亚麻荠CsPDAT基因及其在植物抗旱方面的应用
阅读说明：本技术 亚麻荠CsPDAT基因及其在植物抗旱方面的应用 (Camelina sativa CsPDAT gene and application thereof in drought resistance of plants ) 是由 苑丽霞 李润植 于 2019-10-28 设计创作，主要内容包括：本发明植物抗旱基因技术领域,具体涉及一种亚麻荠CsPDAT基因及其在植物抗旱方面的应用。所述亚麻荠基因CsPDAT为CsPDAT2-A,CsPDAT2-A的核苷酸序列如SEQ ID NO.1所示,氨基酸序列如SEQ ID NO.2所示。CsPDAT基因的功能分析和鉴定为获得植物新品种提供了理论基础,利用该基因进行分子育种有望培养出具有生产应用价值的高油植物；为培育抗旱的转基因作物新品种奠定了理论和生产实践基础。(The invention belongs to the technical field of plant drought-resistant genes, and particularly relates to a camelina sativa CsPDAT gene and application thereof in plant drought resistance. The camelina sativa gene CsPDAT is CsPDAT2-A, the nucleotide sequence of CsPDAT2-A is shown as SEQ ID NO.1, and the amino acid sequence is shown as SEQ ID NO. 2. The function analysis and identification of the CsPDAT gene provide a theoretical basis for obtaining new plant varieties, and the gene is expected to be used for molecular breeding to culture high-oil plants with production and application values; lays a theoretical and production practical foundation for cultivating new varieties of drought-resistant transgenic crops.)
技术领域
本发明属于植物抗旱基因技术领域，具体涉及一种亚麻荠CsPDAT基因及其在植物抗旱方面的应用。
背景技术
亚麻荠(Camelina sativa L.Crantz)是一种在北美及欧洲地区广泛种植的十字花科油料作物，因其具有“低耗、高抗和环保”等优良的生物学特性和农艺性状，种植区域日益扩大。亚麻荠生育期短(80-100d)，株高平均92.4cm，千粒质量平均1.25g，种子含油量高(34％-45％)，我国近年来也开展了亚麻荠的引种、育种及推广种植。亚麻荠种子独特的不饱和脂肪酸组成，以及高水平的ω-3脂肪酸和低含量的芥子油苷，赋予亚麻荠油重要的食品营养价值和工业利用价值。此外，亚麻荠种子也含有占种子干质量约30％的贮藏蛋白质，其是人类食品及动物饲料蛋白质的重要来源。亚麻荠是一种低投入、耐逆境的油料作物，对水肥的要求也比较低。与小麦和油菜相比，亚麻荠需水量和用肥量明显减少，生产投入大约只是油菜的一半。亚麻荠在较为贫瘠的土壤条件下(如，沟壑、路边，荒郊野外、盐碱地等非农边际性土地)也能正常生长。在我国干旱的北方地区推广种植亚麻荠，可缓解北方农业水资源的紧张状况及保护生态环境，具有很高的生产价值。
干旱和水资源短缺已成为世界农业发展的主要制约因素，对农业生产构成了严重的威胁。亚麻荠因具有耐旱、耐贫瘠、抗病和抗虫等优良特性，是十字花科作物遗传改良的重要种质资源，更符合可持续农业的发展要求，是一种较为理想的新型能源作物。在亚麻荠抗旱品种的选育方面，最常用的方法就是常规杂交育种，常规杂交育种是通过不同基因型个体之间的交配而取得双亲基因重新组合的个体的方法。杂交过程并不会产生新基因，且杂交后代会出现性状分离，育种过程缓慢，过程复杂。因此开发能够鉴定亚麻荠抗旱性的基因，对于亚麻荠抗旱品种的选育具有重要意义。
发明内容
本发明提供了一种亚麻荠CsPDAT基因及其在植物抗旱方面的应用，CsPDAT2-A基因能使亚麻荠的抗旱性能提高。
本发明的目的是提供一种亚麻荠基因CsPDAT，所述亚麻荠基因CsPDAT为CsPDAT2-A，CsPDAT2-A的核苷酸序列如SEQ ID NO.1所示，氨基酸序列如SEQ ID NO.2所示。
本发明还提供了一种所述的亚麻荠基因CsPDAT在植物抗旱育种中的应用。
本发明还提供了一种所述的亚麻荠基因CsPDAT在高油脂含量植物育种中的应用。
优选的，根据上述应用，所述亚麻荠基因CsPDAT还包括CsPDAT1-A、CsPDAT1-B、CsPDAT1-C和CsPDAT2-B。
优选的，根据上述应用，所述植物为亚麻荠和烟草。
与现有技术相比，本发明提供的一种亚麻荠CsPDAT基因及其在植物抗旱方面的应用，至少具有以下有益效果：
CsPDAT基因的功能分析和鉴定为获得植物新品种提供了理论基础，利用该基因进行分子育种有望培养出具有生产应用价值的高油植物。为培育抗旱的转基因作物新品种奠定了理论和生产实践基础。
我们的研究结果显示干旱胁迫处理导致CsPDAT2-A mRNA表达增加2倍，同时也分离了这些CsPDAT基因的cDNA克隆，用于烟草叶片的瞬时表达，分析显示五个基因(CsPDAT1-A、CsPDAT1-B、CsPDAT1-C、CsPDAT2-A和CsPDAT2-B)PDAT酶活性和油脂积累大幅增加。
附图说明
图1是干旱胁迫下亚麻荠幼苗中TAG的积累量随时间的变化；
图2是CsPDAT成员在干旱胁迫下幼苗mRNA表达呈现出三种不同的模式；
图3是CsPDAT导入烟草叶片瞬间表达后TAG的含量。
具体实施方式
为了使本领域技术人员更好地理解本发明的技术方案能予以实施，下面结合具体实施例和附图对本发明作进一步说明。
下面各实施例以及上述发明内容中未注明具体条件的试验方法，均按照本领域的常规方法和条件进行，所用的材料若无特殊说明均为市售。
本发明提供了一种能够鉴定植物抗旱性的亚麻荠基因CsPDAT，所述亚麻荠基因CsPDAT为CsPDAT2-A，CsPDAT2-A的核苷酸序列如SEQ ID NO.1所示，氨基酸序列如SEQ IDNO.2所示。CsPDAT2-A在细胞内的第四号染色体上，且有6个外显子，编码662个氨基酸的蛋白。
CsPDAT能够编码磷脂胆碱二酰甘油酰基转移酶，该酶将磷脂酰胆碱(phosphatidylcholine,PC)分子中的酰基导入DAG最后生成TAG(三酰甘油),是参与生成TAG的最后一步酰基化反应的重要酶，该酶表达量越高，则说明亚麻荠的产油脂性能越强。
本发明还提供了一种如SEQ ID NO.1所示CsPDAT2-A基因在植物抗旱性育种、尤其是亚麻荠抗旱育种中的应用。
实施例1CsPDAT基因成员的特征分析
为了鉴定亚麻荠(C.sativa)中编码PDAT的基因，我们使用拟南芥PDAT1(At5g13640)和PDAT2(At3g44830)的氨基酸(AA)序列作为查询序列，对camelina基因组数据库(https://www.ncbi.nlm.nih.gov/genome/？term＝camelina+sativa)进行BLAST搜索。因此，从数据库中识别出5个候选的camelina CsPDAT基因，即CsPDAT1-A(氨基酸序列如SEQ ID NO.3)、CsPDAT1-B(氨基酸序列如SEQ ID NO.4)、CsPDAT1-C(氨基酸序列如SEQ IDNO.5)、CsPDAT2-A和CsPDAT2-B(氨基酸序列如SEQ ID NO.6)，有较低的p值(<2.3E-204)。三个CsPDAT1，即CsPDAT1-A、CsPDAT1-B和CsPDAT1-C，对拟南芥PDAT1(At5g13640)分别表现出97％、96％和95％的氨基酸一致性；而两个CsPDAT2，即CsPDAT2-A和CsPDAT2-B，对拟南芥PDAT2(At3g44830)分别表现出90％和89％的氨基酸序列一致性。表1列出了已鉴定的CsPDAT基因的一般信息。基因结构检测表明，除CsPDAT1-C有7个外显子外，CsPDAT1-A、CsPDAT1-B、CsPDAT2-A和CsPDAT2-B均有6个外显子。所有CsPDAT基因的内含子都有一致的GT-AG连接。通过与已公布的PDATs基因进行综合分析证明，我们选择的CsPDAT1-A、CsPDAT1-B、CsPDAT1-C、CsPDAT2-A和CsPDAT2-B具有PDAT家族的明显特征，即PDATs通过将酰基从PLs转移到DAGs来生成TAG。
表1 CsPDAT基因和编码多肽的基本信息
实施例2 CsPDAT2-A基因在干旱胁迫中发挥重要作用
为了确定CsPDAT家族中哪个成员参与了亚麻荠对干旱胁迫的响应，在干旱胁迫下生长3周龄的亚麻荠幼苗样品分成两批，一组用于总脂提取，另一组用于总RNA提取和qRT-PCR检测。在PEG6000模拟的干旱条件下，幼苗的三酰甘油TAG积累水平逐渐升高，在5天达到最大。图1是干旱胁迫下亚麻荠幼苗中TAG的积累量随时间的变化，图1中PEG6000胁迫处理采用的PEG6000浓度为15g/100ml，空白对照采用蒸馏水替代PEG6000。如果一个CsPDAT家族成员在干旱胁迫条件下对亚麻荠幼苗积累TAG起作用，那么该基因的表达量也应相应发生变化。为了确定这种关系，用qRT-PCR分别检测了CsPDAT1-A、CsPDAT1-B、CsPDAT1-C、CsPDAT2-A和CsPDAT2-B的基因表达，结果如图2所示，CsPDAT成员在干旱胁迫下幼苗mRNA表达与干旱胁迫处理0天的相比较呈现出三种不同的模式：TAG上调(CsPDAT2-A)、TAG下调(CsPDAT2-B)、无显著变化(CsPDAT1-A、CsPDAT1-B、CsPDAT1-C)。图2中，在PEG6000干旱胁迫下，CsPDAT2-A的表达量从第1d开始增加，并在第4d达到峰值，水平是对照(0d)的2倍(P<0.01)。CsPDAT2-B的表达量从1d下降到4d，5d时几乎没有检测到转录本(P<0.01)。CsPDAT1-C表达量在对照和应激条件下变化不大，但不显著(P<0.05)。
此外，我们还分离了这些CsPDAT1-A、CsPDAT1-B、CsPDAT1-C、CsPDAT2-A和CsPDAT2-B基因的cDNA克隆，分别将其导入烟草叶片，具体步骤如下：用RT-PCR方法从亚麻荠发育组织的总RNA中扩增上述基因的cDNA，然后分别***克隆载体。用高保真Taq DNA聚合酶扩增每个ORF序列，表2列出了CsPDAT cDNAs的ORF 5'和3'末端序列的引物。在每个正向和反向引物上分别添加HindIII和BamH1的限制性位点。再将每个CsPDAT基因的相应ORF***到HindIII-BamH1消化的pBI121载体中，以产生35S为启动子驱动每个CsPDAT基因的表达载体。然后将每个CsPDAT表达载体分别转移到农杆菌GV3101中。农杆菌细胞在28℃下培养过夜。使用渗透缓冲液(10mM MES,pH 5.7,10mM MgCl2,and 150mM acetosyringone)收集并重新悬浮细胞，为使CsPDATs瞬时表达随后用真空渗透法分别对4-5周龄的烟草叶片进行处理，7天后采集烟草叶片(感染的和对应未感染的区域)进行脂质分析。
表2用于扩增CsPDAT cDNA中ORFs的引物序列
烟草叶片的TAG瞬时表达情况参见图3。图3中，WT是野生型烟草叶片中TAG积累量，VC表示pBI121空载体烟草叶片中TAG积累量，CsPDAT1-A、CsPDAT1-B、CsPDAT1-C、CsPDAT2-A和CsPDAT2-B分别表示烟草叶片中转化了CsPDAT1-A、CsPDAT1-B、CsPDAT1-C、CsPDAT2-A和CsPDAT2-B基因的TAG积累量。通过对亚麻荠幼苗在干旱胁迫条件下CsPDATs表达模式和TAG积累的综合分析表明，CsPDAT2-A是干旱胁迫下促进TAG生物合成的关键因子。
需要说明的是本发明权利要求书中涉及数值范围时，应理解为每个数值范围的两个端点以及两个端点之间任何一个数值均可选用，由于采用的步骤方法与实施例相同，为了防止赘述，本发明的描述了优选的实施例，但本领域内的技术人员一旦得知了基本创造性概念，则可对这些实施例作出另外的变更和修改。所以，所附权利要求意欲解释为包括优选实施例以及落入本发明范围的所有变更和修改。显然，本领域的技术人员可以对本发明进行各种改动和变型而不脱离本发明的精神和范围。这样，倘若本发明的这些修改和变型属于本发明权利要求及其等同技术的范围之内，则本发明也意图包含这些改动和变型在内。
序列表
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Trp Trp Phe Leu Leu Phe Leu Tyr Asn Ala Met Pro Ala Ser Phe Pro
65 70 75 80
Gln Tyr Val Thr Glu Arg Ile Thr Gly Pro Leu Pro Asp Pro Pro Gly
85 90 95
Val Lys Leu Lys Lys Glu Gly Leu Lys Ala Gln His Pro Val Val Phe
100 105 110
Ile Pro Gly Ile Val Thr Gly Gly Leu Glu Leu Trp Glu Gly Lys Gln
115 120 125
Cys Ala Asp Gly Leu Phe Arg Lys Arg Leu Trp Gly Gly Thr Phe Gly
130 135 140
Glu Val Tyr Lys Arg Pro Leu Cys Trp Val Glu His Met Ser Leu Asp
145 150 155 160
Asn Glu Thr Gly Leu Asp Pro Asp Gly Ile Arg Val Arg Ala Val Ser
165 170 175
Gly Leu Val Ala Ala Asp Tyr Phe Ala Pro Gly Tyr Phe Val Trp Ala
180 185 190
Val Leu Ile Ala Asn Leu Ala His Ile Gly Tyr Glu Glu Lys Asn Met
195 200 205
Tyr Met Ala Ala Tyr Asp Trp Arg Leu Ser Phe Gln Asn Thr Glu Val
210 215 220
Arg Asp Gln Thr Leu Ser Arg Met Lys Ser Asn Ile Glu Leu Met Val
225 230 235 240
Ser Thr Asn Gly Gly Lys Lys Ala Val Ile Val Pro His Ser Met Gly
245 250 255
Val Leu Tyr Phe Leu His Phe Met Lys Trp Val Glu Glu Pro Ala Pro
260 265 270
Met Gly Gly Gly Gly Gly Pro Asp Trp Cys Ala Lys Tyr Ile Lys Ala
275 280 285
Val Met Asn Ile Gly Gly Pro Phe Leu Gly Val Pro Lys Ala Val Ser
290 295 300
Gly Leu Phe Ser Ala Glu Ala Lys Asp Val Ala Val Ala Arg Ala Ile
305 310 315 320
Ala Pro Gly Phe Leu Asp Thr Asp Ile Phe Arg Leu Gln Thr Leu Gln
325 330 335
His Ile Met Arg Met Thr Arg Thr Trp Asp Ser Thr Met Ser Met Leu
340 345 350
Pro Lys Gly Gly Asp Thr Ile Trp Gly Gly Leu Asp Trp Ser Pro Glu
355 360 365
Lys Gly His Thr Cys Cys Gly Lys Lys Gln Lys Arg Asn Glu Thr Cys
370 375 380
Gly Glu Ala Gly Glu Asn Gly Val Ser Lys Ile Lys Pro Ala Asn Tyr
385 390 395 400
Gly Arg Met Ile Ser Phe Gly Lys Glu Ile Ala Glu Ala Ala Pro Ser
405 410 415
Glu Ile Asn Asn Ile Asp Phe Arg Gly Ala Val Lys Gly Gln Ser Phe
420 425 430
Pro Asn His Thr Cys Arg Asp Val Trp Thr Glu Tyr His Asp Met Gly
435 440 445
Ile Ala Gly Ile Lys Ala Ile Ala Glu Tyr Lys Val Tyr Thr Ala Asp
450 455 460
Ala Val Ile Asp Leu Leu His Tyr Val Ala Pro Lys Met Met Ala Arg
465 470 475 480
Gly Ser Ala His Phe Ser Tyr Gly Ile Ala Asp Asp Leu Asp Asp Pro
485 490 495
Lys Tyr Lys Asp Pro Lys Tyr Trp Ser Asn Pro Leu Glu Thr Lys Leu
500 505 510
Pro Asn Ala Pro Glu Met Glu Ile Tyr Ser Leu Tyr Gly Val Gly Ile
515 520 525
Pro Thr Glu Arg Ala Tyr Val Tyr Lys Leu Asn Gln Ser Pro Asp Ser
530 535 540
Cys Ile Pro Phe Gln Ile Phe Thr Ser Ala His Glu Glu Asp Glu Asp
545 550 555 560
Ser Cys Leu Lys Ala Gly Val Tyr Asn Val Asp Gly Asp Glu Thr Val
565 570 575
Pro Val Leu Ser Ala Gly Tyr Met Cys Ala Lys Ala Trp Arg Gly Lys
580 585 590
Thr Arg Phe Asn Pro Ser Gly Ile Lys Thr Tyr Val Arg Glu Tyr Asn
595 600 605
His Ser Pro Pro Ala Asn Leu Leu Glu Gly Arg Gly Thr Gln Ser Gly
610 615 620
Ala His Val Asp Ile Met Gly Asn Phe Ala Leu Ile Glu Asp Ile Met
625 630 635 640
Arg Val Ala Ala Gly Gly Asn Gly Ser Glu Ile Gly His Asp Gln Val
645 650 655
Tyr Ser Gly Ile Tyr Glu Trp Ser Asp Arg Ile Asp Leu Lys Leu
660 665 670
<210> 4
<211> 597
<212> PRT
<213> 人工序列
<400> 4
Met Pro Ala Ser Phe Pro Gln Tyr Val Thr Glu Arg Ile Thr Gly Pro
1 5 10 15
Leu Pro Asp Pro Pro Gly Val Lys Leu Lys Lys Glu Gly Leu Lys Ala
20 25 30
Gln His Pro Val Val Phe Ile Pro Gly Ile Val Thr Gly Gly Leu Glu
35 40 45
Leu Trp Glu Gly Lys Gln Cys Ala Asp Gly Leu Phe Arg Lys Arg Leu
50 55 60
Trp Gly Gly Thr Phe Gly Glu Val Tyr Lys Arg Pro Leu Cys Trp Val
65 70 75 80
Glu His Met Ser Leu Asp Asn Glu Thr Gly Leu Asp Pro Asp Gly Ile
85 90 95
Arg Val Arg Ala Val Ser Gly Leu Val Ala Ala Asp Tyr Phe Ala Pro
100 105 110
Gly Tyr Phe Val Trp Ala Val Leu Ile Ala Asn Leu Ala His Ile Gly
115 120 125
Tyr Glu Glu Lys Asn Met Tyr Met Ala Ala Tyr Asp Trp Arg Leu Ser
130 135 140
Phe Gln Asn Thr Glu Val Arg Asp Gln Thr Leu Ser Arg Met Lys Ser
145 150 155 160
Asn Ile Glu Leu Met Val Ser Thr Asn Gly Gly Lys Lys Ala Val Ile
165 170 175
Val Pro His Ser Met Gly Val Leu Tyr Phe Leu His Phe Met Lys Trp
180 185 190
Val Glu Glu Pro Ala Pro Met Gly Gly Gly Gly Gly Pro Asn Trp Cys
195 200 205
Ala Lys Tyr Ile Lys Ala Val Met Asn Ile Gly Gly Pro Phe Leu Gly
210 215 220
Val Pro Lys Ala Val Ser Gly Leu Phe Ser Ala Glu Ala Lys Asp Val
225 230 235 240
Ala Val Ala Arg Ala Ile Ala Pro Gly Phe Leu Asp Thr Asp Ile Phe
245 250 255
Arg Leu Gln Thr Leu Gln His Ile Met Arg Met Thr Arg Thr Trp Asp
260 265 270
Ser Thr Met Ser Met Leu Pro Lys Gly Gly Asp Thr Ile Trp Gly Gly
275 280 285
Leu Asp Trp Ser Pro Glu Lys Gly His Thr Cys Cys Gly Asn Lys Gln
290 295 300
Lys Arg Asn Glu Thr Cys Gly Glu Ala Gly Glu Asn Gly Val Ser Lys
305 310 315 320
Ile Lys Pro Val Asn Tyr Gly Arg Met Ile Ser Phe Gly Lys Glu Ile
325 330 335
Ala Glu Ala Ala Ser Ser Glu Ile Asn Asn Ile Asp Phe Arg Gly Ala
340 345 350
Val Lys Gly Gln Ser Phe Pro Asn His Thr Cys Arg Asp Val Trp Thr
355 360 365
Glu Tyr His Asp Met Gly Ile Ala Gly Ile Lys Ala Ile Ala Glu Tyr
370 375 380
Lys Val Tyr Thr Ala Asp Ala Val Ile Asp Leu Leu His Tyr Val Ala
385 390 395 400
Pro Lys Met Met Ala Arg Gly Ser Ala His Phe Ser Tyr Gly Ile Ala
405 410 415
Asp Asp Leu Asp Asp Pro Lys Tyr Lys Asp Pro Lys Tyr Trp Ser Asn
420 425 430
Pro Leu Glu Thr Lys Leu Pro Asn Ala Pro Glu Met Glu Ile Phe Ser
435 440 445
Leu Tyr Gly Val Gly Ile Pro Thr Glu Arg Ala Tyr Val Tyr Lys Leu
450 455 460
Asn Gln Ser Pro Asp Ser Cys Ile Pro Phe Gln Ile Phe Thr Ser Ala
465 470 475 480
His Glu Glu Asp Glu Asp Ser Cys Leu Lys Ala Gly Val Tyr Asn Val
485 490 495
Asp Gly Asp Glu Thr Val Pro Val Leu Ser Ala Gly Tyr Met Cys Ala
500 505 510
Lys Ala Trp Arg Gly Lys Thr Arg Phe Asn Pro Ser Gly Ile Lys Thr
515 520 525
Tyr Ile Arg Glu Tyr Asn His Ser Pro Pro Ala Asn Leu Leu Glu Gly
530 535 540
Arg Gly Thr Gln Ser Gly Ala His Val Asp Ile Met Gly Asn Phe Ala
545 550 555 560
Leu Ile Glu Asp Ile Met Arg Val Ala Ala Gly Gly Asn Gly Ser Asp
565 570 575
Ile Gly His Asp Gln Val Tyr Ser Gly Ile Tyr Glu Trp Ser Asp Arg
580 585 590
Ile Asp Leu Lys Leu
595
<210> 5
<211> 671
<212> PRT
<213> 人工序列
<400> 5
Met Pro Leu Ile His Arg Lys Lys Pro Gln Thr Pro Pro Ser Glu Glu
1 5 10 15
Val Val Asn Ala Asp Glu Asp Ser Gln Lys Lys Pro Pro His Glu Ser
20 25 30
Ser Ser Ser Ser Lys Thr His Lys Lys Ser Asn Gly Gly Gly Lys Trp
35 40 45
Ser Cys Val Asp Ser Cys Cys Trp Phe Ile Gly Cys Val Cys Val Thr
50 55 60
Trp Trp Phe Leu Leu Phe Leu Tyr Asn Ala Met Pro Ala Ser Phe Pro
65 70 75 80
Gln Tyr Val Thr Glu Arg Ile Thr Gly Pro Leu Pro Asp Pro Pro Gly
85 90 95
Val Lys Leu Lys Lys Glu Gly Leu Lys Ala Gln His Pro Val Val Phe
100 105 110
Ile Pro Gly Ile Val Thr Gly Gly Leu Glu Leu Trp Glu Gly Lys Gln
115 120 125
Cys Ala Asp Gly Leu Phe Arg Lys Arg Leu Trp Gly Gly Thr Phe Gly
130 135 140
Glu Val Tyr Lys Arg Pro Leu Cys Trp Val Glu His Met Ser Leu Asp
145 150 155 160
Asn Glu Thr Gly Leu Asp Pro Glu Gly Ile Arg Val Arg Ala Val Ser
165 170 175
Gly Leu Val Ala Ala Asp Tyr Phe Ala Pro Gly Tyr Phe Val Trp Ala
180 185 190
Val Leu Ile Ala Asn Leu Ala His Ile Gly Tyr Glu Glu Lys Asn Met
195 200 205
Tyr Met Ala Ala Tyr Asp Trp Arg Leu Ser Phe Gln Asn Thr Glu Val
210 215 220
Arg Asp Gln Thr Leu Ser Arg Met Lys Ser Asn Ile Glu Leu Met Val
225 230 235 240
Ser Thr Asn Gly Gly Lys Lys Ala Val Ile Val Pro His Ser Met Gly
245 250 255
Val Leu Tyr Phe Leu His Phe Met Lys Trp Val Glu Glu Pro Ala Pro
260 265 270
Met Gly Gly Gly Gly Gly Pro Asn Trp Cys Ala Lys Tyr Ile Lys Ala
275 280 285
Val Met Asn Ile Gly Gly Pro Phe Leu Gly Val Pro Lys Ala Val Ser
290 295 300
Gly Leu Phe Ser Ala Glu Ala Lys Asp Val Ala Val Ala Arg Ala Ile
305 310 315 320
Ala Pro Gly Phe Leu Asp Thr Asp Ile Phe Arg Leu Gln Thr Leu Gln
325 330 335
His Ile Met Arg Met Thr Arg Thr Trp Asp Ser Thr Met Ser Met Leu
340 345 350
Pro Lys Gly Gly Asp Thr Ile Trp Gly Gly Leu Asp Trp Ser Pro Glu
355 360 365
Lys Gly His Thr Cys Cys Gly Lys Lys Gln Lys Arg Asn Glu Thr Cys
370 375 380
Gly Glu Ala Gly Glu Asn Gly Val Ser Lys Ile Lys Pro Val Asn Tyr
385 390 395 400
Gly Arg Met Ile Ser Phe Gly Lys Asp Ile Ala Glu Ala Ala Ser Ser
405 410 415
Glu Ile Asn Asn Ile Asp Phe Arg Gly Ala Val Lys Gly Gln Ser Phe
420 425 430
Pro Asn His Thr Cys Arg Asp Val Trp Thr Glu Tyr His Asp Met Gly
435 440 445
Ile Ala Gly Ile Lys Ala Ile Ala Glu Tyr Lys Val Tyr Thr Ala Asp
450 455 460
Ala Val Ile Asp Leu Leu His Tyr Val Ala Pro Lys Met Met Ala Arg
465 470 475 480
Gly Ser Ala His Phe Ser Tyr Gly Ile Ala Asp Asp Leu Asp Asp Pro
485 490 495
Lys Tyr Lys Asp Pro Lys Tyr Trp Ser Asn Pro Leu Glu Thr Lys Leu
500 505 510
Pro Asn Ala Pro Glu Met Glu Ile Phe Ser Leu Tyr Gly Val Gly Ile
515 520 525
Pro Thr Glu Arg Ala Tyr Val Tyr Lys Leu Asn Gln Ser Pro Asp Ser
530 535 540
Cys Ile Pro Phe Gln Ile Phe Thr Ser Ala His Glu Glu Asp Glu Asp
545 550 555 560
Ser Cys Leu Lys Ala Gly Val Tyr Asn Val Asp Gly Asp Glu Thr Val
565 570 575
Pro Val Leu Ser Ser Gly Tyr Met Cys Ala Lys Ala Trp Arg Gly Lys
580 585 590
Thr Arg Phe Asn Pro Ser Gly Ile Lys Thr Tyr Val Arg Glu Tyr Asn
595 600 605
His Ser Pro Pro Ala Asn Leu Leu Glu Gly Arg Gly Thr Gln Ser Gly
610 615 620
Ala His Val Asp Ile Met Gly Asn Phe Ala Leu Ile Glu Asp Ile Met
625 630 635 640
Arg Val Ala Ala Gly Gly Asn Gly Ser Asp Ile Gly His Asp Gln Val
645 650 655
Tyr Ser Gly Ile Tyr Glu Trp Ser Asp Arg Ile Asp Leu Lys Leu
660 665 670
<210> 6
<211> 662
<212> PRT
<213> 人工序列
<400> 6
Met Pro Pro Leu Leu Arg Phe Arg Lys Leu Ser Phe Ser Glu Asp Thr
1 5 10 15
Val Asn Pro Thr Ala Lys Gln Gln Thr Thr Thr Glu Lys Pro Lys Arg
20 25 30
Arg Arg Ala Gly Arg Cys Ser Cys Val Asp Ser Cys Cys Trp Ala Ile
35 40 45
Gly Tyr Leu Cys Thr Ala Trp Trp Leu Leu Leu Phe Leu Tyr His Ser
50 55 60
Val Pro Val Pro Ala Met Leu Gln Ala Pro Glu Ser Pro Gly Thr Arg
65 70 75 80
Leu Ser Arg Asp Gly Val Lys Ala Leu His Pro Val Ile Leu Val Pro
85 90 95
Gly Ile Val Thr Gly Gly Leu Glu Leu Trp Glu Gly Arg Pro Cys Ala
100 105 110
Glu Gly Leu Phe Arg Lys Arg Leu Trp Gly Ala Ser Phe Thr Glu Ile
115 120 125
Leu Arg Arg Pro Leu Cys Trp Leu Glu His Leu Ser Leu Asp Ser Glu
130 135 140
Thr Gly Leu Asp Pro Pro Gly Ile Arg Val Arg Ala Val Pro Gly Leu
145 150 155 160
Val Ala Ala Asp Tyr Phe Val Pro Cys Tyr Phe Ala Trp Ala Val Leu
165 170 175
Ile Glu Asn Leu Ala Lys Ile Gly Tyr Glu Gly Lys Asn Leu His Met
180 185 190
Ala Ser Tyr Asp Trp Arg Leu Ser Phe His Asn Thr Glu Val Arg Asp
195 200 205
Gln Ser Leu Ser Arg Leu Lys Ser Lys Ile Glu Leu Met Tyr Ala Thr
210 215 220
Asn Gly Tyr Lys Lys Val Val Val Val Pro His Ser Met Gly Ala Ile
225 230 235 240
Tyr Phe Leu His Phe Leu Lys Trp Val Glu Thr Pro Leu Pro Asp Gly
245 250 255
Gly Gly Gly Gly Pro Gly Trp Cys Ala Lys His Val Lys Ala Val Val
260 265 270
Asn Ile Gly Pro Ala Phe Leu Gly Val Pro Lys Ala Val Ser Asn Leu
275 280 285
Leu Ser Ala Glu Gly Lys Asp Met Ala Tyr Ala Arg Ser Leu Ala Pro
290 295 300
Gly Leu Leu Asp Ser Glu Leu Leu Lys Leu Gln Thr Leu Glu His Leu
305 310 315 320
Met Arg Met Ser His Ser Trp Asp Ser Ile Val Ser Leu Leu Pro Lys
325 330 335
Gly Gly Glu Ala Ile Trp Gly Asp Leu Asp Ser His Thr Lys Glu Gly
340 345 350
His Ser Phe Ile Asp Ser Lys Arg Lys Leu Ser Gln Ser Asn Leu His
355 360 365
Arg Gln Asn Asp Ser Val Arg Pro Glu Ser Trp Val Lys Glu Pro Ala
370 375 380
Lys Tyr Gly Arg Phe Ile Ala Phe Gly Lys Arg Ala Ser Glu Val Pro
385 390 395 400
Ser Ala Gln Leu Asn Pro Leu Asn Ile Lys Glu Leu Leu Arg Ser Asp
405 410 415
Val Ile Ser Asn Asp Ser Thr Ser Cys Arg Gln Phe Trp Ser Glu Tyr
420 425 430
Ser Glu Ile Ser Arg Glu Ser Ile Val Lys Val Ala Glu Asn Thr Ala
435 440 445
Tyr Thr Ala Thr Thr Val Leu Asp Leu Leu Arg Phe Ile Ala Pro Lys
450 455 460
Met Met Lys Arg Ala Glu Ala His Phe Ser His Gly Ile Ala Glu Asp
465 470 475 480
Leu Asp Asp Pro Lys Tyr Gly His Tyr Lys Tyr Trp Ser Asn Pro Leu
485 490 495
Glu Thr Lys Leu Pro Asp Ala Pro Glu Met Glu Met Tyr Ser Leu Tyr
500 505 510
Gly Val Gly Ile Pro Thr Glu Arg Ser Tyr Ile Tyr Lys Leu Thr Thr
515 520 525
Thr Pro Ser Ser Lys Cys Lys Ser Ser Ile Pro Phe Arg Ile Asp Gly
530 535 540
Ser Val Asp Gly Asp Asp Asp Ile Tyr Leu Lys Gly Gly Thr Arg Phe
545 550 555 560
Val Asp Gly Asp Glu Ser Val Pro Val Ile Ser Ala Gly Phe Met Cys
565 570 575
Ala Lys Gly Trp Arg Gly Lys Thr Arg Phe Asn Pro Ser Gly Met Glu
580 585 590
Thr Tyr Val Arg Glu Tyr Lys His Lys Pro Pro Gly Ser Leu Leu Glu
595 600 605
Ser Arg Gly Met Glu Ser Gly Ser His Val Asp Ile Met Gly Asn Val
610 615 620
Gly Leu Ile Glu Asp Val Leu Arg Ile Ala Ser Gly Ala Ser Gly Gln
625 630 635 640
Glu Ile Gly Gly Asp Arg Val Tyr Ser Asn Val Met Lys Met Ser Glu
645 650 655
Thr Val Ser Ile Lys Leu
660
<210> 7
<211> 22
<212> DNA
<213> 人工序列
<400> 7
aagcttatgc ctcttattca tc 22
<210> 8
<211> 21
<212> DNA
<213> 人工序列
<400> 8
ggatccagtg tcgaagtcca g 21
<210> 9
<211> 19
<212> DNA
<213> 人工序列
<400> 9
caagcttatg cccgccagc 19
<210> 10
<211> 21
<212> DNA
<213> 人工序列
<400> 10
ggatccagtg tcgaagtcca g 21
<210> 11
<211> 22
<212> DNA
<213> 人工序列
<400> 11
caagcttatg cctcttattc ac 22
<210> 12
<211> 21
<212> DNA
<213> 人工序列
<400> 12
ggatccagtg tcgaagtcca g 21
<210> 13
<211> 21
<212> DNA
<213> 人工序列
<400> 13
aagcttatgc ctccacttct c 21
<210> 14
<211> 21
<212> DNA
<213> 人工序列
<400> 14
cggatccagt gttgaattac g 21
<210> 15
<211> 21
<212> DNA
<213> 人工序列
<400> 15
aagcttatgc ctccacttct c 21
<210> 16
<211> 20
<212> DNA
<213> 人工序列
<400> 16
ggatccagtg ttgaactacg 20
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