CN115160418B - 盔形毕赤酵母Peptidase类分泌蛋白及其应用 - Google Patents

Abstract

本发明公开了氨基酸序列如SEQ ID No.1、SEQ ID No.2、SEQ ID No.3、SEQ ID No.4或SEQ ID No.5所示的盔形毕赤酵母Peptidase类分泌蛋白及其在制备诱导柑橘果实抗病性的保鲜剂中的应用，研究结果证实，农杆菌介导的遗传转化在柑橘果实上瞬时表达Peptidase类分泌蛋白，能一定程度抑制柑橘果实绿霉病的发病和病斑直径的增长；本发明从盔形毕赤酵母中挖掘出具有诱导柑橘果实抗病性的Peptidase类分泌蛋白，不仅丰富了盔形毕赤酵母诱导柑橘果实抗病性的机理，拓宽了拮抗酵母在果实保鲜领域的应用方式，也为柑橘果实采后绿霉病的防治提供了新的解决方法。

Description

盔形毕赤酵母Peptidase类分泌蛋白及其应用

技术领域

本发明属于基因工程技术和果实采后病害生物防治技术领域，涉及盔形毕赤酵母(Pichia galeiformis)Peptidase类分泌蛋白及其应用。

背景技术

拮抗酵母是果实采后病害防治中常用的生物防治拮抗菌。已有大量文献报道拮抗酵母能诱导果实抗病性，涉及果实多种抗病基因的表达、激素合成和信号转导途径、抗病相关物质的合成等。然而，拮抗酵母分泌蛋白对果实抗病性的作用还不清楚。

柑橘是芸香科柑橘亚科柑橘属植物，是世界和中国产量最多的水果。新鲜柑橘果实在采后运输、贮藏等过程中极易受微生物侵染，损失可达总产量的30％以上。由真菌指状青霉(Penicillium digitatum)引起的绿霉病是柑橘果实贮藏过程中主要的侵染性病害。目前产业上主要采用咪鲜胺等化学杀菌剂控制果实采后病害，但存在杀菌剂残留、微生物产生耐药性等问题。

发明内容

本发明的目的在于探究和明确拮抗酵母分泌蛋白在诱导果实抗病性中的作用，以期为果实采后病害的防治提供新的解决方案。

经研究，本发明提供如下技术方案：

1.盔形毕赤酵母Peptidase类分泌蛋白，氨基酸序列如SEQ ID No.1、SEQ ID No.2或SEQ ID No.3、SEQ ID No.4或SEQ ID No.5所示。

2.盔形毕赤酵母Peptidase类分泌蛋白的编码基因。

进一步，所述编码基因的核苷酸序列如SEQ ID No.6、SEQ ID No.7、SEQ ID No.8、SEQ ID No.9或SEQ ID No.10所示。

3.含有盔形毕赤酵母Peptidase类分泌蛋白编码基因的重组表达载体。

进一步，所述重组表达载体是将核苷酸序列如SEQ ID No.6、SEQ ID No.7、SEQ IDNo.8、SEQ ID No.9或SEQ ID No.10所示的盔形毕赤酵母Peptidase类分泌蛋白编码基因克隆入植物表达载体pCAMBIA2300的多克隆位点KpnI和PstI之间而得到。

4.含有上述重组表达载体的工程菌。

进一步，所述工程菌是将含有盔形毕赤酵母Peptidase类分泌蛋白编码基因的重组表达载体转入农杆菌GV3101中而得到。

5.盔形毕赤酵母Peptidase类分泌蛋白在制备诱导柑橘果实抗病性的保鲜剂中的应用。

进一步，所述诱导柑橘果实抗病性为诱导柑橘果实绿霉病抗病性。

6.所述工程菌在制备诱导柑橘果实抗病性的保鲜剂中的应用。

进一步，所述诱导柑橘果实抗病性为诱导柑橘果实绿霉病抗病性。

本发明的有益效果在于：本发明提供了盔形毕赤酵母Peptidase类分泌蛋白及其在制备诱导柑橘果实抗病性的保鲜剂中的应用，研究结果证实，农杆菌介导的遗传转化在柑橘果实上瞬时表达Peptidase类分泌蛋白，能一定程度抑制柑橘果实绿霉病的发病和病斑直径的增长。本发明从盔形毕赤酵母中挖掘出具有诱导柑橘果实抗病性的Peptidase类分泌蛋白，不仅丰富了盔形毕赤酵母诱导柑橘果实抗病性的机理，拓宽了拮抗酵母在果实保鲜领域的应用方式，也为柑橘果实采后绿霉病的防治提供了新的解决方法。

附图说明

图1为盔形毕赤酵母Peptidase类分泌蛋白PgPgPeptidase1、PgPgPeptidase2、PgPgPeptidase3、PgPeptidase4、PgPeptidase5编码基因的电泳图，其中M为DNA分子量标准。

图2为重组载体pCAMBIA2300-PgPeptidase1、pCAMBIA2300-PgPeptidase2、pCAMBIA2300-PgPeptidase3、pCAMBIA2300-PgPeptidase4、pCAMBIA2300-PgPeptidase5的电泳图，其中M为DNA分子量标准。

图3为含有PgPeptidase1、PgPeptidase2、PgPeptidase3、PgPeptidase4或PgPeptidase5编码基因的农杆菌工程菌的电泳图，其中M为DNA分子量标准。

图4为盔形毕赤酵母Peptidase类分泌蛋白PgPeptidase1、PgPeptidase2、PgPeptidase3、PgPeptidase4、PgPeptidase5对柑橘果实采后绿霉病的诱导效果；A为发病率，B为病斑直径，*表示与对照组(Control)相比差异显著(P<0.05)；C为柑橘果实在接种指状青霉后于25℃贮藏6天后的绿霉病发病症状。

具体实施方式

为了使本发明的目的、技术方案和有益效果更加清楚，下面对本发明的优选实施例进行详细的描述。

本实施例所用盔形毕赤酵母分离于柑橘果园的柠檬果实表面。

1.引物设计

根据盔形毕赤酵母的基因序列和pCAMBIA2300载体上的多克隆位点KpnI和PstI，设计以下引物，委托生工生物工程(上海)股份有限公司进行合成。

PgPeptidase1-F：caatttactattctagggtaccATGAAACTTTTAACACCCTTGCTGT(SEQ IDNo.11)

PgPeptidase1-R：gtatgggtatctagactgcagTCACATATGCATGTCGCCCAT(SEQ IDNo.12)

PgPeptidase2-F：caatttactattctagggtaccATGAAGCTCTCTTCTTCCCTTT(SEQ IDNo.13)

PgPeptidase2-R：gtatgggtatctagactgcagCTAAACAGAAATAACATCTTCAACG(SEQ IDNo.14)

PgPeptidase3-F：caatttactattctagggtaccATGAAACTGGACTTCCTGACCT(SEQ IDNo.15)

PgPeptidase3-R：gtatgggtatctagactgcagTTAAAAGATTAACTTGTCACCTCTG(SEQ IDNo.16)

PgPeptidase4-F：caatttactattctagggtaccATGAAGATTGCCTTGACCAC(SEQ IDNo.17)

PgPeptidase4-R：gtatgggtatctagactgcagTTAAGTAAAACGGTAATTACCACTG(SEQ IDNo.18)

PgPeptidase5-F：caatttactattctagggtaccATGGTTTGGTTATCCGCTTTAAG(SEQ IDNo.19)

PgPeptidase5-R：gtatgggtatctagactgcagCTATTTGCCTAGAGCATGCAAG(SEQ IDNo.20)

2.盔形毕赤酵母总RNA提取及cDNA合成

用北京酷来搏科技有限公司的真菌RNA提取试剂盒(RE781-50T)提取盔形毕赤酵母总RNA，具体步骤按照试剂盒说明书进行。

所得盔形毕赤酵母总RNA用PrimeScript^TM RT regent Kit试剂盒(Takara)反转录合成cDNA，具体步骤按照试剂盒说明书进行，用酶标仪Take3检测所得cDNA的浓度和质量，-20℃保存备用。

3.盔形毕赤酵母PgPeptidase类分泌蛋白编码基因序列克隆

以反转录合成的cDNA为模板，使用引物PgPeptidase1-F和PgPeptidase1-R，使用诺唯赞高保真酶P505试剂盒，PCR扩增核苷酸序列如SEQ ID No.6所示的PgPeptidase1编码基因序列，具体步骤按照试剂盒说明书进行。

以反转录合成的cDNA为模板，使用引物PgPeptidase2-F和PgPeptidase2-R，使用诺唯赞高保真酶P505试剂盒，PCR扩增核苷酸序列如SEQ ID No.7所示的PgPeptidase2编码基因序列，具体步骤按照试剂盒说明书进行。

以反转录合成的cDNA为模板，使用引物PgPeptidase3-F和PgPeptidase3-R，使用诺唯赞高保真酶P505试剂盒，PCR扩增核苷酸序列如SEQ ID No.8所示的PgPeptidase3编码基因序列，具体步骤按照试剂盒说明书进行。

以反转录合成的cDNA为模板，使用引物PgPeptidase4-F和PgPeptidase4-R，使用诺唯赞高保真酶P505试剂盒，PCR扩增核苷酸序列如SEQ ID No.9所示的PgPeptidase4编码基因序列，具体步骤按照试剂盒说明书进行。

以反转录合成的cDNA为模板，使用引物PgPeptidase5-F和PgPeptidase5-R，使用诺唯赞高保真酶P505试剂盒，PCR扩增核苷酸序列如SEQ ID No.10所示的PgPeptidase5编码基因序列，具体步骤按照试剂盒说明书进行。

PCR产物进行1％琼脂糖凝胶电泳(150V，30min)，结果如图1所示，用无菌刀片切下目的条带，用胶回收试剂盒进行回收，具体步骤按照试剂盒说明书进行，回收产物-20℃保存备用。

4.质粒pCAMBIA2300的KpnI和PstI双酶切

将质粒pCAMBIA2300进行KpnI和PstI双酶切，酶切反应液置37℃金属浴中30min。

酶切产物进行琼脂糖凝胶电泳，用无菌刀片切下目的条带，用胶回收试剂盒进行回收，具体步骤按照试剂盒说明书进行，回收产物-20℃保存备用。

5.重组载体的构建

将回收的线性化质粒pCAMBIA2300与PgPeptidase1编码基因序列使用诺唯赞同源重组酶进行连接，具体步骤按照试剂说明书进行，连接反应液置37℃金属浴中30min。同样的方法将PgPeptidase2、PgPeptidase3、PgPeptidase4、PgPeptidase5编码基因序列分别与线性化质粒pCAMBIA2300连接。

连接产物转化大肠杆菌感受态细胞。用质粒测序引物对转化子进行菌液PCR验证。验证结果为阳性的转化子委托生工生物工程(上海)股份有限公司进行测序。测序结果比对正确的重组质粒即为成功构建的重组载体pCAMBIA2300-PgPeptidase1、pCAMBIA2300-PgPeptidase2、pCAMBIA2300-PgPeptidase3、pCAMBIA2300-PgPeptidase4、pCAMBIA2300-PgPeptidase5。

6.农杆菌工程菌的构建

提取重组载体pCAMBIA2300-PgPeptidase1、pCAMBIA2300-PgPeptidase2、pCAMBIA2300-PgPeptidase3、pCAMBIA2300-PgPeptidase4、pCAMBIA2300-PgPeptidase5(电泳结果如图2所示，以质粒pCAMBIA2300为对照)，分别加入农杆菌GV3101感受态细胞中，轻弹混匀，置冰上5min，液氮5min，37℃5min，冰上5min；加无抗LB液体培养基600μL，28℃、200rpm培育2～3h，4000rpm离心2min，弃上清，将余液均匀涂布于LB平板(含有25μg/L利福平和50μg/L卡那霉素)；挑取单菌落至500μL LB液体培养基(含有25μg/L利福平和50μg/L卡那霉素)中，28℃、200rpm摇菌培养16h后检测目的片段。结果如图3所示，含有PgPeptidase1、PgPeptidase2、PgPeptidase3、PgPeptidase4、PgPeptidase5编码基因的农杆菌即为成功构建的PgPeptidase1、PgPeptidase2、PgPeptidase3、PgPeptidase4、PgPeptidase5农杆菌工程菌。

7.农杆菌工程菌在柑橘果实上瞬时表达分泌蛋白诱导柑橘果实绿霉病抗病性的效果

分别将PgPeptidase1、PgPeptidase2、PgPeptidase3、PgPeptidase4、PgPeptidase5的农杆菌工程菌在LB培养基(含有25μg/L利福平和50μg/L卡那霉素)中培养过夜，当菌液OD₆₀₀约为1时，4000rpm离心5min，将菌体用孵育液(1L孵育液含有5g葡萄糖，1.0663g MES(使用浓度5mM)，0.760g Na₃PO₄·12H₂O(使用浓度2mM)，0.785mL 127.4mM AS母液(使用浓度0.1mM))重悬并调整OD600＝0.8，等量混合基因沉默抑制子P19，28℃避光孵育2～3h，得农杆菌工程菌液；挑选大小均匀一致、色泽均匀、无机械伤和疤痕的柑橘果实，用2％次氯酸钠浸泡2min后用清水冲洗干净，自然风干，用75％酒精擦果实赤道部位，待酒精风干后，用1mL无菌枪头在果实赤道部位对立面各打一个孔，用1mL注射器(去掉针头)向孔中注射约0.5mL农杆菌工程菌液，以含有pCAMBIA2300质粒的农杆菌为对照(control)；接种农杆菌工程菌1d后，在每个孔的右方1cm处再打一个孔，用移液枪接种10μL 1×10⁴CFU/mL的指状青霉(Penicillium digitatum)孢子悬浮液，待菌液吸收后，单果套袋，避光放置于25℃的环境中，从第3天开始统计发病率和病斑直径。

结果如图4所示，含有PgPeptidase1、PgPeptidase2、PgPeptidase3、PgPeptidase4、PgPeptidase5编码基因的农杆菌工程菌在柑橘果实上瞬时表达分泌蛋白PgPeptidase1(氨基酸序列如SEQ ID No.1所示)、PgPeptidase2(氨基酸序列如SEQ IDNo.2所示)、PgPeptidase3(氨基酸序列如SEQ ID No.3所示)、PgPeptidase4(氨基酸序列如SEQ ID No.4所示)、PgPeptidase5(氨基酸序列如SEQ ID No.5所示)，能一定程度抑制柑橘果实绿霉病的发病和病斑直径的增长，其中PgPeptidase1、PgPeptidase2、PgPeptidase4、PgPeptidase5诱导柑橘果实绿霉病抗病性的效果更好。

最后说明的是，以上优选实施例仅用以说明本发明的技术方案而非限制，尽管通过上述优选实施例已经对本发明进行了详细的描述，但本领域技术人员应当理解，可以在形式上和细节上对其作出各种各样的改变，而不偏离本发明权利要求书所限定的范围。

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Lys Asn Glu Val Asp Ala Phe Glu Asp Ser Val Asn Ala Leu Lys Phe

115 120 125

Lys Asn Arg Ala Gly Gly Ile Gly His Val Phe Asp Ser Pro Gly Leu

130 135 140

Arg Gly Tyr Ser Gly Leu Phe Thr Pro Glu Val Ile Asn Leu Leu Arg

145 150 155 160

Arg Ser Glu Asp Val Ala Val Ile Glu Leu Asp Ser Val Val His Ala

165 170 175

Ile Glu Ala Glu Thr Gln Asn Gly Ala Pro Trp Gly Leu Ser Arg Ile

180 185 190

Ser His Arg Glu Ala Leu Gly Leu Gly Asn Tyr Asn Lys Tyr Leu Tyr

195 200 205

Asp Ala Asp Ala Gly Glu Gly Val Thr Ala Tyr Val Ile Asp Thr Gly

210 215 220

Val Asn Ile Glu His Glu Gln Phe Gly Gly Arg Ala Val Trp Gly Ala

225 230 235 240

Thr Ile Pro Ser Asp Asp Thr Asp Thr Asp Gly Asn Gly His Gly Thr

245 250 255

His Cys Ala Gly Thr Ile Gly Ser Glu Asp Tyr Gly Val Ser Lys Lys

260 265 270

Ala Lys Ile Val Ala Val Lys Val Leu Lys Ser Asn Gly Ser Gly Thr

275 280 285

Met Ser Asp Val Val Lys Gly Val Glu Phe Ala Ala Asn Ala His Val

290 295 300

Lys Glu Ala Lys Glu Ala Lys Ser Gly Phe Lys Gly Ser Thr Ala Asn

305 310 315 320

Met Ser Leu Gly Gly Gly Lys Ser Pro Ala Leu Asp Leu Ala Val Asn

325 330 335

Ala Ala Val Lys Ala Gly Leu His Phe Ala Val Ala Ala Gly Asn Asp

340 345 350

Asp Ala Asp Ala Cys Asn Tyr Ser Pro Ala Ala Ala Glu Ser Ala Val

355 360 365

Thr Val Gly Ala Ser Thr Leu Ser Asp Thr Arg Ala Tyr Phe Ser Asn

370 375 380

Tyr Gly Lys Cys Val Asp Ile Phe Ala Pro Gly Leu Asn Val Leu Ser

385 390 395 400

Thr Tyr Ile Gly Ser Asn Thr Ala Thr Ala Ile Leu Ser Gly Thr Ser

405 410 415

Met Ala Ser Pro His Val Cys Gly Leu Leu Thr Tyr Phe Leu Ser Leu

420 425 430

Ala Pro Ser Ser Asp Ser Met Phe Ser Thr Ala Ser Ile Thr Pro Lys

435 440 445

Gln Leu Lys Gln Asn Ile Leu Lys Tyr Gly Thr Lys Asn Ala Leu Ser

450 455 460

Ser Ile Pro Asp Asp Asp Thr Pro Asn Ile Leu Ile Tyr Asn Gly Ala

465 470 475 480

Gly Gly Asp Ile Ser Asp Phe Trp Asn Phe Glu Ser Glu Ser Ser Ser

485 490 495

Ser Asp Asp Leu Gln Arg Ser Ile Glu Val Ala Asn Glu Phe Ala Asp

500 505 510

Thr Gln Leu Ser Lys Ala Lys Gln Glu Ile Lys Val Ile Leu Ser Glu

515 520 525

Ile Thr Gln Val Val Asp Ser Val Glu Asp Val Ile Ser Val

530 535 540

<210> 3

<211> 507

<212> PRT

<213> 盔形毕赤酵母(Pichia galeiformis)

<400> 3

Met Lys Leu Asp Phe Leu Thr Ser Ala Leu Ile Leu Ser Asn Val Ala

1 5 10 15

Asn Ala Ala Ile Leu Pro Gln Ala Ala Leu Asn Leu Glu Asn Gln Ile

20 25 30

Glu His Leu Glu Ser Tyr Leu Glu Leu Ala Asp Ser Pro Ser Phe Ala

35 40 45

Asp Leu Pro Leu Val Asn Ser Thr Glu Leu Gln Ala Leu Ile Thr His

50 55 60

Glu Ala Leu Phe Thr Arg Ala Glu Lys Leu Tyr Gln Ile Ala Gly His

65 70 75 80

Ser Asn Lys Ser Tyr Gly His Pro Thr Arg Val Ile Gly Ser Pro Gly

85 90 95

His Trp Ala Thr Ile Gly Tyr Ile Glu His Glu Leu Lys Lys Leu His

100 105 110

Ala Tyr Tyr Asn Val Thr Thr Gln Ala Phe Lys Ala Phe Met Ser Glu

115 120 125

Ile Phe Ser Phe Asn Ala Thr Ile Asp Asn Glu Val Pro Lys Ser Leu

130 135 140

Val Glu Met Asp Met Ser Pro Pro Thr Pro His Lys Ala Pro Val Tyr

145 150 155 160

Gly Leu Leu Ala Leu Val Ser Asn Ser Gly Cys His Asp Asp Asp Tyr

165 170 175

Ala Asn Val Thr Gly Asn Ile Ala Phe Ile Lys Arg Gly Glu Cys Ala

180 185 190

Phe Gly Asp Lys Ala Gln Leu Ala Ala Lys His Gly Ala Leu Ala Ala

195 200 205

Ile Ile Tyr Asn Asn Glu Pro Gly Glu Ile Arg Gly Thr Leu Gly Val

210 215 220

Pro Pro Glu Gly Val Lys Val Ser Ser Ser Val Gly Ile Asn Gln Glu

225 230 235 240

Glu Gly Glu Lys Tyr Ala Lys Leu Leu Arg Glu Gly Lys Val Leu Asn

245 250 255

Ala Ser Val Gln Val Asp Ser Tyr Val Gly Pro Ile Thr Thr Val Asn

260 265 270

Val Ile Ala Glu Thr Asn His Gly Asp Pro Asn Asn Ile Val Met Leu

275 280 285

Gly Ala His Ser Asp Ser Val Ala Ala Gly Pro Gly Ile Asn Asp Asp

290 295 300

Gly Ser Gly Thr Ile Ser Leu Leu Glu Val Ala Lys Gln Leu Thr Asn

305 310 315 320

Tyr Arg Val Glu Asn Lys Val Arg Phe Ala Phe Trp Ala Ala Glu Glu

325 330 335

Glu Gly Leu Leu Gly Ser Asn Tyr Tyr Ala Asp Asn Leu Thr Pro Gly

340 345 350

Glu Asn Glu Lys Ile Arg Leu Phe Met Asp Tyr Asp Met Met Ala Ser

355 360 365

Pro Asn Tyr Glu Tyr Glu Val Tyr Asp Ala Asn Asn Val Asp Asn Pro

370 375 380

Lys Gly Ser Glu Glu Leu Lys Asn Leu Tyr Ile Asp Tyr Tyr Thr Ser

385 390 395 400

His Gly Leu Asn Tyr Thr Leu Ile Pro Phe Asp Gly Arg Ser Asp Tyr

405 410 415

Val Gly Phe Ile Glu His Gly Ile Pro Gly Gly Gly Ile Ala Ala Gly

420 425 430

Ala Glu Lys Asn Asn Ala Glu Asn Gly Asp Val Leu Asp Lys Cys Tyr

435 440 445

His Gln Leu Cys Asp Asp Leu Ser Asn Ile Ala Trp Asp Ala Phe Leu

450 455 460

Ile Asn Thr Gln Leu Ile Ala His Ser Val Ala Thr Tyr Gly Ala Ser

465 470 475 480

Leu Asp Gly Phe Pro Thr Arg Glu Asn Asp Thr Thr Val Asp Ser Leu

485 490 495

Ala Phe Ala Tyr Arg Gly Asp Lys Leu Ile Phe

500 505

<210> 4

<211> 547

<212> PRT

<213> 盔形毕赤酵母(Pichia galeiformis)

<400> 4

Met Lys Ile Ala Leu Thr Thr Ala Ala Ile Ala Ser Leu Ser Phe Gly

1 5 10 15

Leu Ala Ser Ala Asn Pro Val Leu Gly Tyr Asn Arg Gln Asn Val Leu

20 25 30

Thr Tyr Asn Glu Arg Gln His His Gly His Pro Ile Leu Asp Lys Tyr

35 40 45

Ile Glu Lys Gly Phe Phe Asp Glu Ser Leu Val Asp Glu Thr Thr Ser

50 55 60

Glu Ala Trp Lys Leu Leu Glu Gln Thr Val Asp Ala Ser Lys Ile Glu

65 70 75 80

Gln Glu Leu Lys Arg Tyr Gly Ser Glu Glu Ser Lys Ala Pro Lys Ser

85 90 95

Ala Leu Arg Leu Pro Glu Leu Lys Asp Asn Thr Asn Tyr Glu Tyr Ile

100 105 110

Ser Leu Ser Glu Phe Ser Ser Tyr Gly Leu Arg Ile Lys Lys His Leu

115 120 125

Gly Ser Asp Pro Ser Val Leu Gly Val Asp Lys Val Lys Gln Trp Thr

130 135 140

Gly Tyr Phe Asp Ile Asn Glu Glu Asp Lys His Leu Phe Phe Trp Phe

145 150 155 160

Phe Glu Ser Arg Asn Asp Pro Glu Asn Asp Pro Val Ile Leu Trp Leu

165 170 175

Asn Gly Gly Pro Gly Cys Ser Ser Met Thr Gly Leu Phe Phe Glu Leu

180 185 190

Gly Pro Ser Ser Ile Asn Gly Thr Thr Leu Lys Pro Ile Asn Asn Pro

195 200 205

Tyr Ser Trp Asn Ser Asn Ala Ser Ile Ile Phe Leu Glu Gln Pro Val

210 215 220

Gly Val Gly Tyr Ser Tyr Ala Glu Lys Ser Thr Val Ser Ser Thr Lys

225 230 235 240

Gln Ala Ala Glu Asp Val Phe Ala Phe Leu Gln Leu Phe Phe Ala Lys

245 250 255

Phe Ile Thr Phe Ala Ser His Asp Phe His Ile Ala Gly Glu Ser Tyr

260 265 270

Ala Gly His Tyr Ile Pro Asn Ile Ala Ser Val Ile Ala Asn Lys Glu

275 280 285

Glu Lys Phe Phe Asp Leu Thr Ser Val Met Ile Gly Asn Gly Ile Thr

290 295 300

Asp Ala Leu Ile Gln Tyr Tyr Tyr Tyr Ile Pro Met Ala Cys Asn Ala

305 310 315 320

Thr Glu Ser Gly Tyr Lys Gln Leu Ile Ser Asp Asp Glu Cys Glu Asp

325 330 335

Leu Glu Glu Met Tyr Pro Arg Cys Ala Ala Leu Ile Asn Ser Cys Tyr

340 345 350

Lys Ser Gln Thr Ala Leu Thr Cys Leu Pro Ala Asn Leu Tyr Cys Glu

355 360 365

Arg Met Met Asn Pro Phe Glu Lys Thr Gly Leu Asn Tyr Tyr Asp Val

370 375 380

Arg Lys Pro Cys Leu Gly Asp Gly Cys Tyr Pro Gln Met Asn Ala Ile

385 390 395 400

Asp Glu Tyr Leu Asn Gln Pro Glu Val Met Thr Ala Leu Gly Ser Glu

405 410 415

Val Lys Ser Tyr Val Gly Cys Asp Asn Gln Val Phe Arg Asn Phe Ile

420 425 430

Leu Ser Gly Asp Glu Pro Lys Pro Phe Gln Gln Phe Val Ala Glu Ile

435 440 445

Leu Glu Leu Asn Ile Pro Val Leu Ile Tyr Ala Gly Asp Lys Asp Tyr

450 455 460

Ile Cys Asn Trp Leu Gly Asn Arg Gly Trp Ser His Glu Leu Glu Trp

465 470 475 480

Thr Gly Lys Glu Thr Phe Asn Ala Ala Asn Thr Thr Asn Trp Tyr Ser

485 490 495

Lys Ile Asp Gly Thr His Ala Gly Thr Ala Lys Ser Asn Gly Leu Leu

500 505 510

Thr Phe Leu Arg Val Phe Asp Ala Gly His Met Val Pro Tyr Asp Gln

515 520 525

Pro Ser His Ser Leu Asp Met Val Asn Arg Trp Ile Ser Gly Asn Tyr

530 535 540

Arg Phe Thr

545

<210> 5

<211> 417

<212> PRT

<213> 盔形毕赤酵母(Pichia galeiformis)

<400> 5

Met Val Trp Leu Ser Ala Leu Ser Leu Leu Ala Ile Ala Ala Ala Thr

1 5 10 15

Asn Ala Ala Pro Ala Pro Ala Pro Asn Gly Leu Ile Pro Asp Val Leu

20 25 30

Ser Ala Val Met Asp Ser Ser Glu Asn Leu Gly His Leu Leu Glu Glu

35 40 45

Gly Leu Ser Thr Asp Glu Tyr Lys Phe Lys Phe Pro Ile Phe Ala Glu

50 55 60

Asn Pro Glu Leu Phe Lys Leu His Ser Gln Leu Val Asn Ile Glu Ser

65 70 75 80

Ile Ser Gly Asn Glu Leu Asp Val Ser Asp Phe Leu Gln Lys Tyr Leu

85 90 95

Asn Lys Leu Gly Leu Gly Phe Asp Leu Thr Ile Asp Lys Thr Gly Lys

100 105 110

Asn Ile Tyr Ala Ser Gly Ser Lys Thr Gly Ser Thr Lys Val Leu Leu

115 120 125

Thr Ser His Ile Asp Thr Val Pro Pro Tyr Ile Asn Tyr Phe Ile Glu

130 135 140

Asn Thr Glu Asp Gly Glu Val Ala Ile His Gly Arg Gly Ser Cys Asp

145 150 155 160

Ala Lys Gly Ser Val Ala Ala Gln Ile Ile Ala Ala Lys Glu Leu Leu

165 170 175

Glu Glu Gly Leu Val Thr Thr Asp Asp Leu Ser Leu Leu Phe Val Phe

180 185 190

Gly Glu Glu Val Gly Gly Ala Gly Met Arg Ser Ala Asn Ser Tyr Phe

195 200 205

Ile Glu Lys Gly Ile Asn Trp Asp Ala Val Ile Phe Gly Glu Pro Thr

210 215 220

Glu Asn Lys Leu Ala Val Gly His Lys Gly Ile Tyr Met Ala Glu Leu

225 230 235 240

Glu Val Phe Gly Phe Ala Ser His Ser Gly Tyr Pro Glu Ile Gly Ile

245 250 255

Asp Ala Asp Lys Ile Leu Ile Glu Ile Met His Glu Leu Glu Thr Tyr

260 265 270

Asp Trp Pro Lys Ser Glu Leu Leu Asn Leu Thr Thr Leu Asn Ile Gly

275 280 285

Leu Phe Ser Gly Gly Thr Ala Gly Asn Val Ile Ser Pro Tyr Ala Lys

290 295 300

Cys Ser Leu Leu Met Arg Thr Ala Val Asn Ala Asp Glu Ile Ile Pro

305 310 315 320

Arg Val Lys Asn Ile Val Ser Lys Tyr Gly Ser Gly Ala Lys Asp Val

325 330 335

Lys Leu Asp Val Gln Val Thr Asp Asp Pro Val Ser Leu Asp Tyr Asp

340 345 350

Val Glu Asp Phe Asp Thr Met Ile Ala Ser Tyr Ala Thr Asp Ile Pro

355 360 365

Lys Leu Glu His Arg Gly Phe Lys Arg Tyr Leu Tyr Gly Pro Gly Ser

370 375 380

Ile Leu Val Ala His Gly Asp Asn Glu Tyr Val Thr Ala Ser Ser Leu

385 390 395 400

Leu Gln Ala Val Ala Asp Tyr Lys Lys Leu Val Leu His Ala Leu Gly

405 410 415

Lys

<210> 6

<211> 2571

<212> DNA

<213> 盔形毕赤酵母(Pichia galeiformis)

<400> 6

atgaaacttt taacaccctt gctgttagca ttagcagaat gttgcaaggc atatacattt 60

gatacttcca atataattga ttacaatgtc acccgttctt cctatcatat caatgctgac 120

aatgagacca acttcagcta ctggtctttg ggcgtaacaa actcctcact tacatatgca 180

gacggttcca acataacagt tgctataata ggtgacggtg ttaactccaa ataccttcca 240

tcgcacgatt ccgttatttc ggtctcagac acctatacca attctaccta ccttttctct 300

gaaaacgaac ttaccgaaga cactctggcc gctaaagtct tgctggccaa tgctccaaac 360

actaagatca aatcttacaa gatttttaga aactctgatt caacaacatc tgtcacatcc 420

aaactagtct ctgccgtcct tgctgctgtc gaggataaaa ctgttgactt tatttacatc 480

aacacatggc aggcatatgg aggttggtca gaatcttatc tttctgtttt gctgaacgat 540

gttggtcttc ataagccaat tgttttacca atgggtcaaa acaacggtat tttccaattc 600

tctgatggcg cagcagctgc aaatgttatt gctgttggtg gtgcaggctt tgaaggctta 660

cctggttatg ctaccgatat cgatggcaag atttatggcg tctatgctga gagctcattg 720

agcctatatg agaataacac gttaattctt ggtaagttag actccaattg ccacttaaaa 780

aacaatgtta gcctcgatga aaccgatctg ttattcctcg atgttagttc atgctctgct 840

gatcagattg ttgaggccat taatgattca aatttcaccc aattgttgac caccggagat 900

gaatatcaaa cctacaactt ctctgatgat tctggtattg tatctggtat gatcggaaga 960

gaaaacggcc ttaaaattct gcaacgttta gaagaaggcg ataccatcgt tgctcatggt 1020

agaaacgaag aaaatatgat cattgcagat tatgaatttg acgaattttc gatcaatttt 1080

aactcatcca tgggtccagc acttgatctt catctagcac caaatatcat agcacaaagt 1140

gccttctatt ttgatgactt gagtgtctac gcctctggta cttcattatc tgcagcttat 1200

gttgccggta tcatcgcagc gtatgcttct tctgttaacg gcagctcttg gtccgacggt 1260

atcattgata gaatcaccac ttctggcagt caagtcaact ttagaaactt tacttatact 1320

tcatcggaag ttgaaaaccc aatcatccag ggcggtggta tcatcaatgc cgaagagttt 1380

gttctcaaga attatgatgt aactccaggc tatttttcat tgaaagaggc taacaccact 1440

accgaagtca ctttcaccat cacgaacaat gaaaacgagg caaaaagatt tcacatcagt 1500

ggcttggatg ctatcaccat ctactcgcaa gactctgatg tcgacgcaaa cagtaataat 1560

atgtacccat tcccaattta tggtaacgca acggctgctg aagtattgat cacgaagaac 1620

actgttctcg tccctgcagg taagtctgtg aatgttacca tttccatcac accaccaaat 1680

tactctactg aagaatcccg gctaccgatt tatggtggtt acattcaggt cactgatttg 1740

gccaccgatt ttgtttccaa cattccatac tttggcagta ctgtggagtt gcactctttg 1800

gcaactcaga ctgacgaagc cttctactgg aaagagttcc attatgagcc agacggaaaa 1860

tactgcgata tgtgggtcaa cgggtctgtc ttgaatgaca ccagccatta catgcaaatc 1920

tacttcaacg ataggattgg ttccccactg atcgatattt tagttctgga aaaagattgg 1980

acacaagatg actttgttta tcctccagtt gctggtgaga acaagtttgt tcaacgtatt 2040

caaggaacaa gaatacaagg tggatatcca tggcattacg ctccaagatc taacagcaca 2100

cagtctgagc aatgggatga ttggcacgat ggtttgttag ccgacggtag cgtcttaccc 2160

gccggagagt ataagatcct tttaagaatc ttaaagcaca atgttgagga cccaggcgat 2220

tgggacgagt ggttcaatca catttctcca tggtttgtca tggactatgc aaatgtcacc 2280

gaagttgaac tcgcatcatc gaccatctcc atatcccacc acctcagcac ctccactctg 2340

aggaccgtct ctaaaaccgc atctagcaac accggaacga aggaaaccaa cagtattgtt 2400

actccaacta ccactgcgag tgcaacttcc acttcctcca cggatatgaa tatgtctggc 2460

atggatatgt ctggcatgga tatgtccggc atggatatgg cgatgagttc cacctcgatg 2520

ggtgccacag ccacaaccac ttcgatggat atgggcgaca tgcatatgtg a 2571

<210> 7

<211> 1629

<212> DNA

<213> 盔形毕赤酵母(Pichia galeiformis)

<400> 7

atgaagctct cttcttccct ttgcttctct atcctcactt ctttggcagc caccaaggtc 60

tccgccttgg ttcttccaaa cttgaaggac atgccagcag tcgtgagcac gtctgacagg 120

gctccagcgg ttgtcgaaaa cgctgttgtt gaaagtgttg actcgttccc atccgtcaag 180

ctcgctgatg acgccatctt ggatgtcgag ttcaacgaga acgccattcc aaattccttc 240

attgttatct tgaaggacga catctcggac aacgcattcg aattccacaa gttgtgggtt 300

tctgacgcgc aggttcaatc ttttgctaaa ctgaacaaga acgaggttga cgcctttgaa 360

gactctgtca acgccttgaa attcaagaac agggccggcg gtatcggtca cgttttcgac 420

tcacctggcc taagaggtta ctcgggtttg ttcacacctg aagttatcaa ccttttgaga 480

cgctccgaag acgttgctgt cattgaattg gactctgtcg tgcatgctat tgaggcagaa 540

acccagaatg gtgctccatg gggtctttct agaatctctc acagagaagc tttaggcttg 600

ggtaactaca acaagtacct ctatgatgct gacgctggtg aaggtgttac tgcctacgtt 660

atcgacaccg gtgtcaatat cgaacacgaa caatttggtg gtagagcagt ctggggtgca 720

accatcccat ctgacgacac agacaccgac ggtaacggtc acggtaccca ttgtgctggt 780

accattggtt ccgaagacta tggtgtttct aaaaaggcaa agattgttgc cgtcaaggtc 840

ttgaagtcca atggttccgg taccatgtct gacgttgtca agggtgttga attcgctgca 900

aacgcccacg ttaaagaagc aaaggaagct aagtcaggat tcaagggttc cactgctaat 960

atgtccttag gtggcggtaa gtctccagcc ttagatttgg ctgtcaacgc cgccgtcaag 1020

gcaggattgc actttgccgt cgctgctggt aacgacgacg cggatgcttg taactactct 1080

ccagctgcag cagaaagtgc tgttactgtc ggtgcttcca ccctatctga taccagagct 1140

tacttctcaa actatggtaa gtgtgtcgac atttttgcac ctggtttgaa tgttctctcc 1200

acttacattg gatccaacac tgctactgca atcttgtctg gtacatccat ggcatctcct 1260

cacgtttgtg gtttgttaac atacttcctt tccttggctc cttcctccga ttctatgttc 1320

tccacggcta gcatcactcc aaagcagtta aagcaaaaca ttttgaagta tggtactaag 1380

aatgcattgt cttccatccc agatgatgat actccaaaca ttttgatcta caatggtgca 1440

ggtggtgata tcagcgattt ctggaatttc gaaagtgaat catcatcctc tgatgatctc 1500

caaagatcaa ttgaagttgc aaacgaattt gcagatactc aattgagcaa agccaagcaa 1560

gagatcaagg taatccttag cgaaataacc caagtggtcg attccgttga agatgttatt 1620

tctgtttag 1629

<210> 8

<211> 1524

<212> DNA

<213> 盔形毕赤酵母(Pichia galeiformis)

<400> 8

atgaaactgg acttcctgac ctctgcactt attttgtcta acgttgccaa tgctgcaatt 60

ttgccccaag ctgctctaaa tttagaaaac cagattgagc atttggagag ctacctcgaa 120

ctagcggact ctcctagttt tgcggatttg ccattggtca actccactga attgcaagcc 180

ttgattactc acgaagctct ctttactcgt gcggaaaagc tctaccaaat tgcaggtcat 240

tcaaacaaaa gttacggcca cccaactcgt gttattggtt ctccaggtca ctgggctact 300

atcggctaca ttgagcacga attgaaaaaa ttgcatgcgt attacaacgt cacaactcag 360

gctttcaaag ctttcatgtc agaaattttc agttttaatg ctaccattga taatgaggtt 420

ccaaagagct tggttgagat ggacatgtca cctccaactc cacacaaggc cccagtttac 480

ggcttactag ccttagtatc caactctggt tgccacgatg atgactatgc taacgttact 540

ggtaatattg catttatcaa gagaggtgag tgtgcctttg gcgacaaggc acaattggct 600

gctaagcacg gcgcattggc tgccatcatt tacaataatg aaccgggaga aatcagaggt 660

actttgggtg tccctccaga aggagttaag gtttcttcct ccgtcggtat caaccaggaa 720

gaaggtgaga aatatgctaa gcttttacgt gagggaaagg ttttgaatgc ttctgttcaa 780

gttgactctt atgttggccc aattaccact gttaacgtca ttgcggaaac caatcacggt 840

gatccaaaca atattgtcat gctaggggca cattctgaca gtgttgcagc aggtccaggt 900

attaacgatg atggttccgg tactatctca cttctagaag ttgcaaagca gttaaccaat 960

taccgtgttg aaaacaaagt caggtttgct ttctgggctg ccgaagaaga gggtctttta 1020

ggctctaact actacgctga caacctgacc cctggtgaga atgaaaaaat cagattattc 1080

atggactatg acatgatggc atcaccaaac tacgaatacg aagtctatga cgcaaacaac 1140

gttgacaatc cgaagggcag tgaggaatta aaaaatcttt atattgacta ctacacatcc 1200

catggtctca attatacttt gattccattt gatggccgta gtgactatgt tggcttcatt 1260

gaacatggta ttcccggcgg aggtattgct gcaggtgccg aaaagaacaa tgctgagaac 1320

ggcgacgttt tagataagtg ctaccaccaa ttatgtgacg atttatccaa tattgcttgg 1380

gacgcatttt taatcaacac ccaattaatt gcacacagtg ttgctactta cggtgctagc 1440

ttggatggct tcccaactag agaaaatgat accaccgtcg attcattagc atttgcatac 1500

agaggtgaca agttaatctt ttaa 1524

<210> 9

<211> 1644

<212> DNA

<213> 盔形毕赤酵母(Pichia galeiformis)

<400> 9

atgaagattg ccttgaccac cgcagcaatt gcttcactta gtttcggctt ggcttctgcc 60

aaccccgttt tgggctacaa caggcaaaat gtacttactt acaacgaaag gcaacatcat 120

ggacacccca tccttgataa atacattgag aagggttttt ttgacgagtc tttagtagat 180

gagactacct ctgaggcgtg gaaattgttg gaacaaaccg tggatgcttc aaagatagag 240

caggaactta agcgttacgg ctcagaagaa tccaaggccc ctaaaagtgc gttaagacta 300

ccagaattga aggataacac taattacgaa tatattagct tatcagagtt tagctcatat 360

ggattacgga taaaaaaaca tctaggctca gatccttctg tcttgggtgt cgacaaggta 420

aaacaatgga ctggatactt tgacataaac gaggaggaca aacatttgtt cttttggttc 480

tttgaaagca gaaatgaccc tgaaaatgac ccagttattc tctggttgaa cggaggacct 540

ggatgttcat cgatgactgg attgtttttt gagttgggcc catcttcgat aaatggcaca 600

actttgaagc caatcaacaa tccttactcg tggaattcta atgcttctat tatttttttg 660

gagcagccag taggggtggg ttattcttat gctgaaaaaa gcaccgtttc ctccacaaag 720

caagcggcgg aagatgtgtt tgctttctta cagctctttt ttgcaaagtt catcactttc 780

gcctctcatg attttcatat agccggtgaa tcttatgctg ggcattacat cccaaacatt 840

gcaagtgtga tcgcaaacaa agaagaaaaa tttttcgatt tgacttccgt catgatcgga 900

aatggtatta ccgacgcatt gatacaatac tattattata ttccaatggc ctgtaatgca 960

actgaatcag ggtataaaca attgatttct gacgatgaat gcgaagattt agaagagatg 1020

tacccaagat gtgcggcttt gataaattca tgttataaat ctcaaacagc gttgacttgc 1080

ctaccagcta atctctattg tgaaagaatg atgaacccat ttgagaaaac aggactaaat 1140

tactatgacg ttagaaagcc atgcttgggt gacggttgtt accctcaaat gaatgcaatt 1200

gatgaatatt taaaccaacc agaagttatg actgctttgg gttctgaagt caagtcttac 1260

gttggatgtg acaaccaagt cttcagaaat ttcattcttt caggcgatga acctaaacca 1320

ttccaacaat ttgtcgctga aatcttagaa ctcaacattc cagtattgat ttatgcgggt 1380

gacaaagact acatttgcaa ctggttagga aataggggat ggtcacatga acttgaatgg 1440

accggaaagg aaactttcaa tgctgcgaac acaacgaatt ggtactcaaa aatagatggt 1500

actcacgctg gaactgctaa atcgaatggt ttgttgacct ttttaagagt ctttgatgca 1560

ggacatatgg ttccatatga ccagccatcc cacagtttag acatggtaaa cagatggatc 1620

agtggtaatt accgttttac ttaa 1644

<210> 10

<211> 1254

<212> DNA

<213> 盔形毕赤酵母(Pichia galeiformis)

<400> 10

atggtttggt tatccgcttt aagcttactt gcaatcgcag cagccacgaa tgcggcccca 60

gccccagctc cgaatggact aatcccagat gtgttatcgg cagttatgga ttcaagtgaa 120

aatctgggcc atttgctgga ggaaggcctt agcactgatg aatacaagtt taagttcccc 180

atttttgctg aaaaccctga actttttaaa ctccacagtc aacttgtaaa tattgaatct 240

atcagtggca atgaattgga tgtatctgat ttcctccaaa aatacctcaa taaactagga 300

ctcggtttcg acttgacaat tgacaaaaca ggcaaaaata tttatgcatc aggtagtaaa 360

actggtagta ctaaagtttt attaacatct catatcgaca ctgtaccacc atatattaat 420

tacttcatcg aaaatacaga ggacggtgag gtagcaattc atggccgggg ttcttgtgat 480

gctaaaggtt ctgttgcagc ccagataatt gcagccaagg agcttttaga agaaggtttg 540

gttactactg atgatttgag tttattgttt gtcttcggtg aagaagttgg tggtgctggc 600

atgagaagtg ccaattctta ctttattgaa aagggtatca actgggatgc tgttattttc 660

ggtgagccaa ctgagaataa acttgctgtt ggacacaagg gtatttacat ggccgagctt 720

gaggtctttg gctttgcaag tcactctggc tatccagaga ttggaataga cgcagataag 780

attttaatcg agataatgca cgagcttgag acttacgact ggcctaaatc tgagcttttg 840

aatttaacaa cgctgaatat tggtttattt agtggtggaa ctgctggtaa cgtcatttcc 900

ccctacgcaa agtgctcact tttgatgaga acggcagtaa acgctgatga aataattcca 960

agagtcaaaa acattgtttc taaatacggg tctggtgcga aggatgttaa acttgatgtt 1020

caagtcacgg atgatccagt ctccttggac tatgatgttg aagattttga tacaatgatc 1080

gcatcatatg ctaccgatat tccaaaatta gagcataggg gcttcaagag atatctttat 1140

ggtccaggtt ctattttggt tgcacacggt gacaatgaat acgtcacagc atcaagcttg 1200

ttacaagcag ttgctgacta caaaaaattg gtcttgcatg ctctaggcaa atag 1254

<210> 11

<211> 47

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 11

caatttacta ttctagggta ccatgaaact tttaacaccc ttgctgt 47

<210> 12

<211> 42

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 12

gtatgggtat ctagactgca gtcacatatg catgtcgccc at 42

<210> 13

<211> 44

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 13

caatttacta ttctagggta ccatgaagct ctcttcttcc cttt 44

<210> 14

<211> 46

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 14

gtatgggtat ctagactgca gctaaacaga aataacatct tcaacg 46

<210> 15

<211> 44

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 15

caatttacta ttctagggta ccatgaaact ggacttcctg acct 44

<210> 16

<211> 46

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 16

gtatgggtat ctagactgca gttaaaagat taacttgtca cctctg 46

<210> 17

<211> 42

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 17

caatttacta ttctagggta ccatgaagat tgccttgacc ac 42

<210> 18

<211> 46

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 18

gtatgggtat ctagactgca gttaagtaaa acggtaatta ccactg 46

<210> 19

<211> 45

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 19

caatttacta ttctagggta ccatggtttg gttatccgct ttaag 45

<210> 20

<211> 43

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 20

gtatgggtat ctagactgca gctatttgcc tagagcatgc aag 43

Claims (8)

1.盔形毕赤酵母Peptidase类分泌蛋白，其特征在于，氨基酸序列如SEQ ID No.1所示。

2.权利要求1所述盔形毕赤酵母Peptidase类分泌蛋白的编码基因。

3.如权利要求2所述的编码基因，其特征在于，核苷酸序列如SEQ ID No.6所示。

4.含有权利要求2所述编码基因的重组表达载体。

5.如权利要求4所述的重组表达载体，其特征在于：是将核苷酸序列如SEQ ID No.6所示的盔形毕赤酵母Peptidase类分泌蛋白的编码基因克隆入植物表达载体pCAMBIA2300的多克隆位点KpnI和PstI之间而得到。

6.含有权利要求4所述重组表达载体的工程菌。

7.如权利要求6所述的工程菌，其特征在于，是将所述重组表达载体转入农杆菌GV3101中而得到。

8.权利要求1所述的盔形毕赤酵母Peptidase类分泌蛋白或权利要求6所述的工程菌在制备诱导柑橘果实绿霉病抗病性的保鲜剂中的应用。

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