专利名称：花生同质型乙酰辅酶a羧化酶基因及其编码的蛋白质与克隆方法
技术领域：
本发明涉及一种基因工程技术领域，具体涉及花生同质型乙酰辅酶A羧化酶基因及其编 码的蛋白质以及基因的克隆方法。
背景技术：
乙酰辅酶A羧化酶(Acetyl-CoA Carboxylase, ACCase)属于生物素包含酶，它在生物 体内催化乙酰辅酶A羧化形成丙二酰辅酶A，为脂肪酸和许多次生代谢产物的合成提供底物 (Konishi T, Shinohara K， Yamada K. Acetyl-CoA carboxylase in higher plants: most plants other than gramineae have both the prokaryotic and the eukaryotic forms of this enzyme[J]. Plant Cell Physiol' 1996, 37(1): 17-122.)。
生物体中ACCase有2种类型。一种是异质型(Heteromeric)，也称多亚基或原核型ACCase， 存在于细菌及双子叶植物和非禾本科单子叶植物的细胞质中。异质型ACCase包含4个亚基， 即牛物素羧化酶(Biotin carboxylase, BC)、生物素羧基载体蛋白(Biotin carboxyl Carrier Protein, BCCP)以及羧基转移酶的2个亚基a -CT和P -CT，其中前2个亚基组成BC和BCCP 域，后2个亚基构成CT催化域。另一种ACCase称为同质型(Homomeric)，亦称多功能或真 核型，存在于动物、酵母、藻类及植物的胞质溶胶中，具有一个相对分子量为22(T260的生 物素包含亚基。它的一条多肽链包含着原核型ACCase所有的四个亚基，排列顺序是BC、 BCCP、 CTP、 CTci ，形成了三个功能域(CT0和CTa算一个功能域)。活性状态下的真核型ACCase 呈现出同型二聚体。
这两种同工型ACCase在植物中的定位有两个例外。 一个是油菜的叶绿体中可能同时包含 两禾中同工型ACCase (Schulte W， Topfer R, Stracke R, et al. Multi—functional acetyl—CoA carboxylase from Brassica napus encode by a multi-gene family: indication for p丄astidic calization of at least one isofonu. Proc Natl Acad Sci USA, 1997, 94: 3465-3470)。另一例外是禾本科植物，它们的质体和胞质溶胶中的ACCase都属于真核类型 (Gengenbach. Transgenic plants expressing maize acetyl CoA carboxylase gene andmethod of altering oil content. United States Patent, 6,222,099， 2001—4—24)。
目前已从拟南芥、小麦、油菜、大豆、玉米和苜蓿等多种植物中获得ACCase基因(K R Roesler， B S Shorrosh， and J B Ohlrogge. Structure and expression of an Arabidopsis acetyl-coenzyme A carboxylase gene. Plant Physiol. 1994， 105(2): 611-617; SergeiReverdatto, Vadim Beilinson, and Niels C. Nielsen. A Multisubunit Acetyl Coenzyme A Carboxylase from Soybean. Plant Physiol. 1999, 119 (3) : 961-978; WSchulte, JSchell, and R T6pfer. A gene encoding acetyl-coenzyme A carboxylase from Brassica napus. Plant Physiol. 1994, 106(2): 793-794.)。但花生中尚没有此基因的相关报道。
发明内容
本发明的目的是克服现有技术的不足，提供一种花生同质型乙酰辅酶A羧化酶基因及其 编码的蛋白质及基因的克隆方法。使该基因在花生中获得较好的表达，以改良花生的品质。
本发明的花生同质型乙酰辅酶A羧化酶基因，其序列由SEQ ID NO. 1中从核苷酸第172 —6954位的核苷酸序列构成。所述基因来自花生(力rsc/u's A^o^aes)，命名为屈^Kk e基 因。
本发明第二个目的是提供所述基因编码的蛋白质，具有序列表中SEQ ID NO. 2所不的氨 基酸序列。所述蛋白质命名为AhACCase蛋白质。
同时，本发明还提供所述基因的克隆方法，包括如下步骤-
(1) 提取花生幼苗中的总RNA;
(2) 利用Promega的RT-PCR体系反转录获得cDNA;
(3) 根据cDNA文库中的EST信息设计引物
(4) 进行PCR扩增，并将PCR产物回收后筛选阳性克隆；
(5) 对筛选到的阳性克隆进行序列分析，获得同质型乙酰辅酶A羧化酶基因。 由于同质型乙酰辅酶A羧化酶是一个大的多功能域酶，在一条多肽链上同时包含BC，
BCCP， CT-a和CT-P四个与原核型ACCase相对应的功能域，单体分子量在200kD以上，该 酶活性的提高有利于提高花生种子的含油量。本发明公开的花生同质型乙酰辅酶A羧化酶 "/^0:ase)基因的核苷酸序列及其编码的蛋白序列以及基因克隆方法，可广泛应用于花生 品质的改良、花生的栽培和培养，尤其是花生作为油料作物的开发和利用等领域。
图1是本发明的蛋白质的结构域示意图。
具体实施例方式
下面结合具体实例对本发明作进一步详细的描述。
实施例l克隆花生同质型乙酰辅酶A羧化酶(^^Ofase)基因(1) 选用E12花生幼苗作为实验材料，釆用Pbiozol植物组织RNA提取试剂盒提取花生 幼苗中的总RNA。 RNA含量及质量可采用琼脂糖凝胶电泳检测。
(2) 利用Promega的RT-PCR体系反转录获得cDNA。
(3) 根据cDNA文库中的EST信息设计花生同质型乙酰辅酶A羧化酶基因编码区引物，其
中
正向引物(5' - GAGAACGAGAATGGCTGGTGTT -3') 反向引物(5， - ACAGGTATGCTTGCCTTCTAAC -3，)，
反应体系2. 5 ul 10XPCR缓冲液(含MgClJ， 1.0 ul 10 uM的引物，4.0 u 1 2. 5 mM的dNTPs， 1 ul cDNA样品，0. 5 u 1 La-Taq酶(Takara)， 15 ul双蒸水。PCR反应程 序为95° C预变性5 min， 95。C变性30s， 56。C复性30s， 72。C延伸7 min， 28个循环后， 72 。C延伸10min。
(4) 采用Takara的La-Taq酶进行PCR扩增，PCR产物回收后与MD18-T simple vector (Takara)连接，连接产物转化A cWi Top IO感受态细胞，采用蓝白斑法筛选阳性克隆。
(5) 筛选的阳性克隆经PCR进一步验证后测序，获得同质型乙酰辅酶A羧化酶基因。
实施例2 JA4CCa e蛋白质的序列信息与特性分析
本发明的AWO:a e基因长7315bp，其开放阅读框为6783bp，位于172_6954bp处。BioXM 软件分析表明舶/!6rase共编码2260个氨基酸。根据PR0SITR数据库分析花生^^6Tase基因 编码的2260个氨基酸，发现此序列含Biotin carboxylation domain profile (PS50979, 38-545aa) ; ATP-grasp domain profile (PS50975， 191-383aa) ; Biotinyl/lip。yl domain profile(PS50968'671—745aa);Acetyl—coenzyme A carboxy丄transferase domain N—terminal region profile (PS50980, 162H800aa) ; Acetyl-coenzyme A carboxyltransferase domain C-terminal region profile (PS50989, 1801-2115aa) ; Carbamoyl-phosphate synthase subdomain signature 2 (PS00867 ， 352-359aa) ; ABC transporters family signature (PS00211， 488-502aa) ; Biotin-requiring enzymes attachment site (PS00188, 703-720aa) ; Peroxidases active site signature (PS00436, 1086-1097aa)，如图1所 示。
用Protparam预测编码蛋白的物理化学性质，推测分子式为CU225H17749N31。703337S8。，分子量 为252187.0，等电点为6.03，理论推导半衰期为30h，不稳定参数是40. 18，属于不稳定蛋 白。该蛋白中含量相对较多的氨基酸是Leu (9.9%)， Ala (8.1%)， Glu (7.7%)， Val (7.4%)， Gly (7.0%);该蛋白中不含Pyl和Sec。总的带正电荷的残基为(Arg + Lys) : 251;总的带负电荷的残基为(Asp + Glu) :285。该蛋白亲水性平均数为-O. 259，预测该蛋白为疏水性蛋白。 采用iPSORT进行预测，表明该蛋白不含信号肽序列，为非分泌性蛋白。以Tmpred网站为基 础的跨膜结构预测表明该蛋白含5个跨膜信号区。采用在线HNN网站预测花生AhACCase蛋白 的二级结构，表明该蛋白含有43.98%的a-螺旋，14.47%的e -折叠以及41. 55%的无规则巻 曲。
本发明涉及的序列及记号分列如下: (l)SEQ ID NO. 1的信息
(i) 序列特征
(A) 长度7315bp
(B) 类型核苷酸
(C) 链性:单链
(D) 拓扑结构线性
(ii) 分子类型核苷酸
(iii) 序列描述SEQ ID NO. 1
(2)SEQ ID NO. 2的信息
(i) 序列特征
(A) 长度2260aa
(B) 类型氨基酸
(C) 链性:单链
(D) 拓扑结构线性
(ii) 分子类型蛋白质
(iii)序列描述SEQ ID NO. 2序列表
〈110>山东省花生研究所
〈120〉花生同质型乙酰辅酶A羧化酶基因及其编码的蛋白质与克隆方法 〈160> 2
<170> Patentln version 3. 3
〈210> 1 <211> 7315 <212> 腿
〈213> 花生(Arachis hypogaea) <220>
<221> CDS
<222> (172).. (6954) <400> 1
gggcatgatc atctcttctc ttcccttaat cacttctcac cctcccaaat gcttccctaa 60
ctcccaccaa ttttcgttat taccaaactc cacaacgcaa aaattcctca tttcttaacc 120
ceicacactcc catcactg邻cacataggac ttaaatagaa ggagaacgag a atg get 177
Met Ala 1
ggt gtt ggg cgt gga aat gga tac aca aat ggt gtg gta cct aac agg 225 Gly Val Gly Arg Gly Asn Gly Tyr Thr Asn Gly Val Val Pro Asn Arg 5 10 15
cac cct get aca ata tct gaa gta gat gaa tac tgc aat gca ctt ggg 273 His Pro Ala Thr lie Ser Glu Val Asp Glu Tyr Cys Asn Ala Leu Gly 20 25 30
gga aca agg cca att cat agt ata tta att gca aac aat gga atg gcc 321 Gly Thr Arg Pro lie His Ser lie Leu lie Ala Asn Asn Gly Met Ala 35 40 45 50
gca gtc aag ttt ata cgc agt gtg agg age tgg get tat gag acg ttt 369 Ala Val Lys Phe lie Arg Ser Val Arg Ser Trp Ala Tyr G丄u Thr Phe 55 60 65
ggt acg gag agg get ate ttg ttg gtt gcc atg get act cca gag gac 417 Gly Thr Glu Arg Ala lie Leu Leu Val Ala Met Ala Thr Pro Glu Asp7G 75 80
atg aga atc aat gca gaa cat atc aga ata gcc gat caa ttt gtc gaa 465 Met Arg lie Asn Ala Glu His lie Arg lie Ala Asp Gin Phe VaJ Glu 85 90 95
gta cct ggt ggg acc aat aac aat aac tat gcc aat gtg cag ctt att 513 Val Pro Gly Gly Thr Asn Asn Asn Asn Tyr Ala Asn Val Gin Leu lie 100 105 110
gta gag atg get gag ata act egg gtt gat get gtg tgg cca ggt tgg 561 Val Glu Met Ala Glu lie Thr Arg Val Asp Ala Val Trp Pro Gly Trp 115 120 125 130
ggt cat gca tea gaa aat cct gag ctt cca gat gcg tta aaa gca aaa 609 Gly His Ala Ser Glu Asn Pro Glu Leu Pro Asp Ala Leu Lys Ala Lys 135 140 145
gga att gta ttt ctt gga cct cca get gta tct atg gca gca ctg gga 657 Gly lie Val Phe Leu Gly Pro Pro Ala Val Ser Met Ala Ala Leu Gly 150 155 160
gac aaa att ggt tea tea ttg att get caa gca gca gaa gtg cca acc 705 Asp Lys lie Gly Ser Ser Leu lie Ala Gin Ala Ala G〗u Val Pro Thr 165 170 175
ctt cca tgg agt ggt tct cat gtg aaa att cct ccc gat agt tgc ttg 753 Leu Pro Trp Ser Gly Ser His Val Lys lie Pro Pro Asp Ser Cys Leu 180 185 190
gtt act att cct gat gaa att tac egg gaa gca tgt gtt tat aca aca 801 Val Thr lie Pro Asp Glu lie Tyr Arg Glu Ala Cys Val Tyr Thr Thr 195 200 205 210
gaa gaa gca att gcc agt tgt caa gtt gtc ggt tac cct gca atg atc 849 Glu Glu Ala lie Ala Ser Cys Gin Val Val Gly Tyr Pro Ala Met lie 215 220 225
aaa gca tct tgg ggt ggt ggt ggt aaa ggc ate aga aag gtt cat aat 897 Lys Ala Ser Trp Gly Gly Gly Gly Lys Gly lie Arg Lys Val His Asn 230 235 240
gat gat gag gta agg gca ttg ttc aag caa gtc caa ggt gaa gtt ccg 945 Asp Asp Glu Val Arg Ala Leu Phe Lys Gin Val Gin Gly Glu Val Pro 245 250 255ggc tea cct ata ttt Gly Ser Pro lie Phe 260
at8 atg aag gtt gcc tec cag age cga cat eta lie Met Lys Val Ala Ser Gin Ser Arg His Leu 265 270
gaa gtc cag tta ctt Glu Val Gin Leu Leu 275
tgt gat cag tat gga aat gtt gca gcc ttg cat Cys Asp Gin Tyr Gly Asn Val Ala Ala Leu His 280 285 290
1041
age cgt gat tgc agt Ser Arg Asp Cys Ser 295
gtt caa agg agg cac caa aag att att gaa gag Val Gin Arg Arg His Gin Lys lie lie Glu Glu 300 305
1089
ggt ccc att act gta Gly Pro lie Thr Val 310
get cct cca caa acg gtg aaa caa eta gaa ca_g Ala Pro Pro Gin Thr Val Lys Gin Leu Glu Gin 315 320
1137
gcEi get aga邻g ttg Ala Ala Arg Arg Leu 325
get aaa tct gta aat tat gtt ggg get get act Ala Lys Scr Val Asn Tyr Val G丄y A丄a Ala Thr 330 335
1185
gtt gag tat ctt ttc Val Glu Tyr Leu Phe 340
agt atg gaa act ggc gag tac tac ttt ttg gaa Ser Met Glu Thr Gly G丄u Tyr Tyr Phe Leu Glu 345 350
1233
ttg aac cct cga eta Leu Asn Pro Arg Leu 355
cag gtt gag cac cct gtt act gag tgg ata gcg Gin Val Glu His Pro Val Thr Glu Trp lie Ala 360 365 370
1281
gag ata aat ctg cca Glu lie Asn Leu Pro 375
gca gca caa gtt gca att ggg atg ggt ate cct Ala Ala Gin Val Ala lie Gly Met Gly lie Pro 380 385
1329
Ctt tgg C33 Ctt CCt
Leu Trp Gin Leu Pro 390
gaa sta 8g3 cgt ttc t3t ggg gtg gaa cat ggt Glu lie Arg Arg Phe Tyr Gly Va丄Glu His Gly 395 400
1377
ggg ggg aat gat get Gly Gly Asn Asp Ala 405
tgg agg aaa aca tea get ttg get acc cct ttt Trp Arg Lys Thr Ser Ala Leu Ala Thr Pro Phe 410 415
1425
gat ttt gac aaa gca Asp Phe Asp Lys Ala 420
caa tec aca aag cca aaa ggt cac tgt gtg get Gin Ser Thr Lys Pro Lys Gly His Cys Val Ala 425 430
1473
gtg cga gtg aca agt gag gac cct gat gat ggt ttc aag cct aca agt Val Arg Val Thr Ser Glu Asp Pro Asp Asp Gly Phe Lys Pro Thr Ser
1521435
440
445
450
ggg 338 gtg csg gag ctt age ttt aaa age aag cca ast gtg tgg gca Gly Lys Val Gin Glu Leu Ser Phe Lys Ser Lys Pro Asn Val Trp Ala 455 460 465
1569
tac ttc tct gtt aag tct gga gga gga at3 cac gag ttt tea gat tct Tyr Phe Ser Val Lys Ser Gly Gly Gly lie His Glu Phe Ser Asp Ser 470 475 480
1617
cag ttt ggg cat gtt ttt get ttt gga gaa tct agg get tta get sta Gin Phe Gly His Val Phe Ala Phe Gly Glu Ser Arg Ala Leu Ala lie 485 490 495
1665
gC3 aat 3tg gtt Ct3 ggg Ct3 33g g3g 3tt C33 3tt Cgt gg3 g3g 3tt
Ala Asn Met Val Leu Gly Leu Lys Glu lie Gin lie Arg Gly Glu lie 500 505 510
1713
cgt acc aat gtt gat tat acc att gat ctt ctg aat get tea gac tac Arg Thr Asn Val Asp Tyr Thr lie Asp Leu Leu Asn Ala Ser Asp Tyr 515 520 525 530
1761
aga gac aac aaa att cac acg gga tgg ctg gac 3gt aga att gcg atg Arg Asp Asn Lys lie His Thr Gly Trp Leu Asp Ser Arg lie Ala Met 535 540 545
1809
agg gtt卿gca gag agg cct ccc tgg tat ctt tct gtt gtt gga ggg Arg Val Arg Ala Glu Arg Pro Pro Trp Tyr Leu Ser Val Val Gly Gly 550 555 560
1857
gcc etc tat aaa get tct gca age agt gca get ctg gta tea gac tat Ala Leu Tyr Lys Ala Ser Ala Ser Ser Ala Ala Leu Val Ser Asp Tyr 565 570 575
1905
gtt ggc tat ctg gaa aag gga caa ate cct ccc aag cat ata tct ctt Val Gly Tyr Leu Glu Lys Gly Gin lie Pro Pro Lys His lie Ser Leu 580 585 590
1953
gtg cat tct caa gtg tec ttg aac att gaa gga age aaa tac acg att Val His Ser Gin Val Ser Leu Asn lie Glu Gly Ser Lys Tyr Thr lie 595 600 605 610
2001
gac atg gta cga gga ggg tct gga agt tat aga ttg aga atg aat caa Asp Met Val Arg Gly Gly Ser Gly Ser Tyr Arg Leu Arg Met Asn Gin 615 620 625
2049tcc g犯gta g犯get g8g 3t3 ca^t set tta cgt ga<t gga< ggt ttg ctg 2097 Ser Glu Val Glu Ala Glu lie His Thr Leu Arg Asp Gly Gly Leu Leu 630 635 640
atg cag ttg g3t gga犯c 3gt cat gtt a<ta tat gca gag gaa gaa get 2145 Met Gin Leu Asp Gly Asn Ser His Val lie Tyr Ala Glu Glu Glu Ala 645 650 655
get gga acg cgc ctt eta att gat gga agg act tgc ttg ctt cag aat 2193 Ala Gly Thr Arg Leu Leu lie Asp Gly Arg Thr Cys Leu Leu Gin Asn 660 665 670
gat cat gat cca teg aag tta gtt gca gag aca cca tgc aag ctt atg 2241 Asp His Asp Pro Ser Lys Leu Val Ala Glu Thr Pro Cys Lys Leu Met 675 680 685 690
agg tat ttg gtt gta gat gac age cat att gat get gac aca cct tat 2289 Arg Tyr Leu Val Val Asp Asp Ser His lie Asp Ala Asp Thr Pro Tyr 695 700 705
get gaa gtt gaa gtc atg aag atg tgc atg cca ctt ctt tea cct get 2337 Ala Glu Val Glu Val Met Lys Met Cys Met Pro Uu Leu Scr Pro Ala 710 715 720
tct ggg gtt att cat ttc aaa atg tct gaa ggt caa ccg atg cag get 2385 Ser Gly Val lie His Phe Lys Met Ser Glu Gly Gin Pro Met Gin Ala 725 730 735
ggt gaa eta ata gca agg ctt gat eta gat gat cct tea gca gta aga 2433 Gly Glu Leu lie Ala Arg Leu Asp Leu Asp Asp Pro Ser A丄a Va丄Arg 740 745 750
aag gca gaa ccc ttc sat gga aaa ttc cca gtc ctg ggc cca ccc act 2481 Lys Ala Glu Pro Phe Asn Gly Lys Phe Pro Val Leu Gly Pro Pro Thr 755 760 765 770
gca act tct gat犯a gtt cat cag aaa tgt get gca age tta agt get 2529 Ala Thr Ser Asp Lys Val His Gin Lys Cys Ala Ala Ser Leu Ser Ala 775 780 785
gca cag atg att ctt get ggt tat gag cac aat att gat gag gtt gtg 2577 Ala Gin Met lie Leu Ala Gly Tyr Glu His Asn lie Asp Glu Val Val 790 795 800
caa agt ttg etc aat tgc ctt gat agt cct gaa tta cct ttc ctt caa 2625 Gin Ser Leu Leu Asn Cys Leu Asp Ser Pro Glu Leu Pro Phe Leu Gin805
810
815
tgg caa gag tgc ttt gca gtt eta gca aa_c cgt Trp Gin Glu Cys Phe Ala Val Uu Ala Asn Arg 820 825
ctt ccc aaa gat ctg Leu Pro Lys Asp Leu 830
2673
saa aat gag ttg g肪teg aaa tat aag gag tax; Lys Asn Glu Leu Glu Ser Lys Tyr Lys Glu Tyr 835 840 845
gag agg att tea age Glu Arg lie Ser Ser 850
2721
ttc caa gtt gtt gat ttc cct gcc aaa ctt ttg Phe Gin Val Val Asp Phe Pro Ala Lys Leu Leu 855 860
aag gga att ctt gaa Lys Gly lie Leu Glu 865
2769
get ca_t ttg tec tea tgt ccc aac aa_a gaa_ aaa Ala His Leu Ser Ser Cys Pro Asn Lys Glu Lys 870 875
ggg get c肪g肌鄉 Gly Ala Gin Glu Arg 880
2817
ctg att gaa cct ctg ttg agt ctt gtg aag tec Leu lie Glu Pro Leu Leu Ser Leu Val Lys Ser 885 890
tat gag ggt gg;a卿 Tyr Glu Gly Gly Arg 895
2865
gag age cat get cgt aaa att gtx caa tec ctt Glu Ser His Ala Arg Lys lie Val Gin Ser Leu 900 905
ttt gaa gag ta_t ctt Phe Glu Glu Tyr Leu 910
2913
ttt gtt gaa gaa eta ttt 3gt gat 33t sta csg Phe Val Glu Glu Leu Phe Ser Asp Asn lie Gin 915 920 925
get gat gta att gaa Ala Asp Val lie Glu 930
2961
cgt etc cgt ctt caa tat aag aaa gat ctg ttg Arg Leu Arg Leu Gin Tyr Lys Lys Asp Leu Leu 935 940
aag 3tt gtg gat 3t3 Lys lie Val Asp lie 945
3009
gtt etc tea cat c;ag ggt ate aag a_gc幼a aat Val Leu Ser His Gin Gly lie Lys Ser Lys Asn 950 955
aag ctg ata eta cga Lys Leu lie Leu. Arg 恥O
3057
eta atg gat aaa ctg gtt tac cca aat cct get Leu Met Asp Lys Leu Val Tyr Pro Asn Pro Ala 965 970
gcc tac agg gat caa Ala Tyr Arg Asp Gin 975
3105
tta ate cgc ttc tct caa etc aac cat act aac Leu lie Arg Phe Ser Gin Leu Asn His Thr Asn 980 985
tat tct cag ttg gcc Tyr Ser Gin Leu Ala 990
3153cta幼g gca agt caa ttg ctg gaa caa act aaa ttg agt gaa ctt 3198 Leu Lys Ala Ser Gin Leu Leu Glu Gin Thr Lys Leu Ser Glu Leu 995 1000 1005
cga tec aac att get aga agt ctt tct gag tta gag atg ttc act 3243 Arg Ser Asn工le Ala Arg Ser Leu Ser Glu Leu Glu Met Phe Thr 1010 1015 1020
gag gat ggt gaa aat att gat act ccc犯g agg aaa age get att 3288 Glu Asp Gly Glu Asn lie Asp Thr Pro Lys Arg Lys Ser Ala lie 1025 1030 1035
aat gac cga atg gag gac ctt gtt agt get cct ttg gca gtt gaa 3333 Asn Asp Arg Met Glu Asp Leu Val Ser Ala Pro Leu Ala Val Glu 1040 1045 1050
gat get ttg gtg ggt tta ttt gat cac agt gat cac acc ctt caa 3378 Asp Ala Leu Val Gly Leu Phe Asp His Ser Asp His Thr Leu Gin 1055 1060 1065
aga 3gg gtt gtg gaa act tat ata cgcetc t￡ic cag ccg tat 3423 Arg Arg Val Val Glu Thr Tyr lie Arg Arg Leu Tyr Gin Pro Tyr 1070 1075 1080
ctt gtc aaa ggg agt gtc aga atg cag tgg cac aga tct ggt ctt 3468 Leu Val Lys Gly Ser Val Arg Met Gin Trp His Arg Ser Gly Leu 1085 1090 1095
att get tea tgg gag ttc tta gaa gag tac att gaa agg aag agt 3513 lie Ala Ser Trp Glu Phc Leu Glu Glu Tyr lie Glu Arg Lys Ser 1100 1105 1110
ggg gtt gaa gac caa atg tea gat aaa acg ctg gtg gag aaa cac 3558 Gly Val Glu Asp Gin Met Ser Asp Lys Thr Leu Val Glu Lys His 1115 1120 1125
act gag犯a aaa tgg ggc gta atg gtt gta att tct ctt cac 3603
Thr Glu Lys Lys Trp Gly Val Met Val Val lie Lys Ser Leu His 1130 1135 1140
ttt ttg cct gca att ate act get gcg tta aag geia gca acc aac 3648 Phe Leu Pro Ala lie lie Thr Ala Ala Leu Lys Glu Ala Thr Asn 1145 1150 1155
aat ctt cat gaa gca gtt tea agt get get ggt gaa cca gtt aag 3693 Asn Leu His Glu Ala Val Ser Ser Ala Ala Gly Glu Pro Val Lys1160 1165 1170
cat ggt aat atg atg cat gtt gca tta gtg ggc ate aac aac cag 3738 His Gly Asn Met Met His Val Ala Leu Val Gly lie Asn Asn Gin 1175 1180 1185
atg agt tta ctg caa gac agt ggt gac gag gat cag get caa gaa 3783 Met Ser l>eu Leu Gin Asp Ser Gly Asp Glu Asp Gin Ala Gin Glu 1190 1195 1200
aga a/tx aat aag ttg gcg aaa ata eta aaa geta gag gaa gta ggc 3828 Arg lie Asn Lys Leu Ala Lys lie Leu Lys Glu Glu Glu Val Gly 1205 1210 1215
tec act ate cga ggt act ggt gtt gga gta att age tgt ate ata 3873 Ser Thr lie Arg Gly Thr Gly Val Gly Val lie Ser Cys lie lie 1220 1225 1230
cag agg g^t g犯ggg cgt sec ccg a^tg sgg cac tec ttt cac tgg 3918 G]n Arg Asp Glu Gly Arg Thr Pro Met Arg His Ser Phe His Trp 1235 1240 1245
tea gca gaa aag etc tat tet; cag gag gaa cct ctg ttg cgt cat 3963 Ser Ala Glu Lys Leu Tyr Tyr Gin Glu Glu Pro Leu Leu Arg His 1250 1255 1260
ttg gaa cca ccg eta tec att tat ctt gaa ttg gac aaa ctt aaa, 4008 Leu Glu Pro Pro Leu Ser lie Tyr Leu Glu Leu Asp Lys Leu Lys 1265 1270 1275
ggc tat gag aac ata egg tat act cct tct cga gat cgt caa tgg 4053 Gly Tyr Glu Asn lie Arg Tyr Thr Pro Ser Arg Asp Arg Gin Trp 1280 1285 1290
cat ctg tac act gtt atg gac caa_ aag cct cm cct get caa aga 4098 His Leu Tyr Thr Val Met Asp Gin Lys Pro Gin Pro Ala Gin Arg 1295 1300 1305
atg ttt ctt cga aca ctt tta aga cag cca acc aca aat gaa gga 4143 Met Phe Leu Arg Thr Leu Leu Arg Gin Pro 丁hr Thr Asn Glu Gly 1310 1315 1320
ttc tct tcg tal caa agg acg gat gca gaa aca cct agt acc gaa 4188 Phe Ser Ser Tyr Gin Arg Thr Asp Ala Glu Thr Pro Ser Thr Glu 1325 1330 1335ttg get atg tec ttc act tea agg age att ttt agg tec ttg atg 4233 Leu Ala Met Ser Phe Thr Ser Arg Ser lie Phe Arg Ser Leu Met 1340 1345 1350
get gca atg gag gag ttg gaa ctt朋t tea cac 朋t gec act ate 4278 Ala Ala Met Glu Glu Leu Glu Leu Asn Ser His Asn Ala Thr lie 1355 1360 1365
aga cct gaa cac get cat atg tac etc tat att ata cgc gag cag 4323 Arg Pro Glu His Ala His Met Tyr Leu Tyr lie lie Arg Glu Gin 1370 1375 1380
gaa ata aat gat ctt gtg cct tat ccc aag aga gtt gac ata gat 4368 Glu lie Asn Asp Leu Val Pro Tyr Pro Lys Arg Val Asp lie Asp 1385 1390 1395
gcg ggc caa gaa gaa aca aca gtt gag gca acc ttg gaa gaa eta 4413 Ala Gly Gin Glu Glu Thr Thr Val Glu Ala Thr Leu Glu Glu Leu 1400 1405 1410
gca cat gaa ate cat tea teg gtt ggt gta aga atg cat aga tta 4458 Ala His Glu lie His Ser Ser Val Gly Val Arg Met His Arg Leu 1415 1420 1425
gga gtt gta gtt tgg gaa gtc aag etc tgg ertg gca gec tgt gca 4503 Gly Val Val Val Trp Glu Val Lys Leu Trp Met Ala Ala Cys Ala 1430 1435 1440
cag gca aat ggc gca tgg agg att gtt gta aac aat gtg aca ggt 4548 Gin Ala Asn Gly Ala Trp Arg lie Val Val Asn Asn Val Thr Gly 1445 1450 1455
cat aca tgc act gta cat ata tac cga gaa atg gaa gat acc aat 4593 His Thr Cys Thr Val His lie Tyr Arg Glu Met Glu Asp Thr Asn 1460 1465 1470
acc cat aga gtg gta tac agt tea ate acc gta aag ggt cca ctg 4638 Thr His Arg Val Val Tyr Ser Ser lie Thr Val Lys Gly Pro Leu 1475 1480 1485
cat ggt gta cct gtg aat gaa act tat caa cct ttg gga gtt att 4683 His Gly Val Pro Val Asn Glu Thr Tyr Gin Pro Leu Gly Val lie 1490 1495 1500
gat cga aaa cgt eta tea gca_ aga aag aac agt acc act ttt tgc 4728 Asp Arg Lys Arg Leu Ser Ala Arg Lys Asn Ser Thr Thr Phe Cys1505
1510
1515
tat gat Tyr Asp 1520
ttc ccc ctg gca Phe Pro Leu Ala 1525
ttt gaa aca gcc ttg gaa cag teg tgg Phe Glu Thr Ala Leu Glu Gin Ser Trp 1530
4773
gca ate caa cag ccg gga ttt cga aga cca aaa gat aaa aat ctg Ala lie Gin Gin Pro Gly Phe Arg Arg Pro Lys Asp Lys Asn Leu 1535 1540 1545
4818
tta aag gta aca gag ctt aga ttt get gac aaa gag ggt agt tgg Leu Lys Val Thr Glu Leu Arg Phe Ala Asp Lys Glu Gly Ser Trp 1550 1555 1560
4863
ggt act cct ctt gtt cct gtg gag cat tct get gga etc aat gat Gly Thr Pro Leu Val Pro Val Glu His Ser Ala Gly Leu Asn Asp 1565 1570 1575
4908
gtt ggc atg gta get tgg ttt atg gac atg tgt acc ccc gaa ttc Val Gly Met Val Ala Trp Phe Met Asp Met Cys Thr Pro Glu Phe 1580 1585 1590
4953
cca tec gga agg aca ata ttg gtt gta gca aat gat gtg aca ttc Pro Ser Gly Arg Thr lie Uu Val Val Ala Asn Asp Val Thr Phe 1595 1600 1605
4998
aag get ggt tct ttt ggc cct aga gag gat gca ttc ttc cgt gca Lys Ala Gly Ser Phe Gly Pro Arg Glu Asp Ala Phe Phe Arg Ala 1610 1615 1620
5043
gtt act gat ctt gca tgt gca saa aaa ttg cct tta att tat tta Val Thr Asp Leu Ala Cys Ala Lys Lys Leu Pro Leu lie Tyr Leu 1625 1630 1635
5088
gca gca aat tct ggt gcc cgt tta ggt get gcc gag gaa gtc aaa Ala Ala Asn Ser Gly Ala Arg Leu Gly Ala Ala Glu Glu Val Lys 1640 1645 1650
5133
gcc tgt ttt aaa gtt ggt tgg tct gag gag tec aac cct gag cat Ala Cys Phe Lys Val Gly Trp Ser Glu Glu Ser Asn Pro Glu His 1655 1660 1665
5178
ggt ttt cag tat gta tat tta aca cct gag gat ttt get egg att Gly Phe Gin Tyr Val Tyr Leu Thr Pro Glu Asp Phe Ala Arg lie 1670 1675 1680
5223gga tea tct gtg att gca cac gag eta朋g ctt gaa age ggt gaa 5268 Gly Ser Ser Val lie Ala His Glu Leu Lys Leu Glu Ser Gly Glu 1685 1690 1695
acc aga tgg ata ata gat acc att gtt ggg aaa gag gat ggc ctg 5313 Thr Arg Trp工le lie Asp Thr lie Val Gly Lys Glu Asp Gly Leu 1700 1705 1710
ggg gtt gaa aac ttg agt ggt agt gga gec att get ggt tec tac 5358 Gly Val Glu Asn Leu Ser Gly Ser Gly Ala lie Ala Gly Ser Tyr 1715 1720 1725
tea agg gca tac aag gaa act ttc aca tta aca tat gtg act ggt 5403 Ser Arg Ala Tyr Lys Glu Thr Phe Thr Leu Thr Tyr Val Thr Gly 1730 1735 1740
agg act gtt gga ata ggg get tat ctt get agg ctt gga atg aga 5448 Arg Thr Val Gly lie Gly Ala Tyr Leu Ala Arg Leu Gly Met Arg 1745 1750 1755
tgc ata cag agg ctt gat cag cct ata att ctt act ggt ttc tea 5493 Cys lie Gin Arg Leu Asp Gin Pro lie lie Leu Thr Gly Phe Ser 1760 1765 1770
gca eta aac a肌ctt ctt ggt egg gag gte tac age tct cac atg 5538 Ala Leu Asn Lys Leu Leu Gly Arg Glu Val Tyr Ser Ser His Met 1775 1780 1785
caa ctt ggt gga cct aaa ate atg get act aat gga gtt gta cat 5583 Gin Leu Gly Gly Pro Lys lie Met Ala Thr Asn Gly Val Val Ilis 1790 1795 1800
ctt aca gtt tea gat gac ctt gaa ggt gtt tct get: att ttg aag 5628 Leu Thr Val Ser Asp Asp Leu Glu Gly Val Ser Ala lie Leu Lys 1805 1810 1815
tgg ctt age tac a_tt cct tct cat gta ggt ggc tea ctt ccc ate 5673 Trp Leu Ser Tyr lie Pro Ser His Val Gly Gly Ser Leu Pro lie 1820 1825 1830
gta aag ccc ctt gac cct cct gaa aga cca gtg gag tac tta cca 5718 Val Lys Pro Leu Asp Pro Pro Glu Arg Pro Vs] Glu Tyr Leu Pro 1835 1840 1845
gaa aat tct tgt gat cct cgt get get att tct gga act ttg gat 5763 Glu Asn Ser Cys Asp Pro Arg Ala Ala lie Ser Gly Thr Leu Asp1850 1855 1860
ggt aat gga aga tgg etc ggt gga att ttt gac aag gac age ttc 5808 Gly Asn Gly Arg Trp Leu Gly Gly lie Phe Asp Lys Asp Ser Phe 1865 1870 1875
gtg gag aca eta gaa gga tgg gca agg aca gtc gtt aca gga agg 5853 Val Glu Thr Leu Glu Gly Trp Ala Arg Thr Val Val Thr Gly Arg 1880 1885 1890
gca aag ctt gga gga ate cct gtg ggg att gtt get gta gaa aca 5898 Ala Lys Leu Gly Gly lie Pro Val Gly lie Val Ala Val Glu Thr 1895 1900 1905
cag aca gtg atg caa ata ata cct get gat ccg ggc cag ctt gat 5943 Gin Thr Val Met Gin lie lie Pro Ala Asp Pro Gly Gin Leu Asp 1910 1915 1920
tec cat gag aga gtt gtt cct cag get ggc caa gtg tgg ttc cct 5988 Ser His Glu Arg Val Val Pro Gin Ala Gly Gin Val Trp Phe Pro 1925 1930 1935
gat tct get act aaa aca gcc caa gca ata atg ga_t ttc aac aga 6033 Asp Ser Ala Thr Lys Thr Ala Gin Ala lie Met Asp Phe Asn Arg 1940 1945 1950
gaa gag etc cca ctt ttc att eta gca aac tgg aga ggc ttc tct 6078 Glu Glu Leu Pro Leu Phe lie Leu Ala Asn Trp Arg Gly Phe Ser 1955 1960 1965
ggt ggt cag agg gac ctt ttc gaa gga att etc cag get ggt teg 6123 Gly Gly Gin Arg Asp Leu Phe Glu Gly lie Leu Gin Ala Gly Ser 1970 1975 1980
aca att gtg gag aac ctt aga aca tac肪g cag ccc att ttt gta 6168 Thr lie Val Glu Asn Leu Arg Thr Tyr Lys Gin Pro lie Phe Val 1985 1990 1995
tac ate cca atg atg ggt gaa etc cgt ggt gga gca tgg gtt gtc 6213 Tyr lie Pro Met Met Gly Glu Leu Arg Gly Gly Ala Trp Val Val 2000 2005 2010
gtt gac agt egg ate aat tec gac cac att gaa atg tat gcc gac 6258 Val Asp Ser Arg lie Asn Ser Asp His lie Glu Met Tyr Ala Asp 2015 2020 2025aga aca get aaa gga aat gtc ctt gaa cca gaa ggg atg att gag 6303 Arg Thr Ala Lys Gly Asn Val Leu Glu Pro Glu Gly Met lie Ghi 2030 2035 2040
att aag ttt aga Bcei agg gaa ttg ttg g已g tgc atg ggt aga ctt 6348 lie Lys Phe Arg Thr Arg Glu Leu Leu Glu Cys Met Gly Arg Leu 2045 2050 2055
gst cag sag ttg 3t3 set ctg犯g gca< aaa ctt c解gag get aag 6393 Asp Gin Lys Leu lie Thr Leu Lys Ala Lys Leu Gin Glu Ala Lys 2060 2065 2070
gac aag agg gac acc gag tec ttt gaa tct eta cag cag cag att 6438 Asp Lys Arg Asp Thr Glu Ser Phe Glu Ser Leu Gin Gin Gin工le 2075 2080 2085
aaa tct cgc gaa aaa cag ctt ttg cct ctg tat acc cag ata get 6483 Lys Ser Arg Glu Lys Gin Leu Leu Pro Leu Tyi* Thr Gin lie Ala 2090 2095 2100
acc aaa ttt get gaa ctt cat gat act tec tta aga 8tg get get 6528 Thr Lys Phe Ala Glu Leu His Asp Thr Ser Leu Arg Met Ala Ala 2105 2110 2115
犯g ggg gta 3ta ￡igei caa gtt ctg gac tgg ggt aac teg cgc gcc 6573 Lys Gly Val lie Arg Gin Val Leu Asp Trp Gly Asn Ser Arg Ala 2120 2125 2130
gtc ttc tac egg aga ctg tac agg aga att ggt gag cag tea ctt 6618 Val Phe Tyr Arg Arg Leu Tyr Arg Arg lie Gly Glu Gin Ser Leu 2135 2140 2145
ate aac aat gtg aga gaa get get ggt gac cat ttg tea cat gtt 6663 lie Asn Asn Val Arg Glu Ala Ala Gly Asp His Uu Ser His Val 2150 2155 2160
tec gcc atg gac ttg gtc aaa aac tgg tat ttg agt tec aac att 6708 Ser Ala Met Asp Leu Val Lys Asn Trp Tyr Leu Ser Ser Asn lie 2165 2170 2175
gcc aaa ggt aga aaa gat get tgg ctg gac gat gaa gcc ttc ttc 6753 Ma Lys Gly Arg Lys Asp Ala Trp Leu Asp Asp Glu Ala Phe Phe 2180 2185 2190
￡tgt tgg朋g gaa sat ccel teg aat tat gag gat a肌ctg犯g gaa 6798 Ser Trp Lys Glu Asn Pro Ser Asn Tyr Glu Asp Lys Leu Lys Glu2195 2200 2205
ttg cgt gca cag aaa gtg ttg ctt caa ttg aca aac att ggt gac 6843 Leu Arg Ala Gin Lys Val Leu Leu Gin Leu Thr Asn lie Gly Asp 2210 2215 2220
tcg gtt eta gat ttg caa get ctg cct caa gga ctt get get ctt 6888 Ser Val Leu Asp Leu Gin Ala Leu Pro Gin Gly Leu Ala Ala Leu 2225 2230 2235
tta age aag ttg gag cca tcg agt egg gtg aag ttg gcg gag gaa 6933 Leu Ser Lys Leu Glu Pro Ser Ser Arg Val Lys Leu Ala Glu Glu 2240 2245 2250
ctt cga aaa gta ctt ggt tag aacttagaag ttagaaggca agcatacctg 6984 Leu Arg Lys Val Leu Gly 2255 2260
taatcgtagt ttcactgttt agtgtttact aegttgatag tegcatatat gtatccattt 7044 tattagtgea atccgttggc tatgttgtat catcccttgc ttcaaagtgt atatgagaga 7104 tgttgatagc tttgtaattt tagtctggga atggatcaca accagcacca ccagattcct 7164 tctatttatt tgctggttaa ttcatccatg tcatgtatct caataaattt gtaataattt 7224 gtaacattta ttattatcaa catcattatt tattattatt cttgaataag aaaagttttg 7284 gtc333ttts tsttagcasa 3朋3333aaa a 7315
〈210〉 2 <211> 2260 〈212〉 PRT
<213〉 花生(Arachis hypogaea) 〈400〉 2
Met Ala Gly Val Gly Arg Gly Asn Gly Tyr Thr Asn Gly Val Val Pro 15 10 15
Asn Arg His Pro Ala Thr lie Ser Glu Val Asp Glu Tyr Cys Asn Ala 20 25 30Leu Gly Gly Thr Arg Pro lie His Ser lie Leu lie Ala Asn Asn Gly 35 40 45
Met Ala Ala Val Lys Phe lie Arg Ser Val Arg Ser Trp Ala Tyr Glu 50 55 60
Thr Phe Gly Thr Glu Arg Ala lie Leu Leu Val Ala Met Ala Thr Pro 65 70 75 80
Glu Asp Met Arg lie Asn Ala Glu His lie Arg lie Ala Asp Gin Phe 85 90 95
Val Glu Val Pro Gly Gly Thr Asn Asn Asn Asn Tyr Ala Asn Val Gin 100 105 110
Leu lie Val Glu Met Ala Glu lie Thr Arg Val Asp Ala Val Trp Pro 115 120 125
Gly Trp Gly His Ala Ser Glu Asn Pro Glu Leu Pro Asp Ala Leu Lys 130 135 140
Ala Lys Gly lie Val Phe Leu Gly Pro Pro Ala Val Ser Met Ala Ala 145 150 155 160
Leu Gly Asp Lys lie Gly Ser Ser Leu lie Ala Gin Ala Ala Glu Val 165 170 175
Pro Thr Leu Pro Trp Ser Gly Ser His Val Lys lie Pro Pro Asp Ser 180 185 190
Cys Leu Val Thr lie Pro Asp Glu lie Tyr Arg Glu Ala Cys Val Tyr 195 200 205
Thr Thr Glu Glu Ala lie Ala Ser Cys Gin Val Val Gly Tyr Pro Ala 210 215 220Met lie Lys Ala Ser Trp Gly Gly Gly Gly Lys Gly lie Arg Lys Val 225 230 235 240
His Asn Asp Asp Glu Val Arg Ala Leu Phe Lys Gin Val Gin Gly Glu 245 250 255
Val Pro Gly Ser Pro lie Phe lie Met Lys Val Ala Ser Gin Ser Arg 260 265 270
His L>eu Glu Val Gin Leu Leu Cys Asp Gin Tyr Gly Asn Val Ala Ala 275 280 285
Leu His Ser Arg Asp Cys Ser Val Gin Arg Arg His Gin Lys lie lie 290 295 300
Glu Glu Gly Pro lie Thr Val Ala Pro Pro Gin Thr Val Lys Gin Leu 305 310 315 320
Glu Gin Ala Ala Arg Arg Leu Ala Lys Ser Val Asn Tyr Val Gly Ala 325 330 335
Ala Thr Val Glu Tyr Leu Phe Ser Met Glu Thr Gly Glu Tyr Tyr Phe 340 345 350
Leu Glu Leu Asn Pro Arg Leu Gin Val Glu His Pro Val Thr Glu Trp 355 360 365
lie Ala Glu lie Asn Leu Pro Ala Ala Gin Val Ala lie Gly Met Gly 370 375 380
lie Pro Leu Trp Gin Leu Pro Glu lie Arg Arg Phe Tyr Gly Val Glu 385 390 395 400His Gly Gly Gly Asn Asp Ala Trp Arg Lys Thr Ser Ala Leu Ala Thr 405 410 415
Pro Phe Asp Phe Asp Lys Ala Gin Ser Thr Lys Pro Lys Gly His Cys 420 425 430
Val Ala Val Arg Val Thr Ser Glu Asp Pro Asp Asp Gly Phe Lys Pro 435 440 445
Thr Ser Gly Lys Val Gin Glu Leu Ser Phe Lys Ser Lys Pro Asn Val 450 455 460
Trp Ala Tyr Phe Ser Val Lys Ser Gly Gly Gly lie His Glu Phe Ser 465 470 475 480
Asp Ser Gin Phe Gly His Val Phe Ala Phe Gly Glu Ser Arg Ala Leu 485 490 495
Ala lie Ala Asn Met Val Leu Gly Leu Lys Glu lie Gin lie Arg Gly 500 505 510
Glu lie Arg Thr Asn Val Asp Tyr Thr lie Asp Leu Leu Asn Ala Ser 515 520 525
Asp Tyr Arg Asp Asn Lys lie His Thr Gly Trp Leu Asp Ser Arg lie 530 535 540
Ala Met Arg Val Arg Ala Glu Arg Pro Pro Trp Tyr Leu Ser Val Val 545 550 555 560
Gly Gly Ala Leu Tyr Lys Ala Ser Ala Ser Ser Ala Ala Leu Val Ser 565 570 575
Asp Tyr Val Gly Tyr Leu Glu Lys Gly Gin lie Pro Pro Lys His lie 580 585 590Ser Leu Val His Ser Gin Val Ser Leu Asn lie Glu Gly Ser Lys Tyr 595 600 605
Thr lie Asp Met Val Arg Gly Gly Ser Gly Ser Tyr Arg Leu Arg Met 610 615 620
Asn Gin Ser Glu Val Glu Ala Glu lie His Thr Leu Arg Asp Gly Gly 625 630 635 640
Leu Leu Met Gin Leu Asp Gly Asn Ser His Val lie Tyr Ala Glu Glu 645 650 655
Glu Ala Ala Gly Thr Arg Leu Leu lie Asp Gly Arg Thr Cys Leu Leu 660 665 670
Gin Asn Asp His Asp Pro Ser Lys Leu Val Ala Glu Thr Pro Cys Lys 675 680 685
Leu Met Arg Tyr Leu Val Val Asp Asp Ser His lie Asp Ala Asp Thr 690 695 700
Pro Tyr Ala Glu Val Glu Val Met Lys Met Cys Met Pro Leu Leu Ser 705 710 715 720
Pro Ala Ser Gly Val lie His Phe Lys Met Ser Glu Gly Gin Pro Met 725 730 735
Gin Ala Gly Glu Leu lie Ala Arg Leu Asp Leu Asp Asp Pro Ser Ala 740 745 750
Val Arg Lys Ala Glu Pro Phe Asn Gly Lys Phe Pro Val Leu Gly Pro 755 760 765
Pro Thr Ala Thr Ser Asp Lys Val His Gin Lys Cys Ala Ala Ser Leu770
775
780
Ser Ala Ala Gin Met lie Leu Ala Gly Tyr Glu His Asn lie Asp Glu 785 790 795 800
Val Val Gin Ser Leu Leu Asn Cys Leu Asp Ser Pro Glu Leu Pro Phe 805 810 815
Leu Gin Trp Gin Glu Cys Phe Ala Val Leu Ala Asn Arg Leu Pro Lys 820 825 830
Asp Leu Lys Asn Glu Leu Glu Ser Lys Tyr Lys Glu Tyr Glu Arg lie 835 840 845
Ser Ser Phe Gin Val Val Asp Phe Pro Ala Lys Leu Leu Lys Gly lie 850 855 860
Leu Glu Ala His Leu Ser Ser Cys Pro Asn Lys Glu Lys Gly Ala Gin 865 870 875 880
Glu Arg Leu lie Glu Pro Leu Leu Ser Leu Val Lys Ser Tyr Glu Gly 885 890 895
Gly Arg Glu Ser His Ala Arg Lys lie Val Gin Ser Leu Phe Glu Glu 900 905 910
Tyr Leu Phe Val Glu Glu Leu Phe Ser Asp Asn lie Gin Ala Asp Val 915 920 925
lie Glu Arg Leu Arg Leu Gin Tyr Lys Lys Asp Leu Leu Lys lie Val 930 935 940
Asp lie Val Leu Ser His Gin Gly lie Lys Ser Lys Asn Lys Leu lie 945 950 955 960Leu Arg Leu Met Asp Lys Leu Val Tyr Pro Asn Pro Ala Ala Tyr Arg 965 970 975
Asp Gin Leu lie Arg Phe Ser Gin Leu Asn His Thr Asn Tyr Ser Gin 980 985 990
Leu Ala Leu Lys Ala Ser Gin Leu Leu Glu Gin Thr Lys Leu Ser Glu 995 1000 1005
Leu Arg Ser Asn lie Ala Arg Ser Leu Ser Glu Leu Glu Met Phe 1010 1015 1020
Thr Glu Asp Gly Glu Asn lie Asp Thr Pro Lys Arg Lys Ser Ala 1025 1030 1035
lie Asn Asp Arg Met Glu Asp Leu Val Ser Ala Pro Leu Ala Val 1040 1045 1050
Glu Asp Ala Leu Val Gly Leu Phe Asp His Ser Asp His Thr Leu 1055 1060 1065
Gin Arg Arg Val Val Glu Thr Tyr lie Arg Arg Leu Tyr Gin Pro 1070 1075 1080
Tyr Leu Val Lys Gly Ser Val Arg Met Gin Trp His Arg Ser Gly 1085 1090 1095
Leu lie Ala Ser Trp Glu Phe Leu Glu Glu Tyr lie Glu Arg Lys 1100 1105 1110
Ser Gly Val Glu Asp Gin Met Ser Asp Lys Thr Leu Val Glu Lys 1115 1120 1125
His Thr Glu Lys Lys Trp Gly Val Met Val Val lie Lys Ser Leu1130 1135 1140
His Phe Leu Pro Ala lie lie Thr Ala Ala Leu Lys Glu Ala Thr 1145 1150 1155
Asn Asn Leu His Glu Ala Val Ser Ser Ala Ala Gly Glu Pro Val 1160 1165 1170
Lys His Gly Asn Met Met His Val Ala Leu Val Gly lie Asn Asn 1175 1180 1185
Gin Met Ser Leu Leu Gin Asp Ser Gly Asp Glu Asp Gin Ala Gin 1190 1195 1200
Glu Arg lie Asn Lys Leu Ala Lys lie Leu Lys Glu Glu Glu Val 1205 1210 1215
Gly Ser Thr lie Arg Gly Thr Gly Val Gly Val lie Ser Cys lie 1220 1225 1230
lie Gin Arg Asp Glu Gly Arg Thr Pro Met Arg His Ser Phe His 1235 1240 1245
Trp Ser Ala Glu Lys Leu Tyr Tyr Gin Glu Glu Pro Leu Leu Arg 1250 '1255 1260
His Leu Glu Pro Pro Leu Ser lie Tyr Leu Glu Leu Asp Lys Leu 1265 1270 1275
Lys Gly Tyr Glu Asn lie Arg Tyr Thr Pro Ser Arg Asp Arg Gin 1280 1285 1290
Trp His Leu Tyr Thr Val Met Asp Gin Lys Pro Gin Pro Ala Gin 1295 1300 1305Arg Met Phe Leu Arg Thr Leu Leu Arg Gin Pro Thr Thr Asn Glu 1310 1315 1320
Gly Phe Ser Ser Tyr Gin Arg Thr Asp Ala Glu Thr Pro Ser Thr 1325 1330 1335
Glu Leu Ala Met Ser Phe Thr Ser Arg Ser lie Phe Arg Ser Leu 1340 1345 1350
Met Ala Ala Met Glu Glu Leu Glu Leu Asn Ser His Asn Ala Thr 1355 1360 1365
lie Arg Pro Glu His Ala His Met Tyr Leu Tyr lie lie Arg Glu 1370 1375 1380
Gin Glu lie Asn Asp Leu Val Pro Tyr Pro Lys Arg Val Asp lie 1385 1390 1395
Asp Ala Gly Gin Glu Glu Thr Thr Val Glu Ala Thr Leu Glu Glu 1400 1405 1410
Leu Ala His Glu lie His Ser Ser Val Gly Val Arg Met His Arg 1415 1420 1425
Leu Gly Val Val Val Trp Glu Val Lys Leu Trp Met Ala Ala Cys 1430 1435 1440
Ala Gin Ala Asn Gly Ala Trp Arg lie Val Val Asn Asn Val Thr 1445 1450 1455
Gly His Thr Cys Thr Val His lie Tyr Arg Glu Met Glu Asp Thr 1460 1465 1470
Asn Thr His Arg Val Val Tyr Ser Ser lie Thr Val Lys Gly Pro1475
1480
1485
Leu His Gly Val Pro Val Asn Glu Thr Tyr Gin Pro Leu Gly Val 1490 1495 1500
lie Asp Arg Lys Arg Leu Ser Ala Arg Lys Asn Ser Thr Thr Phe 1505 1510 1515
Cys Tyr Asp Phe Pro Leu Ala Phe Glu Thr Ala Leu Glu Gin Ser 1520 1525 1530
Trp Ala lie Gin Gin Pro Gly Phe Arg Arg Pro Lys Asp Lys Asn 1535 1540 1545
Leu Leu Lys Val Thr Glu Leu Arg Phe Ala Asp Lys Glu Gly Ser 1550 1555 1560
Trp Gly Thr Pro Leu Val Pro Val Glu His Ser Ala Gly Leu Asn 1565 1570 1575
Asp Val Gly Met Val Ala T卬 Phe Met Asp Met Cys Thr Pro Glu 1580 1585 1590
Phe Pro Ser Gly Arg Thr lie Leu Val Val Ala Asn Asp Val Thr 1595 1600 1605
Phe Lys Ala Gly Ser Phe Gly Pro Arg Glu Asp Ala Phe Phe Arg 1610 1615 1620
Ala Val Thr Asp Leu Ala Cys Ala Lys Lys Leu Pro Leu lie Tyr 1625 1630 1635
Leu Ala Ala Asn Ser Gly Ala Arg Leu Gly Ala Ala Glu Glu Val 1640 1645 1650Lys Ala Cys Phe Lys Val Gly Trp Ser Glu Glu Ser Asn Pro Glu 1655 1660 1665
His Gly Phe Gin Tyr Val Tyr Leu Thr Pro Glu Asp Phe Ala Arg 1670 1675 1680
lie Gly Ser Ser Val lie Ala His Glu Leu Lys Leu Glu Ser Gly 1685 1690 1695
Glu Thr Arg Trp lie lie Asp Thr lie Val Gly Lys Glu Asp Gly 1700 1705 1710
Leu Gly Val Glu Asn Leu Ser Gly Ser Gly Ala lie Ala Gly Ser 1715 1720 1725
Tyr Ser Arg Ala Tyr Lys Glu Thr Phe Thr Leu Thr Tyr Val Thr 1730 1735 1740
Gly Arg Thr Val Gly lie Gly Ala Tyr Leu Ala Arg Leu Gly Met 1745 1750 1755
Arg Cys lie Gin Arg Leu Asp Gin Pro lie lie Leu Thr Gly Phe 1760 1765 1770
Ser Ala Leu Asn Lys Leu Leu Gly Arg Glu Val Tyr Ser Ser His 1775 1780 1785
Met Gin Leu Gly Gly Pro Lys lie Met Ala Thr Asn Gly Val Val 1790 1795 1800
His Leu Thr Val Ser Asp Asp Leu Glu Gly Val Ser Ala lie Leu 1805 1810 1815
Lys Trp Leu Ser Tyr lie Pro Ser His Val Gly Gly Ser Leu Pro1820 1825 1830
lie Val Lys Pro Leu Asp Pro Pro Glu Arg Pro Val Glu Tyr Leu 1835 1840 1845
Pro Glu Asn Ser Cys Asp Pro Arg Ala Ala lie Ser Gly Thr Leu 1850 1855 I860
Asp Gly Asn Gly Arg Trp Leu Gly Gly lie Phe Asp Lys Asp Ser 1865 1870 1875
Phe Val Glu Thr Leu Glu Gly Trp Ala Arg Thr Val Val Thr Gly 1880 1885 1890
Arg Ala Lys Leu Gly Gly lie Pro Val Gly lie Val Ala Val Glu 1895 1900 1905
Thr Gin Thr Val Met Gin lie lie Pro Ala Asp Pro Gly Gin Leu 1910 1915 1920
Asp Ser His Glu Arg Val Val Pro Gin Ala Gly Gin Val Trp Phe 1925 1930 1935
Pro Asp Ser Ala Thr Lys Thr Ala Gin Ala lie Met Asp Phe Asn 1940 1945 1950
Arg Glu Glu Leu Pro Leu Phe lie Leu Ala Asn Trp Arg Gly Phe 1955 1960 1965
Ser Gly Gly Gin Arg Asp Leu Phe Glu Gly lie Leu Gin Ala Gly 1970 1975 1980
Ser Thr lie Val Glu Asn Leu Arg Thr Tyr Lys Gin Pro lie Phe 1985 1990 1995Val Tyr lie Pro Met Met Gly Glu Leu Arg Gly Gly Ala Trp Val 2000 2005 2010
Val Val Asp Ser Arg lie Asn Ser Asp His lie Glu Met Tyr Ala 2015 2020 2025
Asp Arg Thr Ala Lys Gly Asn Val Leu Glu Pro Glu Gly Met lie 2030 2035 2040
Glu lie Lys Phe Arg Thr Arg Glu Leu Leu Glu Cys Met Gly Arg 2045 2050 2055
Leu Asp Gin Lys Leu工le Thr Leu Lys Ala Lys Leu Gin Glu Ala 2060 2065 2070
Lys Asp Lys Arg Asp Thr Glu Ser Phe Glu Ser Leu Gin Gin Gin 2075 2080 2085
lie Lys Ser Arg Glu Lys Gin Leu Leu Pro Leu Tyr Thr Gin lie 2090 2095 2100
Ala Thr Lys Phe Ala Glu Leu His Asp Thr Ser Leu Arg Met Ala 2105 2110 2115
Ala Lys Gly Val lie Arg Gin Val Leu Asp Trp Gly Asn Ser Arg 2120 2125 2130
Ala Val Phe Tyr Arg Arg Leu Tyr Arg Arg lie Gly Glu Gin Ser 2135 2140 2145
Leu lie Asn Asn Val Arg Glu Ala Ala Gly Asp His Leu Ser His 2150 2155 2160
Val Ser Ala Met Asp Leu Val Lys Asn Trp Tyr Leu Ser Ser Asn2165
2170
2175
lie Ala Lys Gly Arg Lys Asp Ala Trp Leu Asp Asp Glu Ala Phe 2180 2185 2190
Phe Ser Trp Lys Glu Asn Pro Ser Asn Tyr Glu Asp Lys Leu Lys 2195 2200 2205
Glu Leu Arg Ala Gin Lys Val Leu Leu Gin Leu Thr Asn lie Gly 2210 2215 2220
Asp Ser Val Leu Asp Leu Gin Ala Leu Pro Gin Gly Leu Ala Ala 2225 2230 2235
Leu Leu Ser Lys Leu Glu Pro Ser Ser Arg Val Lys Leu Ala Glu 2240 2245 2250
Glu Leu Arg Lys Val Leu Gly 2255 2260
权利要求
1.一种花生同质型乙酰辅酶A羧化酶基因，其特征在于所述基因序列由SEQ ID NO.1中从核苷酸第172-6954位的核苷酸序列构成。
2. 根据权利要求1所述的花生同质型乙酰辅酶A羧化酶基因，其特征在于所述基因编码的 蛋白质具有序列表中SEQ ID NO. 2所示的氨基酸序列。
3. —种花生同质型乙酰辅酶A羧化酶基因的克隆方法，包括如下步骤(1) 提取花生幼苗中的总R織；(2) 利用Promega的RT-PCR体系反转录获得cDNA;(3) 根据cDNA文库中的EST信息设计引物(4) 进行PCR扩增，并将PCR产物回收后筛选阳性克隆；(5) 对筛选到的阳性克隆进行序列分析，获得同质型乙酰辅酶A羧化酶基因。
全文摘要
本发明公开了一种花生同质型乙酰辅酶A羧化酶基因及其编码的蛋白质与克隆方法。所述基因序列由SEQ ID NO.1中从核苷酸第172-6954位的核苷酸序列所构成；所述基因编码的蛋白质具有序列表中SEQ ID NO.2所示的氨基酸序列。本发明的花生同质型乙酰辅酶A羧化酶(AhACCase)基因的核苷酸序列及其编码的蛋白序列以及基因克隆方法，可广泛应用于花生品质的改良、花生的栽培和培养，尤其是花生作为油料作物的开发和利用等领域。
文档编号C12P19/34GK101580844SQ20091011955
公开日2009年11月18日 申请日期2009年3月13日 优先权日2009年3月13日
发明者和亚男, 杨庆利, 潘丽娟, 禹山林, 迟晓元, 平 闵 申请人:山东省花生研究所