLeuLysThrLeuGlyLeuThrValGluAspAspSer
130135140
ValMetLysArgAlaThrValValGlySerGlyGlyGlnPheProVal
145150155160
GlyLysAspSerLysGluValGlnLeuPheLeuGlyAsnAlaGlyThr
165170175
AlaMetArgProLeuThrAlaAlaValIleAlaAlaGlyGlyAsnAla
180185190
SerTyrIleLeuAspGlyValProArgMetArgGluArgProIleGly
195200205
AspLeuValValGlyLeuLysGlnLeuGlyAlaAspValAspCysIle
210215220
LeuGlyThrAspCysProProValArgAlaAsnAlaHisGlyGlyLeu
225230235240
ProGlyGlyLysValLysLeuSerGlySerIleSerSerGlnTyrLeu
245250255
ThrAlaLeuLeuMetAlaAlaProLeuAlaLeuGlyAspValGluIle
260265270
GluIleMetAspLysLeuIleSerValProTyrValGluMetThrLeu
275280285
LysLeuMetGluArgPheGlyValSerValGluHisSerSerSerTrp
290295300
AspArgPhePheIleLysGlyGlyGlnLysTyrLysSerProGlyAsn
305310315320
AlaTyrValGluGlyAspAlaSerSerAlaSerTyrPheLeuAlaGly
325330335
AlaAlaValThrGlyGlyThrValThrValGluGlyCysGlyThrSer
340345350
SerLeuGlnGlyAspValLysPheAlaGluValLeuGluLysMetGly
355360365
AlaLysValThrTrpThrGluAsnSerValThrValThrGlyProPro
370375380
GlnAspProSerLysLysLysArgLeuArgAlaValAspValAsnMet
385390395400
AsnLysMetProAspValAlaMetThrLeuAlaValValAlaLeuTyr
405410415
AlaAspGlyProThrAlaIleArgAspValAlaSerTrpArgValLys
420425430
GluThrGluArgMetIleAlaIleCysThrGluLeuArgLysLeuGly
435440445
AlaThrValGluGluGlyProAspTyrCysValIleThrProProGlu
450455460
LysLeuAsnAlaAsnAlaIleAspThrTyrAspAspHisArgMetAla
465470475480
MetAlaPheSerLeuAlaAlaCysAlaAspValProValThrIleArg
485490495
AspProGlyCysThrArgLysThrPheProAspTyrPheGluValLeu
500505510
GlnArgPheThrThrHis
515
<210>5
<211>1557
<212>DNA
<213>阔叶土麦冬种（Liriopeplatyphylla）
<220>
<400>SEQIDNO:5
atggagcaagcgatcatggctaagggggtcgccaccaacctcagcctctccacttcctcc60
cttgggaatccgaagctcatcgtggcctcttcttctcctccttcttcttcgattcggatc120
ggatcggggctgaaactagggttttcggttggtttgaaggggggagttgggatgaggagg180
gcggttacggtttcggcgtcggtggcggcggcggagaagccgtcgacggtgccggagatc240
gtgctgcagccgatcaaggagatctcggggacgatcaagctcccgggatccaagtcgttg300
tccaatcggattctgcttcttgccgcgctcgctgagggaactactgttgtggacaatttg360
ttggacagtgatgacattcgctatatgcttgctgcacttaaaacgcttgggctcgctgta420
gaagatgatagtgtcatgaaacgtgcaactgttgtgggatctggtggtcaatttcctgtt480
gggaaagattccaaagaggttcaactttttctaggaaatgcagggactgcaatgcgtcct540
ctgacagctgctgttattgctgctggtggaaatgcaagttacatacttgatggggtacca600
cgaatgagggagaggcctataggggacttggttgttggcttgaagcagttaggtgcagat660
gttgattgcattttgggatctgactgtcctcctgttcgtgccaatgcacatggaggtctt720
ccaggaggaaaggtgaaactctctggatcaattagcagtcagtatttgactgcgttgctt780
atggcagcgcccttagctcttggagatgtggagattgagatcatggataaacttatttca840
gtcccatatgttgaaatgacgcttaaactgatggaacgttttggtgttagtgtggagcat900
tccagtagctgggataggttcttcatcaagggtggtcaaaagtacaagtcccctggaaat960
gcttatgtggaaggtgatgcttcgagcgctagttattttctcgcaggtgcagcggtcact1020
ggcggcacggtaactgttgagggttgtggcacgagcagtttgcagggagatgtaaaattc1080
gctgaagttcttgagaaaatgggggcaaaggttacatggacagagaatagcgtcacagta1140
acaggcccaccgcaagatccttccaagaagaagcgattgcgagccattgatgtcaatatg1200
aataaaatgcctgatgttgccatgactctagctgttgttgctctttatgctgatggtccg1260
actgccattagagacgttgcttcttggcgagtaaaggagaccgaacgaatgattgctatt1320
tgtacagaacttagaaagctgggagcaacagtggaggaaggacccgattactgcgtgatc1380
accccaccggagaagctgaacgcgactgcaatcgacacctacgacgatcacaggatggct1440
atggcgttctctctcgcagcctgtggggatgtacccgtcacgatcagagaccccggttgc1500
acccgcaaaactttcccggactattttgaggtattgcagaggttcacaacgcactaa1557
<210>6
<211>518
<212>PRT
<213>阔叶土麦冬种（Liriopeplatyphylla）
<220>
<221>ACT_SITE
<222>(146,186,234,275)
<223>Met,Ile,Ala,Met
<220>
<221>SIGNAL
<222>(1)...(65)
<220>
<221>CHAIN
<222>(66)...(518)
<400>SEQIDNO:6
MetGluGlnAlaIleMetAlaLysGlyValAlaThrAsnLeuSerLeu
151015
SerThrSerSerLeuGlyAsnProLysLeuIleValAlaSerSerSer
202530
ProProSerSerSerIleArgIleGlySerGlyLeuLysLeuGlyPhe
354045
SerValGlyLeuLysGlyGlyValGlyMetArgArgAlaValThrVal
505560
SerAlaSerValAlaAlaAlaGluLysProSerThrValProGluIle
65707580
ValLeuGlnProIleLysGluIleSerGlyThrIleLysLeuProGly
859095
SerLysSerLeuSerAsnArgIleLeuLeuLeuAlaAlaLeuAlaGlu
100105110
GlyThrThrValValAspAsnLeuLeuAspSerAspAspIleArgTyr
115120125
MetLeuAlaAlaLeuLysThrLeuGlyLeuAlaValGluAspAspSer
130135140
ValMetLysArgAlaThrValValGlySerGlyGlyGlnPheProVal
145150155160
GlyLysAspSerLysGluValGlnLeuPheLeuGlyAsnAlaGlyThr
165170175
AlaMetArgProLeuThrAlaAlaValIleAlaAlaGlyGlyAsnAla
180185190
SerTyrIleLeuAspGlyValProArgMetArgGluArgProIleGly
195200205
AspLeuValValGlyLeuLysGlnLeuGlyAlaAspValAspCysIle
210215220
LeuGlySerAspCysProProValArgAlaAsnAlaHisGlyGlyLeu
225230235240
ProGlyGlyLysValLysLeuSerGlySerIleSerSerGlnTyrLeu
245250255
ThrAlaLeuLeuMetAlaAlaProLeuAlaLeuGlyAspValGluIle
260265270
GluIleMetAspLysLeuIleSerValProTyrValGluMetThrLeu
275280285
LysLeuMetGluArgPheGlyValSerValGluHisSerSerSerTrp
290295300
AspArgPhePheIleLysGlyGlyGlnLysTyrLysSerProGlyAsn
305310315320
AlaTyrValGluGlyAspAlaSerSerAlaSerTyrPheLeuAlaGly
325330335
AlaAlaValThrGlyGlyThrValThrValGluGlyCysGlyThrSer
340345350
SerLeuGlnGlyAspValLysPheAlaGluValLeuGluLysMetGly
355360365
AlaLysValThrTrpThrGluAsnSerValThrValThrGlyProPro
370375380
GlnAspProSerLysLysLysArgLeuArgAlaIleAspValAsnMet
385390395400
AsnLysMetProAspValAlaMetThrLeuAlaValValAlaLeuTyr
405410415
AlaAspGlyProThrAlaIleArgAspValAlaSerTrpArgValLys
420425430
GluThrGluArgMetIleAlaIleCysThrGluLeuArgLysLeuGly
435440445
AlaThrValGluGluGlyProAspTyrCysValIleThrProProGlu
450455460
LysLeuAsnAlaThrAlaIleAspThrTyrAspAspHisArgMetAla
465470475480
MetAlaPheSerLeuAlaAlaCysGlyAspValProValThrIleArg
485490495
AspProGlyCysThrArgLysThrPheProAspTyrPheGluValLeu
500505510
GlnArgPheThrThrHis
515
<210>7
<211>443
<212>PRT
<213>土麦冬种（LiriopesSpicatae）
<400>SEQIDNO:7
MetValProGluIleValLeuGlnProIleLysGluIleSerGlyThr
151015
IleLysLeuProGlySerLysSerLeuSerAsnArgIleLeuLeuLeu
202530
AlaAlaLeuAlaGluGlyThrThrValValAspAsnLeuLeuAspSer
354045
AspAspIleArgTyrMetLeuAlaAlaLeuLysThrLeuGlyLeuThr
505560
ValGluAspAspSerValMetLysArgAlaThrValValGlySerGly
65707580
GlyGlnPheProValGlyLysAspSerLysGluValGlnLeuPheLeu
859095
GlyAsnAlaGlyThrAlaMetArgProLeuThrAlaAlaValIleAla
100105110
AlaGlyGlyAsnAlaSerTyrIleLeuAspGlyValProArgMetArg
115120125
GluArgProIleGlyAspLeuValValGlyLeuLysGlnLeuGlyAla
130135140
AspValAspCysIleLeuGlyThrAspCysProProValArgAlaAsn
145150155160
AlaHisGlyGlyLeuProGlyGlyLysValLysLeuSerGlySerIle
165170175
SerSerGlnTyrLeuThrAlaLeuLeuMetAlaAlaProLeuAlaLeu
180185190
GlyAspValGluIleGluIleMetAspLysLeuIleSerValProTyr
195200205
ValGluMetThrLeuLysLeuMetGluArgPheGlyValSerValGlu
210215220
HisSerSerSerTrpAspArgPhePheIleLysGlyGlyGlnLysTyr
225230235240
LysSerProGlyAsnAlaTyrValGluGlyAspAlaSerSerAlaSer
245250255
TyrPheLeuAlaGlyAlaAlaValThrGlyGlyThrValThrValGlu
260265270
GlyCysGlyThrSerSerLeuGlnGlyAspValLysPheAlaGluVal
275280285
LeuGluLysMetGlyAlaLysValThrTrpThrGluAsnSerValThr
290295300
ValThrGlyProProGlnAspProSerLysLysLysArgLeuArgAla
305310315320
ValAspValAsnMetAsnLysMetProAspValAlaMetThrLeuAla
325330335
ValValAlaLeuTyrAlaAspGlyProThrAlaIleArgAspValAla
340345350
SerTrpArgValLysGluThrGluArgMetIleAlaIleCysThrGlu
355360365
LeuArgLysLeuGlyAlaThrValGluGluGlyProAspTyrCysVal
370375380
IleThrProProGluLysLeuAsnAlaAsnAlaIleAspThrTyrAsp
385390395400
AspHisArgMetAlaMetAlaPheSerLeuAlaAlaCysAlaAspVal
405410415
ProValThrIleArgAspProGlyCysThrArgLysThrPheProAsp
420425430
TyrPheGluValLeuGlnArgPheThrThrHis
435440445
<210>8
<211>15
<212>PRT
<213>人工序列
<400>SEQIDNO:8
GlyGlyAspValArgPheAlaGluValLeuGluLysMetGlyAla
151015
<210>9
<211>15
<212>PRT
<213>人工序列
<400>SEQIDNO:9
GlyGlyAspValLysTyrAlaGluValLeuGluLysMetGlyAla
151015
<210>10
<211>15
<212>PRT
<213>人工序列
<400>SEQIDNO:10
GlyGlyAspValLysPheAlaGluValHisGluLysMetGlyAla
151015
<210>11
<211>15
<212>PRT
<213>人工序列
<400>SEQIDNO:11
GlyGlyAspValArgTyrAlaGluValLeuGluLysMetGlyAla
151015
<210>12
<211>15
<212>PRT
<213>人工序列
<400>SEQIDNO:12
GlyGlyAspValArgPheAlaGluValHisGluLysMetGlyAla
151015
<210>13
<211>15
<212>PRT
<213>人工序列
<400>SEQIDNO:13
GlyGlyAspValLysTyrAlaGluValHisGluLysMetGlyAla
151015
<210>14
<211>15
<212>PRT
<213>人工序列
<400>SEQIDNO:14
GlyGlyAspValArgTyrAlaGluValHisGluLysMetGlyAla
151015
<210>15
<211>18
<212>PRT
<213>人工序列
<400>SEQIDNO:15
GluProValThrIleArgAspProGlyCysThrArgLysThrPhePro
151015
AspTyr
202530
<210>16
<211>18
<212>PRT
<213>人工序列
<400>SEQIDNO:16
ValProValThrIleGlyAspProGlyCysThrArgLysThrPhePro
151015
AspTyr
202530
<210>17
<211>18
<212>PRT
<213>人工序列
<400>SEQIDNO:17
ValProValThrIleArgValProGlyCysThrArgLysThrPhePro
151015
AspTyr
202530
<210>18
<211>18
<212>PRT
<213>人工序列
<400>SEQIDNO:18
ValProValThrIleArgAspProCysCysThrArgLysThrPhePro
151015
AspTyr
202530
<210>19
<211>18
<212>PRT
<213>人工序列
<400>SEQIDNO:19
ValProValThrIleArgAspProGlyTyrThrArgLysThrPhePro
151015
AspTyr
202530
<210>20
<211>18
<212>PRT
<213>人工序列
<400>SEQIDNO:20
GluProValThrIleGlyAspProGlyCysThrArgLysThrPhePro
151015
AspTyr
202530
<210>21
<211>18
<212>PRT
<213>人工序列
<400>SEQIDNO:21
GluProValThrIleArgValProGlyCysThrArgLysThrPhePro
151015
AspTyr
202530
<210>22
<211>18
<212>PRT
<213>人工序列
<400>SEQIDNO:22
ValProValThrIleGlyValProGlyCysThrArgLysThrPhePro
151015
AspTyr
202530
<210>23
<211>18
<212>PRT
<213>人工序列
<400>SEQIDNO:23
ValProValThrIleGlyAspProCysCysThrArgLysThrPhePro
151015
AspTyr
202530
<210>24
<211>18
<212>PRT
<213>人工序列
<400>SEQIDNO:24
ValProValThrIleGlyAspProGlyTyrThrArgLysThrPhePro
151015
AspTyr
202530
<210>25
<211>19
<212>PRT
<213>人工序列
<400>SEQIDNO:25
ValProValThrIleArgValProGlyTyrThrArgLysThrPhePro
151015
AspTyr
202530
<210>26
<211>18
<212>PRT
<213>人工序列
<400>SEQIDNO:26
GluProValThrIleGlyValProGlyCysThrArgLysThrPhePro
151015
AspTyr
202530
<210>27
<211>18
<212>PRT
<213>人工序列
<400>SEQIDNO:27
GluProValThrIleArgAspProCysTyrThrArgLysThrPhePro
151015
AspTyr
202530
<210>28
<211>18
<212>PRT
<213>人工序列
<400>SEQIDNO:28
ValProValThrIleGlyValProCysCysThrArgLysThrPhePro
151015
AspTyr
202530
<210>29
<211>443
<212>PRT
<213>土麦冬种（LiriopesSpicatae）
<220>
<221>ACT_SITE
<222>(37，67，276，350，421)
<223>Val，Asn，Ser，Gly，Gly
<400>SEQIDNO:29
MetValProGluIleValLeuGlnProIleLysGluIleSerGlyThr
151015
IleLysLeuProGlySerLysSerLeuSerAsnArgIleLeuLeuLeu
202530
AlaAlaLeuAlaValGlyThrThrValValAspAsnLeuLeuAspSer
354045
AspAspIleArgTyrMetLeuAlaAlaLeuLysThrLeuGlyLeuThr
505560
ValGluAsnAspSerValMetLysArgAlaThrValValGlySerGly
65707580
GlyGlnPheProValGlyLysAspSerLysGluValGlnLeuPheLeu
859095
GlyAsnAlaGlyThrAlaMetArgProLeuThrAlaAlaValIleAla
100105110
AlaGlyGlyAsnAlaSerTyrIleLeuAspGlyValProArgMetArg
115120125
GluArgProIleGlyAspLeuValValGlyLeuLysGlnLeuGlyAla
130135140
AspValAspCysIleLeuGlyThrAspCysProProValArgAlaAsn
145150155160
AlaHisGlyGlyLeuProGlyGlyLysValLysLeuSerGlySerIle
165170175
SerSerGlnTyrLeuThrAlaLeuLeuMetAlaAlaProLeuAlaLeu
180185190
GlyAspValGluIleGluIleMetAspLysLeuIleSerValProTyr
195200205
ValGluMetThrLeuLysLeuMetGluArgPheGlyValSerValGlu
210215220
HisSerSerSerTrpAspArgPhePheIleLysGlyGlyGlnLysTyr
225230235240
LysSerProGlyAsnAlaTyrValGluGlyAspAlaSerSerAlaSer
245250255
TyrPheLeuAlaGlyAlaAlaValThrGlyGlyThrValThrValGlu
260265270
GlyCysGlySerSerSerLeuGlnGlyAspValLysPheAlaGluVal
275280285
LeuGluLysMetGlyAlaLysValThrTrpThrGluAsnSerValThr
290295300
ValThrGlyProProGlnAspProSerLysLysLysArgLeuArgAla
305310315320
ValAspValAsnMetAsnLysMetProAspValAlaMetThrLeuAla
325330335
ValValAlaLeuTyrAlaAspGlyProThrAlaIleArgGlyValAla
340345350
SerTrpArgValLysGluThrGluArgMetIleAlaIleCysThrGlu
355360365
LeuArgLysLeuGlyAlaThrValGluGluGlyProAspTyrCysVal
370375380
IleThrProProGluLysLeuAsnAlaAsnAlaIleAspThrTyrAsp
385390395400
AspHisArgMetAlaMetAlaPheSerLeuAlaAlaCysAlaAspVal
405410415
ProValThrIleGlyAspProGlyCysThrArgLysThrPheProAsp
420425430
TyrPheGluValLeuGlnArgPheThrThrHis
435440445
<210>30
<211>443
<212>PRT
<213>土麦冬种（LiriopesSpicatae）
<220>
<221>ACT_SITE
<222>(10，202，271，308，397)
<223>Thr，Gln，Ala，Ser，Val
<400>SEQIDNO:30
MetValProGluIleValLeuGlnProThrLysGluIleSerGlyThr
151015
IleLysLeuProGlySerLysSerLeuSerAsnArgIleLeuLeuLeu
202530
AlaAlaLeuAlaGluGlyThrThrValValAspAsnLeuLeuAspSer
354045
AspAspIleArgTyrMetLeuAlaAlaLeuLysThrLeuGlyLeuThr
505560
ValGluAspAspSerValMetLysArgAlaThrValValGlySerGly
65707580
GlyGlnPheProValGlyLysAspSerLysGluValGlnLeuPheLeu
859095
GlyAsnAlaGlyThrAlaMetArgProLeuThrAlaAlaValIleAla
100105110
AlaGlyGlyAsnAlaSerTyrIleLeuAspGlyValProArgMetArg
115120125
GluArgProIleGlyAspLeuValValGlyLeuLysGlnLeuGlyAla
130135140
AspValAspCysIleLeuGlyThrAspCysProProValArgAlaAsn
145150155160
AlaHisGlyGlyLeuProGlyGlyLysValLysLeuSerGlySerIle
165170175
SerSerGlnTyrLeuThrAlaLeuLeuMetAlaAlaProLeuAlaLeu
180185190
GlyAspValGluIleGluIleMetAspGlnLeuIleSerValProTyr
195200205
ValGluMetThrLeuLysLeuMetGluArgPheGlyValSerValGlu
210215220
HisSerSerSerTrpAspArgPhePheIleLysGlyGlyGlnLysTyr
225230235240
LysSerProGlyAsnAlaTyrValGluGlyAspAlaSerSerAlaSer
245250255
TyrPheLeuAlaGlyAlaAlaValThrGlyGlyThrValThrAlaGlu
260265270
GlyCysGlyThrSerSerLeuGlnGlyAspValLysPheAlaGluVal
275280285
LeuGluLysMetGlyAlaLysValThrTrpThrGluAsnSerValThr
290295300
ValThrGlySerProGlnAspProSerLysLysLysArgLeuArgAla
305310315320
ValAspValAsnMetAsnLysMetProAspValAlaMetThrLeuAla
325330335
ValValAlaLeuTyrAlaAspGlyProThrAlaIleArgAspValAla
340345350
SerTrpArgValLysGluThrGluArgMetIleAlaIleCysThrGlu
355360365
LeuArgLysLeuGlyAlaThrValGluGluGlyProAspTyrCysVal
370375380
IleThrProProGluLysLeuAsnAlaAsnAlaIleValThrTyrAsp
385390395400
AspHisArgMetAlaMetAlaPheSerLeuAlaAlaCysAlaAspVal
405410415
ProValThrIleGlyAspProGlyCysThrArgLysThrPheProAsp
420425430
TyrPheGluValLeuGlnArgPheThrThrHis
435440445
<210>31
<211>443
<212>PRT
<213>土麦冬种（LiriopesSpicatae）
<220>
<221>ACT_SITE
<222>(27，82，90，93，198，347，366，421，425)
<223>Tyr，Leu，Asn，His，Val，Gly，Arg，Gly，Tyr
<400>SEQIDNO:31
MetValProGluIleValLeuGlnProIleLysGluIleSerGlyThr
151015
IleLysLeuProGlySerLysSerLeuSerTyrArgIleLeuLeuLeu
202530
AlaAlaLeuAlaGluGlyThrThrValValAspAsnLeuLeuAspSer
354045
AspAspIleArgTyrMetLeuAlaAlaLeuLysThrLeuGlyLeuThr
505560
ValGluAspAspSerValMetLysArgAlaThrValValGlySerGly
65707580
GlyLeuPheProValGlyLysAspSerAsnGluValHisLeuPheLeu
859095
GlyAsnAlaGlyThrAlaMetArgProLeuThrAlaAlaValIleAla
100105110
AlaGlyGlyAsnAlaSerTyrIleLeuAspGlyValProArgMetArg
115120125
GluArgProIleGlyAspLeuValValGlyLeuLysGlnLeuGlyAla
130135140
AspValAspCysIleLeuGlyThrAspCysProProValArgAlaAsn
145150155160
AlaHisGlyGlyLeuProGlyGlyLysValLysLeuSerGlySerIle
165170175
SerSerGlnTyrLeuThrAlaLeuLeuMetAlaAlaProLeuAlaLeu
180185190
GlyAspValGluIleValIleMetAspLysLeuIleSerValProTyr
195200205
ValGluMetThrLeuLysLeuMetGluArgPheGlyValSerValGlu
210215220
HisSerSerSerTrpAspArgPhePheIleLysGlyGlyGlnLysTyr
225230235240
LysSerProGlyAsnAlaTyrValGluGlyAspAlaSerSerAlaSer
245250255
TyrPheLeuAlaGlyAlaAlaValThrGlyGlyThrValThrValGlu
260265270
GlyCysGlyThrSerSerLeuGlnGlyAspValLysPheAlaGluVal
275280285
LeuGluLysMetGlyAlaLysValThrTrpThrGluAsnSerValThr
290295300
ValThrGlyProProGlnAspProSerLysLysLysArgLeuArgAla
305310315320
ValAspValAsnMetAsnLysMetProAspValAlaMetThrLeuAla
325330335
ValValAlaLeuTyrAlaAspGlyProThrGlyIleArgAspValAla
340345350
SerTrpArgValLysGluThrGluArgMetIleAlaIleArgThrGlu
355360365
LeuArgLysLeuGlyAlaThrValGluGluGlyProAspTyrCysVal
370375380
IleThrProProGluLysLeuAsnAlaAsnAlaIleAspThrTyrAsp
385390395400
AspHisArgMetAlaMetAlaPheSerLeuAlaAlaCysAlaAspVal
405410415
ProValThrIleGlyAspProGlyTyrThrArgLysThrPheProAsp
420425430
TyrPheGluValLeuGlnArgPheThrThrHis
435440445
<210>32
<211>443
<212>PRT
<213>土麦冬种（LiriopesSpicatae）
<220>
<221>ACT_SITE
<222>(69，208，254，284，299，421)
<223>Arg，Phe，Gly，Arg，Ile，Gly
<400>SEQIDNO:32
MetGluProGluIleValLeuGlnProIleLysGluIleSerGlyThr
151015
IleLysLeuProGlySerLysSerLeuSerAsnArgIleLeuLeuLeu
202530
AlaAlaLeuAlaGluGlyThrThrValValAspAsnLeuLeuAspSer
354045
AspAspIleArgTyrMetLeuAlaAlaLeuLysThrLeuGlyLeuThr
505560
ValGluAspAspArgValMetLysArgAlaThrValValGlySerGly
65707580
GlyGlnPheProValGlyLysAspSerLysGluValGlnLeuPheLeu
859095
GlyAsnAlaGlyThrAlaMetArgProLeuThrAlaAlaValIleAla
100105110
AlaGlyGlyAsnAlaSerTyrIleLeuAspGlyValProArgMetArg
115120125
GluArgProIleGlyAspLeuValValGlyLeuLysGlnLeuGlyAla
130135140
AspValAspCysIleLeuGlyThrAspCysProProValArgAlaAsn
145150155160
AlaHisGlyGlyLeuProGlyGlyLysValLysLeuSerGlySerIle
165170175
SerSerGlnTyrLeuThrAlaLeuLeuMetAlaAlaProLeuAlaLeu
180185190
GlyAspValGluIleGluIleMetAspLysLeuIleSerValProPhe
195200205
ValGluMetThrLeuLysLeuMetGluArgPheGlyValSerValGlu
210215220
HisSerSerSerTrpAspArgPhePheIleLysGlyGlyGlnLysTyr
225230235240
LysSerProGlyAsnAlaTyrValGluGlyAspAlaSerGlyAlaSer
245250255
TyrPheLeuAlaGlyAlaAlaValThrGlyGlyThrValThrValGlu
260265270
GlyCysGlyThrSerSerLeuGlnGlyAspValArgPheAlaGluVal
275280285
LeuGluLysMetGlyAlaLysValThrTrpIleGluAsnSerValThr
290295300
ValThrGlyProProGlnAspProSerLysLysLysArgLeuArgAla
305310315320
ValAspValAsnMetAsnLysMetProAspValAlaMetThrLeuAla
325330335
ValValAlaLeuTyrAlaAspGlyProThrAlaIleArgAspValAla
340345350
SerTrpArgValLysGluThrGluArgMetIleAlaIleCysThrGlu
355360365
LeuArgLysLeuGlyAlaThrValGluGluGlyProAspTyrCysVal
370375380
IleThrProProGluLysLeuAsnAlaAsnAlaIleAspThrTyrAsp
385390395400
AspHisArgMetAlaMetAlaPheSerLeuAlaAlaCysAlaAspVal
405410415
ProValThrIleGlyAspProGlyCysThrArgLysThrPheProAsp
420425430
TyrPheGluValLeuGlnArgPheThrThrHis
435440445
<210>33
<211>443
<212>PRT
<213>土麦冬种（LiriopesSpicatae）
<220>
<221>ACT_SITE
<222>(70，100，284，421)
<223>Ala，Trp，Arg，Gly
<400>SEQIDNO:33
MetValProGluIleValLeuGlnProIleLysGluIleSerGlyThr
151015
IleLysLeuProGlySerLysSerLeuSerAsnArgIleLeuLeuLeu
202530
AlaAlaLeuAlaGluGlyThrThrValValAspAsnLeuLeuAspSer
354045
AspAspIleArgTyrMetLeuAlaAlaLeuLysThrLeuGlyLeuThr
505560
ValGluAspAspSerAlaMetLysArgAlaThrValValGlySerGly
65707580
GlyGlnPheProValGlyLysAspSerLysGluValGlnLeuPheLeu
859095
GlyAsnAlaTrpThrAlaMetArgProLeuThrAlaAlaValIleAla
100105110
AlaGlyGlyAsnAlaSerTyrIleLeuAspGlyValProArgMetArg
115120125
GluArgProIleGlyAspLeuValValGlyLeuLysGlnLeuGlyAla
130135140
AspValAspCysIleLeuGlyThrAspCysProProValArgAlaAsn
145150155160
AlaHisGlyGlyLeuProGlyGlyLysValLysLeuSerGlySerIle
165170175
SerSerGlnTyrLeuThrAlaLeuLeuMetAlaAlaProLeuAlaLeu
180185190
GlyAspValGluIleGluIleMetAspLysLeuIleSerValProTyr
195200205
ValGluMetThrLeuLysLeuMetGluArgPheGlyValSerValGlu
210215220
HisSerSerSerTrpAspArgPhePheIleLysGlyGlyGlnLysTyr
225230235240
LysSerProGlyAsnAlaTyrValGluGlyAspAlaSerSerAlaSer
245250255
TyrPheLeuAlaGlyAlaAlaValThrGlyGlyThrValThrValGlu
260265270
GlyCysGlyThrSerSerLeuGlnGlyAspValArgPheAlaGluVal
275280285
LeuGluLysMetGlyAlaLysValThrTrpThrGluAsnSerValThr
290295300
ValThrGlyProProGlnAspProSerLysLysLysArgLeuArgAla
305310315320
ValAspValAsnMetAsnLysMetProAspValAlaMetThrLeuAla
325330335
ValValAlaLeuTyrAlaAspGlyProThrAlaIleArgAspValAla
340345350
SerTrpArgValLysGluThrGluArgMetIleAlaIleCysThrGlu
355360365
LeuArgLysLeuGlyAlaThrValGluGluGlyProAspTyrCysVal
370375380
IleThrProProGluLysLeuAsnAlaAsnAlaIleAspThrTyrAsp
385390395400
AspHisArgMetAlaMetAlaPheSerLeuAlaAlaCysAlaAspVal
405410415
ProValThrIleGlyAspProGlyCysThrArgLysThrPheProAsp
420425430
TyrPheGluValLeuGlnArgPheThrThrHis
435440445
<210>34
<211>443
<212>PRT
<213>土麦冬种（LiriopesSpicatae）
<220>
<221>ACT_SITE
<222>(69,284，421)
<223>Arg，Arg，Gly
<400>SEQIDNO:34
MetValProGluIleValLeuGlnProIleLysGluIleSerGlyThr
151015
IleLysLeuProGlySerLysSerLeuSerTyrArgIleLeuLeuLeu
202530
AlaAlaLeuAlaGluGlyThrThrValValAspAsnLeuLeuAspSer
354045
AspAspIleArgTyrMetLeuAlaAlaLeuLysThrLeuGlyLeuThr
505560
ValGluAspAspArgValMetLysArgAlaThrValValGlySerGly
65707580
GlyLeuPheProValGlyLysAspSerAsnGluValHisLeuPheLeu
859095
GlyAsnAlaGlyThrAlaMetArgProLeuThrAlaAlaValIleAla
100105110
AlaGlyGlyAsnAlaSerTyrIleLeuAspGlyValProArgMetArg
115120125
GluArgProIleGlyAspLeuValValGlyLeuLysGlnLeuGlyAla
130135140
AspValAspCysIleLeuGlyThrAspCysProProValArgAlaAsn
145150155160
AlaHisGlyGlyLeuProGlyGlyLysValLysLeuSerGlySerIle
165170175
SerSerGlnTyrLeuThrAlaLeuLeuMetAlaAlaProLeuAlaLeu
180185190
GlyAspValGluIleValIleMetAspLysLeuIleSerValProTyr
195200205
ValGluMetThrLeuLysLeuMetGluArgPheGlyValSerValGlu
210215220
HisSerSerSerTrpAspArgPhePheIleLysGlyGlyGlnLysTyr
225230235240
LysSerProGlyAsnAlaTyrValGluGlyAspAlaSerSerAlaSer
245250255
TyrPheLeuAlaGlyAlaAlaValThrGlyGlyThrValThrValGlu
260265270
GlyCysGlyThrSerSerLeuGlnGlyAspValArgPheAlaGluVal
275280285
LeuGluLysMetGlyAlaLysValThrTrpThrGluAsnSerValThr
290295300
ValThrGlyProProGlnAspProSerLysLysLysArgLeuArgAla
305310315320
ValAspValAsnMetAsnLysMetProAspValAlaMetThrLeuAla
325330335
ValValAlaLeuTyrAlaAspGlyProThrGlyIleArgAspValAla
340345350
SerTrpArgValLysGluThrGluArgMetIleAlaIleArgThrGlu
355360365
LeuArgLysLeuGlyAlaThrValGluGluGlyProAspTyrCysVal
370375380
IleThrProProGluLysLeuAsnAlaAsnAlaIleAspThrTyrAsp
385390395400
AspHisArgMetAlaMetAlaPheSerLeuAlaAlaCysAlaAspVal
405410415
ProValThrIleGlyAspProGlyTyrThrArgLysThrPheProAsp
420425430
TyrPheGluValLeuGlnArgPheThrThrHis
435440445
<210>35
<211>443
<212>PRT
<213>土麦冬种（LiriopesSpicatae）
<220>
<221>ACT_SITE
<222>(284，421)
<223>Arg，Gly
<400>SEQIDNO:35
MetValProGluIleValLeuGlnProIleLysGluIleSerGlyThr
151015
IleLysLeuProGlySerLysSerLeuSerAsnArgIleLeuLeuLeu
202530
AlaAlaLeuAlaGluGlyThrThrValValAspAsnLeuLeuAspSer
354045
AspAspIleArgTyrMetLeuAlaAlaLeuLysThrLeuGlyLeuThr
505560
ValGluAspAspSerValMetLysArgAlaThrValValGlySerGly
65707580
GlyGlnPheProValGlyLysAspSerLysGluValGlnLeuPheLeu
859095
GlyAsnAlaGlyThrAlaMetArgProLeuThrAlaAlaValIleAla
100105110
AlaGlyGlyAsnAlaSerTyrIleLeuAspGlyValProArgMetArg
115120125
GluArgProIleGlyAspLeuValValGlyLeuLysGlnLeuGlyAla
130135140
AspValAspCysIleLeuGlyThrAspCysProProValArgAlaAsn
145150155160
AlaHisGlyGlyLeuProGlyGlyLysValLysLeuSerGlySerIle
165170175
SerSerGlnTyrLeuThrAlaLeuLeuMetAlaAlaProLeuAlaLeu
180185190
GlyAspValGluIleGluIleMetAspLysLeuIleSerValProTyr
195200205
ValGluMetThrLeuLysLeuMetGluArgPheGlyValSerValGlu
210215220
HisSerSerSerTrpAspArgPhePheIleLysGlyGlyGlnLysTyr
225230235240
LysSerProGlyAsnAlaTyrValGluGlyAspAlaSerSerAlaSer
245250255
TyrPheLeuAlaGlyAlaAlaValThrGlyGlyThrValThrValGlu
260265270
GlyCysGlyThrSerSerLeuGlnGlyAspValArgPheAlaGluVal
275280285
LeuGluLysMetGlyAlaLysValThrTrpThrGluAsnSerValThr
290295300
ValThrGlyProProGlnAspProSerLysLysLysArgLeuArgAla
305310315320
ValAspValAsnMetAsnLysMetProAspValAlaMetThrLeuAla
325330335
ValValAlaLeuTyrAlaAspGlyProThrAlaIleArgAspValAla
340345350
SerTrpArgValLysGluThrGluArgMetIleAlaIleCysThrGlu
355360365
LeuArgLysLeuGlyAlaThrValGluGluGlyProAspTyrCysVal
370375380
IleThrProProGluLysLeuAsnAlaAsnAlaIleAspThrTyrAsp
385390395400
AspHisArgMetAlaMetAlaPheSerLeuAlaAlaCysAlaAspVal
405410415
ProValThrIleGlyAspProGlyCysThrArgLysThrPheProAsp
420425430
TyrPheGluValLeuGlnArgPheThrThrHis
435440445
<210>36
<211>443
<212>PRT
<213>土麦冬种（LiriopesSpicatae）
<220>
<221>ACT_SITE
<222>(69，211，247，284，348，421)
<223>Arg，Val，Cys，Arg，Val，Gly
<400>SEQIDNO:36
MetValProGluIleValLeuGlnProIleLysGluIleSerGlyThr
151015
IleLysLeuProGlySerLysSerLeuSerAsnArgIleLeuLeuLeu
202530
AlaAlaLeuAlaGluGlyThrThrValValAspAsnLeuLeuAspSer
354045
AspAspIleArgTyrMetLeuAlaAlaLeuLysThrLeuGlyLeuThr
505560
ValGluAspAspArgValMetLysArgAlaThrValValGlySerGly
65707580
GlyGlnPheProValGlyLysAspSerLysGluValGlnLeuPheLeu
859095
GlyAsnAlaGlyThrAlaMetArgProLeuThrAlaAlaValIleAla
100105110
AlaGlyGlyAsnAlaSerTyrIleLeuAspGlyValProArgMetArg
115120125
GluArgProIleGlyAspLeuValValGlyLeuLysGlnLeuGlyAla
130135140
AspValAspCysIleLeuGlyThrAspCysProProValArgAlaAsn
145150155160
AlaHisGlyGlyLeuProGlyGlyLysValLysLeuSerGlySerIle
165170175
SerSerGlnTyrLeuThrAlaLeuLeuMetAlaAlaProLeuAlaLeu
180185190
GlyAspValGluIleGluIleMetAspLysLeuIleSerValProTyr
195200205
ValGluValThrLeuLysLeuMetGluArgPheGlyValSerValGlu
210215220
HisSerSerSerTrpAspArgPhePheIleLysGlyGlyGlnLysTyr
225230235240
LysSerProGlyAsnAlaCysValGluGlyAspAlaSerSerAlaSer
245250255
TyrPheLeuAlaGlyAlaAlaValThrGlyGlyThrValThrValGlu
260265270
GlyCysGlyThrSerSerLeuGlnGlyAspValArgPheAlaGluVal
275280285
LeuGluLysMetGlyAlaLysValThrTrpThrGluAsnSerValThr
290295300
ValThrGlyProProGlnAspProSerLysLysLysArgLeuArgAla
305310315320
ValAspValAsnMetAsnLysMetProAspValAlaMetThrLeuAla
325330335
ValValAlaLeuTyrAlaAspGlyProThrAlaValArgAspValAla
340345350
SerTrpArgValLysGluThrGluArgMetIleAlaIleCysThrGlu
355360365
LeuArgLysLeuGlyAlaThrValGluGluGlyProAspTyrCysVal
370375380
IleThrProProGluLysLeuAsnAlaAsnAlaIleAspThrTyrAsp
385390395400
AspHisArgMetAlaMetAlaPheSerLeuAlaAlaCysAlaAspVal
405410415
ProValThrIleGlyAspProGlyCysThrArgLysThrPheProAsp
420425430
TyrPheGluValLeuGlnArgPheThrThrHis
435440445
<210>37
<211>443
<212>PRT
<213>土麦冬种（LiriopesSpicatae）
<220>
<221>ACT_SITE
<222>(48，137，169，235，248，421)
<223>Cys，Ile，Glu，Met，Arg，Gly
<400>SEQIDNO:37
MetValProGluIleValLeuGlnProIleLysGluIleSerGlyThr
151015
IleLysLeuProGlySerLysSerLeuSerAsnArgIleLeuLeuLeu
202530
AlaAlaLeuAlaGluGlyThrThrValValAspAsnLeuLeuAspCys
354045
AspAspIleArgTyrMetLeuAlaAlaLeuLysThrLeuGlyLeuThr
505560
ValGluAspAspSerValMetLysArgAlaThrValValGlySerGly
65707580
GlyGlnPheProValGlyLysAspSerLysGluValGlnLeuPheLeu
859095
GlyAsnAlaGlyThrAlaMetArgProLeuThrAlaAlaValIleAla
100105110
AlaGlyGlyAsnAlaSerTyrIleLeuAspGlyValProArgMetArg
115120125
GluArgProIleGlyAspLeuValIleGlyLeuLysGlnLeuGlyAla
130135140
AspValAspCysIleLeuGlyThrAspCysProProValArgAlaAsn
145150155160
AlaHisGlyGlyLeuProGlyGlyGluValLysLeuSerGlySerIle
165170175
SerSerGlnTyrLeuThrAlaLeuLeuMetAlaAlaProLeuAlaLeu
180185190
GlyAspValGluIleGluIleMetAspLysLeuIleSerValProTyr
195200205
ValGluMetThrLeuLysLeuMetGluArgPheGlyValSerValGlu
210215220
HisSerSerSerTrpAspArgPhePheIleMetGlyGlyGlnLysTyr
225230235240
LysSerProGlyAsnAlaTyrValGluGlyAspAlaSerSerAlaSer
245250255
TyrPheLeuAlaGlyAlaAlaValThrGlyGlyThrValThrValGlu
260265270
GlyCysGlyThrSerSerLeuGlnGlyAspValArgPheAlaGluVal
275280285
LeuGluLysMetGlyAlaLysValThrTrpThrGluAsnSerValThr
290295300
ValThrGlyProProGlnAspProSerLysLysLysArgLeuArgAla
305310315320
ValAspValAsnMetAsnLysMetProAspValAlaMetThrLeuAla
325330335
ValValAlaLeuTyrAlaAspGlyProThrAlaIleArgAspValAla
340345350
SerTrpArgValLysGluThrGluArgMetIleAlaIleCysThrGlu
355360365
LeuArgLysLeuGlyAlaThrValGluGluGlyProAspTyrCysVal
370375380
IleThrProProGluLysLeuAsnAlaAsnAlaIleAspThrTyrAsp
385390395400
AspHisArgMetAlaMetAlaPheSerLeuAlaAlaCysAlaAspVal
405410415
ProValThrIleGlyAspProGlyCysThrArgLysThrPheProAsp
420425430
TyrPheGluValLeuGlnArgPheThrThrHis
435440445
<210>38
<211>443
<212>PRT
<213>土麦冬种（LiriopesSpicatae）
<220>
<221>ACT_SITE
<222>(120，284，332，340，389，421)
<223>Leu，Arg，Val，Pro，Val，Gly
<400>SEQIDNO:38
MetValProGluIleValLeuGlnProIleLysGluIleSerGlyThr
151015
IleLysLeuProGlySerLysSerLeuSerAsnArgIleLeuLeuLeu
202530
AlaAlaLeuAlaGluGlyThrThrValValAspAsnLeuLeuAspSer
354045
AspAspIleArgTyrMetLeuAlaAlaLeuLysThrLeuGlyLeuThr
505560
ValGluAspAspSerValMetLysArgAlaThrValValGlySerGly
65707580
GlyGlnPheProValGlyLysAspSerLysGluValGlnLeuPheLeu
859095
GlyAsnAlaGlyThrAlaMetArgProLeuThrAlaAlaValIleAla
100105110
AlaGlyGlyAsnAlaSerTyrLeuLeuAspGlyValProArgMetArg
115120125
GluArgProIleGlyAspLeuValValGlyLeuLysGlnLeuGlyAla
130135140
AspValAspCysIleLeuGlyThrAspCysProProValArgAlaAsn
145150155160
AlaHisGlyGlyLeuProGlyGlyLysValLysLeuSerGlySerIle
165170175
SerSerGlnTyrLeuThrAlaLeuLeuMetAlaAlaProLeuAlaLeu
180185190
GlyAspValGluIleGluIleMetAspLysLeuIleSerValProTyr
195200205
ValGluMetThrLeuLysLeuMetGluArgPheGlyValSerValGlu
210215220
HisSerSerSerTrpAspArgPhePheIleLysGlyGlyGlnLysTyr
225230235240
LysSerProGlyAsnAlaTyrValGluGlyAspAlaSerSerAlaSer
245250255
TyrPheLeuAlaGlyAlaAlaValThrGlyGlyThrValThrValGlu
260265270
GlyCysGlyThrSerSerLeuGlnGlyAspValArgPheAlaGluVal
275280285
LeuGluLysMetGlyAlaLysValThrTrpThrGluAsnSerValThr
290295300
ValThrGlyProProGlnAspProSerLysLysLysArgLeuArgAla
305310315320
ValAspValAsnMetAsnLysMetProAspValValMetThrLeuAla
325330335
ValValAlaProTyrAlaAspGlyProThrAlaIleArgAspValAla
340345350
SerTrpArgValLysGluThrGluArgMetIleAlaIleCysThrGlu
355360365
LeuArgLysLeuGlyAlaThrValGluGluGlyProAspTyrCysVal
370375380
IleThrProProValLysLeuAsnAlaAsnAlaIleAspThrTyrAsp
385390395400
AspHisArgMetAlaMetAlaPheSerLeuAlaAlaCysAlaAspVal
405410415
ProValThrIleGlyAspProGlyCysThrArgLysThrPheProAsp
420425430
TyrPheGluValLeuGlnArgPheThrThrHis
435440445
<210>39
<211>443
<212>PRT
<213>土麦冬种（LiriopesSpicatae）
<220>
<221>ACT_SITE
<222>(66，224，284，353，421)
<223>Gly，Gly，Arg，Thr，Gly
<400>SEQIDNO:39
MetValProGluIleValLeuGlnProIleLysGluIleSerGlyThr
151015
IleLysLeuProGlySerLysSerLeuSerAsnArgIleLeuLeuLeu
202530
AlaAlaLeuAlaGluGlyThrThrValValAspAsnLeuLeuAspSer
354045
AspAspIleArgTyrMetLeuAlaAlaLeuLysThrLeuGlyLeuThr
505560
ValGlyAspAspSerValMetLysArgAlaThrValValGlySerGly
65707580
GlyGlnPheProValGlyLysAspSerLysGluValGlnLeuPheLeu
859095
GlyAsnAlaGlyThrAlaMetArgProLeuThrAlaAlaValIleAla
100105110
AlaGlyGlyAsnAlaSerTyrIleLeuAspGlyValProArgMetArg
115120125
GluArgProIleGlyAspLeuValValGlyLeuLysGlnLeuGlyAla
130135140
AspValAspCysIleLeuGlyThrAspCysProProValArgAlaAsn
145150155160
AlaHisGlyGlyLeuProGlyGlyLysValLysLeuSerGlySerIle
165170175
SerSerGlnTyrLeuThrAlaLeuLeuMetAlaAlaProLeuAlaLeu
180185190
GlyAspValGluIleGluIleMetAspLysLeuIleSerValProTyr
195200205
ValGluMetThrLeuLysLeuMetGluArgPheGlyValSerValGly
210215220
HisSerSerSerTrpAspArgPhePheIleLysGlyGlyGlnLysTyr
225230235240
LysSerProGlyAsnAlaTyrValGluGlyAspAlaSerSerAlaSer
245250255
TyrPheLeuAlaGlyAlaAlaValThrGlyGlyThrValThrValGlu
260265270
GlyCysGlyThrSerSerLeuGlnGlyAspValArgPheAlaGluVal
275280285
LeuGluLysMetGlyAlaLysValThrTrpThrGluAsnSerValThr
290295300
ValThrGlyProProGlnAspProSerLysLysLysArgLeuArgAla
305310315320
ValAspValAsnMetAsnLysMetProAspValAlaMetThrLeuAla
325330335
ValValAlaLeuTyrAlaAspGlyProThrAlaIleArgAspValAla
340345350
ThrTrpArgValLysGluThrGluArgMetIleAlaIleCysThrGlu
355360365
LeuArgLysLeuGlyAlaThrValGluGluGlyProAspTyrCysVal
370375380
IleThrProProGluLysLeuAsnAlaAsnAlaIleAspThrTyrAsp
385390395400
AspHisArgMetAlaMetAlaPheSerLeuAlaAlaCysAlaAspVal
405410415
ProValThrIleGlyAspProGlyCysThrArgLysThrPheProAsp
420425430
TyrPheGluValLeuGlnArgPheThrThrHis
435440445
<210>40
<211>443
<212>PRT
<213>土麦冬种（LiriopesSpicatae）
<220>
<221>ACT_SITE
<222>(66，284，371，401，421)
<223>Val，Arg，Met，Gly，Gly
<400>SEQIDNO:40
MetValProGluIleValLeuGlnProIleLysGluIleSerGlyThr
151015
IleLysLeuProGlySerLysSerLeuSerAsnArgIleLeuLeuLeu
202530
AlaAlaLeuAlaGluGlyThrThrValValAspAsnLeuLeuAspSer
354045
AspAspIleArgTyrMetLeuAlaAlaLeuLysThrLeuGlyLeuThr
505560
ValValAspAspSerValMetLysArgAlaThrValValGlySerGly
65707580
GlyGlnPheProValGlyLysAspSerLysGluValGlnLeuPheLeu
859095
GlyAsnAlaGlyThrAlaMetArgProLeuThrAlaAlaValIleAla
100105110
AlaGlyGlyAsnAlaSerTyrIleLeuAspGlyValProArgMetArg
115120125
GluArgProIleGlyAspLeuValValGlyLeuLysGlnLeuGlyAla
130135140
AspValAspCysIleLeuGlyThrAspCysProProValArgAlaAsn
145150155160
AlaHisGlyGlyLeuProGlyGlyLysValLysLeuSerGlySerIle
165170175
SerSerGlnTyrLeuThrAlaLeuLeuMetAlaAlaProLeuAlaLeu
180185190
GlyAspValGluIleGluIleMetAspLysLeuIleSerValProTyr
195200205
ValGluMetThrLeuLysLeuMetGluArgPheGlyValSerValGlu
210215220
HisSerSerSerTrpAspArgPhePheIleLysGlyGlyGlnLysTyr
225230235240
LysSerProGlyAsnAlaTyrValGluGlyAspAlaSerSerAlaSer
245250255
TyrPheLeuAlaGlyAlaAlaValThrGlyGlyThrValThrValGlu
260265270
GlyCysGlyThrSerSerLeuGlnGlyAspValArgPheAlaGluVal
275280285
LeuGluLysMetGlyAlaLysValThrTrpThrGluAsnSerValThr
290295300
ValThrGlyProProGlnAspProSerLysLysLysArgLeuArgAla
305310315320
ValAspValAsnMetAsnLysMetProAspValAlaMetThrLeuAla
325330335
ValValAlaLeuTyrAlaAspGlyProThrAlaIleArgAspValAla
340345350
SerTrpArgValLysGluThrGluArgMetIleAlaIleCysThrGlu
355360365
LeuArgMetLeuGlyAlaThrValGluGluGlyProAspTyrCysVal
370375380
IleThrProProGluLysLeuAsnAlaAsnAlaIleAspThrTyrAsp
385390395400
GlyHisArgMetAlaMetAlaPheSerLeuAlaAlaCysAlaAspVal
405410415
ProValThrIleGlyAspProGlyCysThrArgLysThrPheProAsp
420425430
TyrPheGluValLeuGlnArgPheThrThrHis
435440445
<210>41
<211>443
<212>PRT
<213>土麦冬种（LiriopesSpicatae）
<220>
<221>ACT_SITE
<222>(169，265，272，284，301，310，421)
<223>Arg，Ala，Gly，Arg，Tyr，Arg，Gly
<400>SEQIDNO:41
MetValProGluIleValLeuGlnProIleLysGluIleSerGlyThr
151015
IleLysLeuProGlySerLysSerLeuSerAsnArgIleLeuLeuLeu
202530
AlaAlaLeuAlaGluGlyThrThrValValAspAsnLeuLeuAspSer
354045
AspAspIleArgTyrMetLeuAlaAlaLeuLysThrLeuGlyLeuThr
505560
ValGluAspAspSerValMetLysArgAlaThrValValGlySerGly
65707580
GlyGlnPheProValGlyLysAspSerLysGluValGlnLeuPheLeu
859095
GlyAsnAlaGlyThrAlaMetArgProLeuThrAlaAlaValIleAla
100105110
AlaGlyGlyAsnAlaSerTyrIleLeuAspGlyValProArgMetArg
115120125
GluArgProIleGlyAspLeuValValGlyLeuLysGlnLeuGlyAla
130135140
AspValAspCysIleLeuGlyThrAspCysProProValArgAlaAsn
145150155160
AlaHisGlyGlyLeuProGlyGlyArgValLysLeuSerGlySerIle
165170175
SerSerGlnTyrLeuThrAlaLeuLeuMetAlaAlaProLeuAlaLeu
180185190
GlyAspValGluIleGluIleMetAspLysLeuIleSerValProTyr
195200205
ValGluMetThrLeuLysLeuMetGluArgPheGlyValSerValGlu
210215220
HisSerSerSerTrpAspArgPhePheIleLysGlyGlyGlnLysTyr
225230235240
LysSerProGlyAsnAlaTyrValGluGlyAspAlaSerSerAlaSer
245250255
TyrPheLeuAlaGlyAlaAlaValAlaGlyGlyThrValThrValGly
260265270
GlyCysGlyThrSerSerLeuGlnGlyAspValArgPheAlaGluVal
275280285
LeuGluLysMetGlyAlaLysValThrTrpThrGluTyrSerValThr
290295300
ValThrGlyProProArgAspProSerLysLysLysArgLeuArgAla
305310315320
ValAspValAsnMetAsnLysMetProAspValAlaMetThrLeuAla
325330335
ValValAlaLeuTyrAlaAspGlyProThrAlaIleArgAspValAla
340345350
SerTrpArgValLysGluThrGluArgMetIleAlaIleCysThrGlu
355360365
LeuArgLysLeuGlyAlaThrValGluGluGlyProAspTyrCysVal
370375380
IleThrProProGluLysLeuAsnAlaAsnAlaIleAspThrTyrAsp
385390395400
AspHisArgMetAlaMetAlaPheSerLeuAlaAlaCysAlaAspVal
405410415
ProValThrIleGlyAspProGlyCysThrArgLysThrPheProAsp
420425430
TyrPheGluValLeuGlnArgPheThrThrHis
435440445
<210>42
<211>443
<212>PRT
<213>土麦冬种（LiriopesSpicatae）
<220>
<221>ACT_SITE
<222>(27，35，48，202，284，421，434)
<223>Tyr，His，Gly，Asn，Arg，Gly，Leu
<400>SEQIDNO:42
MetValProGluIleValLeuGlnProIleLysGluIleSerGlyThr
151015
IleLysLeuProGlySerLysSerLeuSerTyrArgIleLeuLeuLeu
202530
AlaAlaHisAlaGluGlyThrThrValValAspAsnLeuLeuAspGly
354045
AspAspIleArgTyrMetLeuAlaAlaLeuLysThrLeuGlyLeuThr
505560
ValGluAspAspSerValMetLysArgAlaThrValValGlySerGly
65707580
GlyGlnPheProValGlyLysAspSerLysGluValGlnLeuPheLeu
859095
GlyAsnAlaGlyThrAlaMetArgProLeuThrAlaAlaValIleAla
100105110
AlaGlyGlyAsnAlaSerTyrIleLeuAspGlyValProArgMetArg
115120125
GluArgProIleGlyAspLeuValValGlyLeuLysGlnLeuGlyAla
130135140
AspValAspCysIleLeuGlyThrAspCysProProValArgAlaAsn
145150155160
AlaHisGlyGlyLeuProGlyGlyLysValLysLeuSerGlySerIle
165170175
SerSerGlnTyrLeuThrAlaLeuLeuMetAlaAlaProLeuAlaLeu
180185190
GlyAspValGluIleGluIleMetAspAsnLeuIleSerValProTyr
195200205
ValGluMetThrLeuLysLeuMetGluArgPheGlyValSerValGlu
210215220
HisSerSerSerTrpAspArgPhePheIleLysGlyGlyGlnLysTyr
225230235240
LysSerProGlyAsnAlaTyrValGluGlyAspAlaSerSerAlaSer
245250255
TyrPheLeuAlaGlyAlaAlaValThrGlyGlyThrValThrValGlu
260265270
GlyCysGlyThrSerSerLeuGlnGlyAspValArgPheAlaGluVal
275280285
LeuGluLysMetGlyAlaLysValThrTrpThrGluAsnSerValThr
290295300
ValThrGlyProProGlnAspProSerLysLysLysArgLeuArgAla
305310315320
ValAspValAsnMetAsnLysMetProAspValAlaMetThrLeuAla
325330335
ValValAlaLeuTyrAlaAspGlyProThrAlaIleArgAspValAla
340345350
SerTrpArgValLysGluThrGluArgMetIleAlaIleCysThrGlu
355360365
LeuArgLysLeuGlyAlaThrValGluGluGlyProAspTyrCysVal
370375380
IleThrProProGluLysLeuAsnAlaAsnAlaIleAspThrTyrAsp
385390395400
AspHisArgMetAlaMetAlaPheSerLeuAlaAlaCysAlaAspVal
405410415
ProValThrIleGlyAspProGlyCysThrArgLysThrPheProAsp
420425430
TyrLeuGluValLeuGlnArgPheThrThrHis
435440445
<210>43
<211>443
<212>PRT
<213>土麦冬种（LiriopesSpicatae）
<220>
<221>ACT_SITE
<222>(44，169，251，284，308，353，394，421)
<223>Lys，Thr，Asn，Arg，Thr，Thr，Tyr，Gly
<400>SEQIDNO:43
MetValProGluIleValLeuGlnProIleLysGluIleSerGlyThr
151015
IleLysLeuProGlySerLysSerLeuSerAsnArgIleLeuLeuLeu
202530
AlaAlaLeuAlaGluGlyThrThrValValAspLysLeuLeuAspSer
354045
AspAspIleArgTyrMetLeuAlaAlaLeuLysThrLeuGlyLeuThr
505560
ValGluAspAspSerValMetLysArgAlaThrValValGlySerGly
65707580
GlyGlnPheProValGlyLysAspSerLysGluValGlnLeuPheLeu
859095
GlyAsnAlaGlyThrAlaMetArgProLeuThrAlaAlaValIleAla
100105110
AlaGlyGlyAsnAlaSerTyrIleLeuAspGlyValProArgMetArg
115120125
GluArgProIleGlyAspLeuValValGlyLeuLysGlnLeuGlyAla
130135140
AspValAspCysIleLeuGlyThrAspCysProProValArgAlaAsn
145150155160
AlaHisGlyGlyLeuProGlyGlyThrValLysLeuSerGlySerIle
165170175
SerSerGlnTyrLeuThrAlaLeuLeuMetAlaAlaProLeuAlaLeu
180185190
GlyAspValGluIleGluIleMetAspLysLeuIleSerValProTyr
195200205
ValGluMetThrLeuLysLeuMetGluArgPheGlyValSerValGlu
210215220
HisSerSerSerTrpAspArgPhePheIleLysGlyGlyGlnLysTyr
225230235240
LysSerProGlyAsnAlaTyrValGluGlyAsnAlaSerSerAlaSer
245250255
TyrPheLeuAlaGlyAlaAlaValThrGlyGlyThrValThrValGlu
260265270
GlyCysGlyThrSerSerLeuGlnGlyAspValArgPheAlaGluVal
275280285
LeuGluLysMetGlyAlaLysValThrTrpThrGluAsnSerValThr
290295300
ValThrGlyThrProGlnAspProSerLysLysLysArgLeuArgAla
305310315320
ValAspValAsnMetAsnLysMetProAspValAlaMetThrLeuAla
325330335
ValValAlaLeuTyrAlaAspGlyProThrAlaIleArgAspValAla
340345350
ThrTrpArgValLysGluThrGluArgMetIleAlaIleCysThrGlu
355360365
LeuArgLysLeuGlyAlaThrValGluGluGlyProAspTyrCysVal
370375380
IleThrProProGluLysLeuAsnAlaTyrAlaIleAspThrTyrAsp
385390395400
AspHisArgMetAlaMetAlaPheSerLeuAlaAlaCysAlaAspVal
405410415
ProValThrIleGlyAspProGlyCysThrArgLysThrPheProAsp
420425430
TyrPheGluValLeuGlnArgPheThrThrHis
435440445
<210>44
<211>443
<212>PRT
<213>土麦冬种（LiriopesSpicatae）
<220>
<221>ACT_SITE
<222>(284，285，289，325，378，398，421)
<223>Arg，Tyr，His，Leu，Asp，Ser，Gly
<400>SEQIDNO:44
MetValProGluIleValLeuGlnProIleLysGluIleSerGlyThr
151015
IleLysLeuProGlySerLysSerLeuSerAsnArgIleLeuLeuLeu
202530
AlaAlaLeuAlaGluGlyThrThrValValAspAsnLeuLeuAspSer
354045
AspAspIleArgTyrMetLeuAlaAlaLeuLysThrLeuGlyLeuThr
505560
ValGluAspAspSerValMetLysArgAlaThrValValGlySerGly
65707580
GlyGlnPheProValGlyLysAspSerLysGluValGlnLeuPheLeu
859095
GlyAsnAlaGlyThrAlaMetArgProLeuThrAlaAlaValIleAla
100105110
AlaGlyGlyAsnAlaSerTyrIleLeuAspGlyValProArgMetArg
115120125
GluArgProIleGlyAspLeuValValGlyLeuLysGlnLeuGlyAla
130135140
AspValAspCysIleLeuGlyThrAspCysProProValArgAlaAsn
145150155160
AlaHisGlyGlyLeuProGlyGlyLysValLysLeuSerGlySerIle
165170175
SerSerGlnTyrLeuThrAlaLeuLeuMetAlaAlaProLeuAlaLeu
180185190
GlyAspValGluIleGluIleMetAspLysLeuIleSerValProTyr
195200205
ValGluMetThrLeuLysLeuMetGluArgPheGlyValSerValGlu
210215220
HisSerSerSerTrpAspArgPhePheIleLysGlyGlyGlnLysTyr
225230235240
LysSerProGlyAsnAlaTyrValGluGlyAspAlaSerSerAlaSer
245250255
TyrPheLeuAlaGlyAlaAlaValThrGlyGlyThrValThrValGlu
260265270
GlyCysGlyThrSerSerLeuGlnGlyAspValArgTyrAlaGluVal
275280285
HisGluLysMetGlyAlaLysValThrTrpThrGluAsnSerValThr
290295300
ValThrGlyProProGlnAspProSerLysLysLysArgLeuArgAla
305310315320
ValAspValAsnLeuAsnLysMetProAspValAlaMetThrLeuAla
325330335
ValValAlaLeuTyrAlaAspGlyProThrAlaIleArgAspValAla
340345350
SerTrpArgValLysGluThrGluArgMetIleAlaIleCysThrGlu
355360365
LeuArgLysLeuGlyAlaThrValGluAspGlyProAspTyrCysVal
370375380
IleThrProProGluLysLeuAsnAlaAsnAlaIleAspSerTyrAsp
385390395400
AspHisArgMetAlaMetAlaPheSerLeuAlaAlaCysAlaAspVal
405410415
ProValThrIleGlyAspProGlyCysThrArgLysThrPheProAsp
420425430
TyrPheGluValLeuGlnArgPheThrThrHis
435440445
<210>45
<211>443
<212>PRT
<213>土麦冬种（LiriopesSpicatae）
<220>
<221>ACT_SITE
<222>(49，120，186，270，284，304，416，421)
<223>Val，Val，Thr，Ile，Arg，Lys，Glu，Gly
<400>SEQIDNO:45
MetValProGluIleValLeuGlnProIleLysGluIleSerGlyThr
151015
IleLysLeuProGlySerLysSerLeuSerAsnArgIleLeuLeuLeu
202530
AlaAlaLeuAlaGluGlyThrThrValValAspAsnLeuLeuAspSer
354045
ValAspIleArgTyrMetLeuAlaAlaLeuLysThrLeuGlyLeuThr
505560
ValGluAspAspSerValMetLysArgAlaThrValValGlySerGly
65707580
GlyGlnPheProValGlyLysAspSerLysGluValGlnLeuPheLeu
859095
GlyAsnAlaGlyThrAlaMetArgProLeuThrAlaAlaValIleAla
100105110
AlaGlyGlyAsnAlaSerTyrValLeuAspGlyValProArgMetArg
115120125
GluArgProIleGlyAspLeuValValGlyLeuLysGlnLeuGlyAla
130135140
AspValAspCysIleLeuGlyThrAspCysProProValArgAlaAsn
145150155160
AlaHisGlyGlyLeuProGlyGlyLysValLysLeuSerGlySerIle
165170175
SerSerGlnTyrLeuThrAlaLeuLeuThrAlaAlaProLeuAlaLeu
180185190
GlyAspValGluIleGluIleMetAspLysLeuIleSerValProTyr
195200205
ValGluMetThrLeuLysLeuMetGluArgPheGlyValSerValGlu
210215220
HisSerSerSerTrpAspArgPhePheIleLysGlyGlyGlnLysTyr
225230235240
LysSerProGlyAsnAlaTyrValGluGlyAspAlaSerSerAlaSer
245250255
TyrPheLeuAlaGlyAlaAlaValThrGlyGlyThrValIleValGlu
260265270
GlyCysGlyThrSerSerLeuGlnGlyAspValArgPheAlaGluVal
275280285
LeuGluLysMetGlyAlaLysValThrTrpThrGluAsnSerValLys
290295300
ValThrGlyProProGlnAspProSerLysLysLysArgLeuArgAla
305310315320
ValAspValAsnMetAsnLysMetProAspValAlaMetThrLeuAla
325330335
ValValAlaLeuTyrAlaAspGlyProThrAlaIleArgAspValAla
340345350
SerTrpArgValLysGluThrGluArgMetIleAlaIleCysThrGlu
355360365
LeuArgLysLeuGlyAlaThrValGluGluGlyProAspTyrCysVal
370375380
IleThrProProGluLysLeuAsnAlaAsnAlaIleAspThrTyrAsp
385390395400
AspHisArgMetAlaMetAlaPheSerLeuAlaAlaCysAlaAspGlu
405410415
ProValThrIleGlyAspProGlyCysThrArgLysThrPheProAsp
420425430
TyrPheGluValLeuGlnArgPheThrThrHis
435440445
<210>46
<211>443
<212>PRT
<213>土麦冬种（LiriopesSpicatae）
<220>
<221>ACT_SITE
<222>(51，59，95，284，316，421)
<223>Val，Arg，Leu，Arg，Arg，Gly
<400>SEQIDNO:46
MetValProGluIleValLeuGlnProIleLysGluIleSerGlyThr
151015
IleLysLeuProGlySerLysSerLeuSerAsnArgIleLeuLeuLeu
202530
AlaAlaLeuAlaGluGlyThrThrValValAspAsnLeuLeuAspSer
354045
AspAspValArgTyrMetLeuAlaAlaLeuArgThrLeuGlyLeuThr
505560
ValGluAspAspSerValMetLysArgAlaThrValValGlySerGly
65707580
GlyGlnPheProValGlyLysAspSerLysGluValGlnLeuLeuLeu
859095
GlyAsnAlaGlyThrAlaMetArgProLeuThrAlaAlaValIleAla
100105110
AlaGlyGlyAsnAlaSerTyrIleLeuAspGlyValProArgMetArg
115120125
GluArgProIleGlyAspLeuValValGlyLeuLysGlnLeuGlyAla
130135140
AspValAspCysIleLeuGlyThrAspCysProProValArgAlaAsn
145150155160
AlaHisGlyGlyLeuProGlyGlyLysValLysLeuSerGlySerIle
165170175
SerSerGlnTyrLeuThrAlaLeuLeuMetAlaAlaProLeuAlaLeu
180185190
GlyAspValGluIleGluIleMetAspLysLeuIleSerValProTyr
195200205
ValGluMetThrLeuLysLeuMetGluArgPheGlyValSerValGlu
210215220
HisSerSerSerTrpAspArgPhePheIleLysGlyGlyGlnLysTyr
225230235240
LysSerProGlyAsnAlaTyrValGluGlyAspAlaSerSerAlaSer
245250255
TyrPheLeuAlaGlyAlaAlaValThrGlyGlyThrValThrValGlu
260265270
GlyCysGlyThrSerSerLeuGlnGlyAspValArgPheAlaGluVal
275280285
LeuGluLysMetGlyAlaLysValThrTrpThrGluAsnSerValThr
290295300
ValThrGlyProProGlnAspProSerLysLysArgArgLeuArgAla
305310315320
ValAspValAsnMetAsnLysMetProAspValAlaMetThrLeuAla
325330335
ValValAlaLeuTyrAlaAspGlyProThrAlaIleArgAspValAla
340345350
SerTrpArgValLysGluThrGluArgMetIleAlaIleCysThrGlu
355360365
LeuArgLysLeuGlyAlaThrValGluGluGlyProAspTyrCysVal
370375380
IleThrProProGluLysLeuAsnAlaAsnAlaIleAspThrTyrAsp
385390395400
AspHisArgMetAlaMetAlaPheSerLeuAlaAlaCysAlaAspVal
405410415
ProValThrIleGlyAspProGlyCysThrArgLysThrPheProAsp
420425430
TyrPheGluValLeuGlnArgPheThrThrHis
435440445
<210>47
<211>443
<212>PRT
<213>土麦冬种（LiriopesSpicatae）
<220>
<221>ACT_SITE
<222>(73，88，154，178，234，250，284，358，421)
<223>Pro，Val，Ser，Cys，Asn，Glu，Arg，Gly，Gly
<400>SEQIDNO:47
MetValProGluIleValLeuGlnProIleLysGluIleSerGlyThr
151015
IleLysLeuProGlySerLysSerLeuSerAsnArgIleLeuLeuLeu
202530
AlaAlaLeuAlaGluGlyThrThrValValAspAsnLeuLeuAspSer
354045
AspAspIleArgTyrMetLeuAlaAlaLeuLysThrLeuGlyLeuThr
505560
ValGluAspAspSerValMetLysProAlaThrValValGlySerGly
65707580
GlyGlnPheProValGlyLysValSerLysGluValGlnLeuPheLeu
859095
GlyAsnAlaGlyThrAlaMetArgProLeuThrAlaAlaValIleAla
100105110
AlaGlyGlyAsnAlaSerTyrIleLeuAspGlyValProArgMetArg
115120125
GluArgProIleGlyAspLeuValValGlyLeuLysGlnLeuGlyAla
130135140
AspValAspCysIleLeuGlyThrAspSerProProValArgAlaAsn
145150155160
AlaHisGlyGlyLeuProGlyGlyLysValLysLeuSerGlySerIle
165170175
SerCysGlnTyrLeuThrAlaLeuLeuMetAlaAlaProLeuAlaLeu
180185190
GlyAspValGluIleGluIleMetAspLysLeuIleSerValProTyr
195200205
ValGluLysThrLeuLysLeuMetGluArgPheGlyValSerValGlu
210215220
HisSerSerSerTrpAspArgPhePheAsnLysGlyGlyGlnLysTyr
225230235240
LysSerProGlyAsnAlaTyrValGluGlyGluAlaSerSerAlaSer
245250255
TyrPheLeuAlaGlyAlaAlaValThrGlyGlyThrValThrValGlu
260265270
GlyCysGlyThrSerSerLeuGlnGlyAspValArgPheAlaGluVal
275280285
LeuGluLysMetGlyAlaLysValThrTrpThrGluAsnSerValThr
290295300
ValThrGlyProProGlnAspProSerLysLysLysArgLeuArgAla
305310315320
ValAspValAsnMetAsnLysMetProAspValAlaMetThrLeuAla
325330335
ValValAlaLeuTyrAlaAspGlyProThrAlaIleArgAspValAla
340345350
SerTrpArgValLysGlyThrGluArgMetIleAlaIleCysThrGlu
355360365
LeuArgLysLeuGlyAlaThrValGluGluGlyProAspTyrCysVal
370375380
IleThrProProGluLysLeuAsnAlaAsnAlaIleAspThrTyrAsp
385390395400
AspHisArgMetAlaMetAlaPheSerLeuAlaAlaCysAlaAspVal
405410415
ProValThrIleGlyAspProGlyCysThrArgLysThrPheProAsp
420425430
TyrPheGluValLeuGlnArgPheThrThrHis
435440445
<210>48
<211>443
<212>PRT
<213>土麦冬种（LiriopesSpicatae）
<220>
<221>ACT_SITE
<222>(24，34，74，133，284，323，387，389，416，421，422)
<223>Thr，Ser，Val，Arg，Arg，Asp，Thr，Val，Leu，Gly，Val
<400>SEQIDNO:48
MetValProGluIleValLeuGlnProIleLysGluIleSerGlyThr
151015
IleLysLeuProGlySerLysThrLeuSerAsnArgIleLeuLeuLeu
202530
AlaSerLeuAlaGluGlyThrThrValValAspAsnLeuLeuAspSer
354045
AspAspIleArgTyrMetLeuAlaAlaLeuLysThrLeuGlyLeuThr
505560
ValGluAspAspSerValMetLysArgValThrValValGlySerGly
65707580
GlyGlnPheProValGlyLysAspSerLysGluValGlnLeuPheLeu
859095
GlyAsnAlaGlyThrAlaMetArgProLeuThrAlaAlaValIleAla
100105110
AlaGlyGlyAsnAlaSerTyrIleLeuAspGlyValProArgMetArg
115120125
GluArgProIleArgAspLeuValValGlyLeuLysGlnLeuGlyAla
130135140
AspValAspCysIleLeuGlyThrAspCysProProValArgAlaAsn
145150155160
AlaHisGlyGlyLeuProGlyGlyLysValLysLeuSerGlySerIle
165170175
SerSerGlnTyrLeuThrAlaLeuLeuMetAlaAlaProLeuAlaLeu
180185190
GlyAspValGluIleGluIleMetAspLysLeuIleSerValProTyr
195200205
ValGluMetThrLeuLysLeuMetGluArgPheGlyValSerValGlu
210215220
HisSerSerSerTrpAspArgPhePheIleLysGlyGlyGlnLysTyr
225230235240
LysSerProGlyAsnAlaTyrValGluGlyAspAlaSerSerAlaSer
245250255
TyrPheLeuAlaGlyAlaAlaValThrGlyGlyThrValThrValGlu
260265270
GlyCysGlyThrSerSerLeuGlnGlyAspValArgPheAlaGluVal
275280285
LeuGluLysMetGlyAlaLysValThrTrpThrGluAsnSerValThr
290295300
ValThrGlyProProGlnAspProSerLysLysLysArgLeuArgAla
305310315320
ValAspAspAsnMetAsnLysMetProAspValAlaMetThrLeuAla
325330335
ValValAlaLeuTyrAlaAspGlyProThrAlaIleArgAspValAla
340345350
SerTrpArgValLysGluThrGluArgMetIleAlaIleCysThrGlu
355360365
LeuArgLysLeuGlyAlaThrValGluGluGlyProAspTyrCysVal
370375380
IleThrThrProValLysLeuAsnAlaAsnAlaIleAspThrTyrAsp
385390395400
AspHisArgMetAlaMetAlaPheSerLeuAlaAlaCysAlaAspLeu
405410415
ProValThrIleGlyValProGlyCysThrArgLysThrPheProAsp
420425430
TyrPheGluValLeuGlnArgPheThrThrHis
435440445
<210>49
<211>443
<212>PRT
<213>土麦冬种（LiriopesSpicatae）
<220>
<221>ACT_SITE
<222>(117，212，284，421，441)
<223>Thr，Ser，Arg，Gly，Pro
<400>SEQIDNO:49
MetValProGluIleValLeuGlnProIleLysGluIleSerGlyThr
151015
IleLysLeuProGlySerLysSerLeuSerAsnArgIleLeuLeuLeu
202530
AlaAlaLeuAlaGluGlyThrThrValValAspAsnLeuLeuAspSer
354045
AspAspIleArgTyrMetLeuAlaAlaLeuLysThrLeuGlyLeuThr
505560
ValGluAspAspSerValMetLysArgAlaThrValValGlySerGly
65707580
GlyGlnPheProValGlyLysAspSerLysGluValGlnLeuPheLeu
859095
GlyAsnAlaGlyThrAlaMetArgProLeuThrAlaAlaValIleAla
100105110
AlaGlyGlyAsnThrSerTyrIleLeuAspGlyValProArgMetArg
115120125
GluArgProIleGlyAspLeuValValGlyLeuLysGlnLeuGlyAla
130135140
AspValAspCysIleLeuGlyThrAspCysProProValArgAlaAsn
145150155160
AlaHisGlyGlyLeuProGlyGlyLysValLysLeuSerGlySerIle
165170175
SerSerGlnTyrLeuThrAlaLeuLeuMetAlaAlaProLeuAlaLeu
180185190
GlyAspValGluIleGluIleMetAspLysLeuIleSerValProTyr
195200205
ValGluMetSerLeuLysLeuMetGluArgPheGlyValSerValGlu
210215220
HisSerSerSerTrpAspArgPhePheIleLysGlyGlyGlnLysTyr
225230235240
LysSerProGlyAsnAlaTyrValGluGlyAspAlaSerSerAlaSer
245250255
TyrPheLeuAlaGlyAlaAlaValThrGlyGlyThrValThrValGlu
260265270
GlyCysGlyThrSerSerLeuGlnGlyAspValArgPheAlaGluVal
275280285
LeuGluLysMetGlyAlaLysValThrTrpThrGluAsnSerValThr
290295300
ValThrGlyProProGlnAspProSerLysLysLysArgLeuArgAla
305310315320
ValAspValAsnMetAsnLysMetProAspValAlaMetThrLeuAla
325330335
ValValAlaLeuTyrAlaAspGlyProThrAlaIleArgAspValAla
340345350
SerTrpArgValLysGluThrGluArgMetIleAlaIleCysThrGlu
355360365
LeuArgLysLeuGlyAlaThrValGluGluGlyProAspTyrCysVal
370375380
IleThrProProGluLysLeuAsnAlaAsnAlaIleAspThrTyrAsp
385390395400
AspHisArgMetAlaMetAlaPheSerLeuAlaAlaCysAlaAspVal
405410415
ProValThrIleGlyAspProGlyCysThrArgLysThrPheProAsp
420425430
TyrPheGluValLeuGlnArgPheProThrHis
435440445
<210>50
<211>443
<212>PRT
<213>土麦冬种（LiriopesSpicatae）
<220>
<221>ACT_SITE
<222>(75，87，268，284，359，362，421)
<223>Ser，Arg，Ser，Arg，Ser，Lys，Gly
<400>SEQIDNO:50
MetValProGluIleValLeuGlnProIleLysGluIleSerGlyThr
151015
IleLysLeuProGlySerLysSerLeuSerAsnArgIleLeuLeuLeu
202530
AlaAlaLeuAlaGluGlyThrThrValValAspAsnLeuLeuAspSer
354045
AspAspIleArgTyrMetLeuAlaAlaLeuLysThrLeuGlyLeuThr
505560
ValGluAspAspSerValMetLysArgAlaSerValValGlySerGly
65707580
GlyGlnPheProValGlyArgAspSerLysGluValGlnLeuPheLeu
859095
GlyAsnAlaGlyThrAlaMetArgProLeuThrAlaAlaValIleAla
100105110
AlaGlyGlyAsnAlaSerTyrIleLeuAspGlyValProArgMetArg
115120125
GluArgProIleGlyAspLeuValValGlyLeuLysGlnLeuGlyAla
130135140
AspValAspCysIleLeuGlyThrAspCysProProValArgAlaAsn
145150155160
AlaHisGlyGlyLeuProGlyGlyLysValLysLeuSerGlySerIle
165170175
SerSerGlnTyrLeuThrAlaLeuLeuMetAlaAlaProLeuAlaLeu
180185190
GlyAspValGluIleGluIleMetAspLysLeuIleSerValProTyr
195200205
ValGluMetThrLeuLysLeuMetGluArgPheGlyValSerValGlu
210215220
HisSerSerSerTrpAspArgPhePheIleLysGlyGlyGlnLysTyr
225230235240
LysSerProGlyAsnAlaTyrValGluGlyAspAlaSerSerAlaSer
245250255
TyrPheLeuAlaGlyAlaAlaValThrGlyGlySerValThrValGlu
260265270
GlyCysGlyThrSerSerLeuGlnGlyAspValArgPheAlaGluVal
275280285
LeuGluLysMetGlyAlaLysValThrTrpThrGluAsnSerValThr
290295300
ValThrGlyProProGlnAspProSerLysLysLysArgLeuArgAla
305310315320
ValAspValAsnMetAsnLysMetProAspValAlaMetThrLeuAla
325330335
ValValAlaLeuTyrAlaAspGlyProThrAlaIleArgAspValAla
340345350
SerTrpArgValLysGluSerGluArgLysIleAlaIleCysThrGlu
355360365
LeuArgLysLeuGlyAlaThrValGluGluGlyProAspTyrCysVal
370375380
IleThrProProGluLysLeuAsnAlaAsnAlaIleAspThrTyrAsp
385390395400
AspHisArgMetAlaMetAlaPheSerLeuAlaAlaCysAlaAspVal
405410415
ProValThrIleGlyAspProGlyCysThrArgLysThrPheProAsp
420425430
TyrPheGluValLeuGlnArgPheThrThrHis
435440445
<210>51
<211>443
<212>PRT
<213>土麦冬种（LiriopesSpicatae）
<220>
<221>ACT_SITE
<222>(68，284，349，421，424)
<223>Gly，Arg，Gly，Gly，Cys
<400>SEQIDNO:51
MetValProGluIleValLeuGlnProIleLysGluIleSerGlyThr
151015
IleLysLeuProGlySerLysSerLeuSerAsnArgIleLeuLeuLeu
202530
AlaAlaLeuAlaGluGlyThrThrValValAspAsnLeuLeuAspSer
354045
AspAspIleArgTyrMetLeuAlaAlaLeuLysThrLeuGlyLeuThr
505560
ValGluAspGlySerValMetLysArgAlaThrValValGlySerGly
65707580
GlyGlnPheProValGlyLysAspSerLysGluValGlnLeuPheLeu
859095
GlyAsnAlaGlyThrAlaMetArgProLeuThrAlaAlaValIleAla
100105110
AlaGlyGlyAsnAlaSerTyrIleLeuAspGlyValProArgMetArg
115120125
GluArgProIleGlyAspLeuValValGlyLeuLysGlnLeuGlyAla
130135140
AspValAspCysIleLeuGlyThrAspCysProProValArgAlaAsn
145150155160
AlaHisGlyGlyLeuProGlyGlyLysValLysLeuSerGlySerIle
165170175
SerSerGlnTyrLeuThrAlaLeuLeuMetAlaAlaProLeuAlaLeu
180185190
GlyAspValGluIleGluIleMetAspLysLeuIleSerValProTyr
195200205
ValGluMetThrLeuLysLeuMetGluArgPheGlyValSerValGlu
210215220
HisSerSerSerTrpAspArgPhePheIleLysGlyGlyGlnLysTyr
225230235240
LysSerProGlyAsnAlaTyrValGluGlyAspAlaSerSerAlaSer
245250255
TyrPheLeuAlaGlyAlaAlaValThrGlyGlyThrValThrValGlu
260265270
GlyCysGlyThrSerSerLeuGlnGlyAspValArgPheAlaGluVal
275280285
LeuGluLysMetGlyAlaLysValThrTrpThrGluAsnSerValThr
290295300
ValThrGlyProProGlnAspProSerLysLysLysArgLeuArgAla
305310315320
ValAspValAsnMetAsnLysMetProAspValAlaMetThrLeuAla
325330335
ValValAlaLeuTyrAlaAspGlyProThrAlaIleGlyAspValAla
340345350
SerTrpArgValLysGluThrGluArgMetIleAlaIleCysThrGlu
355360365
LeuArgLysLeuGlyAlaThrValGluGluGlyProAspTyrCysVal
370375380
IleThrProProGluLysLeuAsnAlaAsnAlaIleAspThrTyrAsp
385390395400
AspHisArgMetAlaMetAlaPheSerLeuAlaAlaCysAlaAspVal
405410415
ProValThrIleGlyAspProCysCysThrArgLysThrPheProAsp
420425430
TyrPheGluValLeuGlnArgPheThrThrHis
435440445
<210>52
<211>443
<212>PRT
<213>土麦冬种（LiriopesSpicatae）
<220>
<221>ACT_SITE
<222>(75，110，284，372，381，421)
<223>Ser，Ala，Arg，Gln，Val，Gly
<400>SEQIDNO:52
MetValProGluIleValLeuGlnProIleLysGluIleSerGlyThr
151015
IleLysLeuProGlySerLysSerLeuSerAsnArgIleLeuLeuLeu
202530
AlaAlaLeuAlaGluGlyThrThrValValAspAsnLeuLeuAspSer
354045
AspAspIleArgTyrMetLeuAlaAlaLeuLysThrLeuGlyLeuThr
505560
ValGluAspAspSerValMetLysArgAlaSerValValGlySerGly
65707580
GlyGlnPheProValGlyLysAspSerLysGluValGlnLeuPheLeu
859095
GlyAsnAlaGlyThrAlaMetArgProLeuThrAlaAlaAlaIleAla
100105110
AlaGlyGlyAsnAlaSerTyrIleLeuAspGlyValProArgMetArg
115120125
GluArgProIleGlyAspLeuValValGlyLeuLysGlnLeuGlyAla
130135140
AspValAspCysIleLeuGlyThrAspCysProProValArgAlaAsn
145150155160
AlaHisGlyGlyLeuProGlyGlyLysValLysLeuSerGlySerIle
165170175
SerSerGlnTyrLeuThrAlaLeuLeuMetAlaAlaProLeuAlaLeu
180185190
GlyAspValGluIleGluIleMetAspLysLeuIleSerValProTyr
195200205
ValGluMetThrLeuLysLeuMetGluArgPheGlyValSerValGlu
210215220
HisSerSerSerTrpAspArgPhePheIleLysGlyGlyGlnLysTyr
225230235240
LysSerProGlyAsnAlaTyrValGluGlyAspAlaSerSerAlaSer
245250255
TyrPheLeuAlaGlyAlaAlaValThrGlyGlyThrValThrValGlu
260265270
GlyCysGlyThrSerSerLeuGlnGlyAspValArgPheAlaGluVal
275280285
LeuGluLysMetGlyAlaLysValThrTrpThrGluAsnSerValThr
290295300
ValThrGlyProProGlnAspProSerLysLysLysArgLeuArgAla
305310315320
ValAspValAsnMetAsnLysMetProAspValAlaMetThrLeuAla
325330335
ValValAlaLeuTyrAlaAspGlyProThrAlaIleArgAspValAla
340345350
SerTrpArgValLysGluThrGluArgMetIleAlaIleCysThrGlu
355360365
LeuArgLysGlnGlyAlaThrValGluGluGlyProValTyrCysVal
370375380
IleThrProProGluLysLeuAsnAlaAsnAlaIleAspThrTyrAsp
385390395400
AspHisArgMetAlaMetAlaPheSerLeuAlaAlaCysAlaAspVal
405410415
ProValThrIleGlyAspProGlyCysThrArgLysThrPheProAsp
420425430
TyrPheGluValLeuGlnArgPheThrThrHis
435440445

Claims (10)

1.一种具有草甘膦耐性的基因，该基因的核苷酸序列为SEQIDNO：3。
2.一种具有草甘膦耐性的蛋白质多肽，该蛋白质多肽的氨基酸序列为SEQIDNO：4。
3.一种具有草甘膦耐性的基因，该基因的核苷酸序列为编码权利要求2所述蛋白质多肽的核苷酸序列。
4.一种具有草甘膦耐性的基因，其特征在于：所述基因编码的蛋白质的氨基酸序列为SEQIDNO：29-52中任意一种。
5.一种具有草甘膦耐性的蛋白质，其特征在于：所述蛋白质的氨基酸序列为SEQIDNO：29-52中任意一种。
6.一种具有草甘膦耐性的基因，该基因的核苷酸序列为编码权利要求5所述蛋白质的核苷酸序列。
7.包含权利要求1、3、4或6所述基因的表达载体，所述表达载体为原核表达载体或植物转化质粒。
8.权利要求1、3、4或6所述基因在制备抗/耐草甘膦植株方面的用途。
9.根据权利要求8所述基因在制备抗/耐草甘膦植株方面的用途，其特征在于：包括以下步骤：
（1）利用DNA重组技术，构建含有所述基因的植物转化质粒；
（2）将步骤（1）中构建的植物转化质粒通过基因枪或农杆菌浸染方法或花粉介导法导入植物组织，利用草甘膦培养基筛选含有所述基因的植物细胞；
（3）将步骤（2）中筛选的植物细胞进行分化获得转化芽，经过生根培养获得植物种苗，从而得到所述抗/耐草甘膦植株。
10.权利要求1、3、4或6所述基因作为植物转基因筛选标记的用途。
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