CN113151293B - 抗逆基因线路AcDwEm及其提高作物耐盐抗旱耐高温的应用 - Google Patents

Abstract

本发明利用合成生物学方法设计创建了一种具有提高宿主细胞抵抗高盐、干旱，高温胁迫能力的功能线路AcDwEm。本发明构建了该抗逆功能线路的重组载体，通过农杆菌介导侵染转化的方法将其在模式植物油菜和水稻中整合重建。实验证明，所述功能模块在模式植物宿主细胞中表达后，能显著增强作物的耐高盐、抗干旱和抗高温的能力，可用于农作物新品种抗逆性改良。

Description

抗逆基因线路AcDwEm及其提高作物耐盐抗旱耐高温的应用

技术领域

本发明属于合成生物学领域，涉及一种多模块抗逆功能线路具有提高生物抵抗干旱和高盐胁迫能力的应用。

背景技术

土壤盐碱化、频繁干旱和长时期高温是全球农业最具破坏性的非生物胁迫，通过对种子萌发、植物生长发育、植物活力和作物产量的不利影响大幅降低农业生产力。

目前，在全世界范围内越来越广泛应用基因工程策略培育抗逆品种。但由于作物耐盐抗旱耐高温性是一个复杂的性状，同时受到多个基因和因素的影响，因此单基因转化操作并不理想，培育的耐逆性提高植物在无压力条件下表现不佳。

进入新世纪以来，新一代合成生物学的原始创新与集成应用加快突破，全基因组设计育种技术促进传统农业品种升级换代，孕育新一轮农业科技革命和产业变革。因此，运用现代合成生物学设计方法，通过人工设计蛋白质功能元件、启动子，并通过多个基因组合方式，人工构建特异性响应高盐胁迫信号、干旱信号和高温胁迫信号的应答功能模块，或有望能够创建出提高生物抵抗干旱和高盐胁迫的能力的抗逆功能体系。

发明内容

本发明的目的是创建一种能够提高生物抵抗干旱和高盐胁迫的能力的抗逆功能线路。

本发明利用现代合成生物学设计方法，优化改造抗逆元件。通过蛋白质功能元件的人工设计、启动子的组织特异性和逆境响应设计，人工构建特异性响应高温胁迫信号的应答功能模块、抗逆功能稳定器模块和组织特异性高效抗逆功能模块，组装形成智能响应定向表达的全新抗逆功能线路，命名为AcDwEm。

通过如下研究，首次鉴定了抗逆功能线路AcDwEm具有提高模式植物抗旱耐盐耐高温能力，可用于新一代抗逆作物新品种的培育。具体研究工作如下：

1、人工设计抗逆功能线路AcDwEm的构建

通过合成生物学设计逆境胁迫应答功能模块，设计构建特异性响应高温胁迫信号的应答功能模块、抗逆功能稳定器模块和组织特异性高效抗逆功能模块，组装形成智能响应定向表达的全新抗逆功能线路，命名为AcDwEm。利用人工化学合成的方法获得了抗逆功能线路AcDwEm全长核酸序列。将抗逆线路AcDwEm连接于pBI-121载体上，构建植物表达载体pBI-AcDwEm，将该表达载体转化根癌农杆菌EHA105(详见实施例1)；

2、转抗逆功能线路AcDwEm油菜与水稻的获得

通过农杆菌介导的转基因植物构建方法，将抗逆功能线路AcDwEm与模式植物油菜和水稻整合重组，通过抗性筛选和PCR验证的方法，培养得到稳定遗传的阳性转基因植株(详见实施例2,4)。

3、转抗逆功能线路AcDwEm油菜的耐盐抗旱性能分析

分别以NaCl和聚乙二醇PEG-6000作为添加物质来模拟盐胁迫和干旱胁迫，采取浇灌的方式进行胁迫处理。将获得的已鉴定为阳性的转基因种子与野生型种子培养出苗，进行逆境处理。每天为植株浇灌等量的胁迫液，分别在胁迫处理的0,1,3,7,14,21d取样拍照，观测生长状态测定生理指标。

4、转抗逆功能线路AcDwEm水稻的耐高温性能分析

将野生型水稻与阳性转基因水稻种子萌发出苗，进行高温处理。培养环境设置，45℃光照14小时，45℃黑暗条件10小时，处理7天，观测植株生长状态。

实验结果表明：正常条件下，抗逆功能线路AcDwEm对宿主植株生长发育无影响，逆境条件下具有显著提高油菜与水稻耐盐抗旱耐高温能力的功能，可用于新一代抗逆作物新品种的培育

序列表信息

SEQ ID NO.1：抗逆功能线路AcDwEm的核苷酸序列。

SEQ ID NO.2：功能模块1的核苷酸序列。

SEQ ID NO.3：功能模块1的编码蛋白的氨基酸序列。

SEQ ID NO.4：功能模块2的核苷酸序列。

SEQ ID NO.5：功能模块2的编码蛋白的氨基酸序列。

SEQ ID NO.6：功能模块3的核苷酸序列。

SEQ ID NO.7：功能模块3的编码蛋白的氨基酸序列。

附图说明：

图1抗逆线路AcDwEm载体构建图；

图2转基因油菜Bn-AcDwEm和非转基因油菜(WT)耐盐抗旱实验结果比较；

图3转基因水稻Os-AcDwEm和非转基因野生型水稻耐高温实验结果比较。

具体实施方式

以下实施例中所举的质粒、菌株、模式植物只用于对本发明作进一步详细说明，并不对本发明的实质内容加以限制。凡未注明具体实验条件的，均为按照本领域技术人员熟知的常规条件或按照制造厂商所建议的条件。实施例中所举的质粒、菌株、植株来源如下：

克隆载体pJET：为ThermoFisher公司市售产品；

穿梭载体：pBI-121：本实验室保存；

根癌农杆菌EHA105：本实验室保存；

水稻材料：水稻种子ZH11为本实验室保存。

甘蓝型油菜材料：油菜种子84100-18为本实验室保存。

实施例1抗逆功能线路AcDwEm的设计与重组根癌农杆菌的构建

一、实验材料

克隆载体pJET：为ThermoFisher公司市售产品；

穿梭载体：pBI-121：本实验室保存；

根癌农杆菌EHA105：本实验室保存。

二、实验方法

1.通过合成生物学设计逆境胁迫应答功能模块，设计构建特异性响应高温胁迫信号的应答功能模块、抗逆功能稳定器模块和组织特异性高效抗逆功能模块，组装形成智能响应定向表达的全新抗逆功能线路，命名为AcDwEm。利用人工化学合成的方法获得了抗逆功能线路AcDwEm全长核酸序列。其大小为3737bp，将其克隆于载体pJET上，构建了含有完整抗逆功能线路的重组克隆质粒pJET-AcDwEm，并测序验证；然后通过EcoRI 和HindIII双酶切获得含有粘性末端的抗逆线路AcDwEm片段及穿梭载体pBI-121载体片段，将抗逆线路AcDwEm连接于pBI-121载体上，构建植物表达载体pBI-AcDwEm，将该表达载体转化根癌农杆菌EHA105，利用卡那霉素抗生素抗性筛选阳性重组菌株，并通过菌落PCR测序验证。

三、实验结果

利用人工化学合成的方法获得了抗逆功能线路AcDwEm全长核酸序列，成功构建将含有功能线路SyAcDwEm的植物表达载体pBI-AcDwEm，并转化根癌农杆菌EHA105。经PCR、酶切，测序验证插入序列正确，将该菌株命名为EHA-AcDwEm。

四、实验结论

完成表达抗逆功能线路AcDwEm的重组根癌农杆菌EHA-AcDwEm的构建。

实施例2农杆菌介导的转抗逆功能线路AcDwEm油菜的获得

一、实验材料

重组菌株EHA-AcDwEm：实施例1获得

甘蓝型油菜材料：油菜种子84100-18为本实验室保存。

二、实验方法

去油菜种子，分别用75％乙醇和0.1％的HgCl2浸泡消毒，均匀放置于植物组织培养基，24℃组织培养室培养一周。用消毒手术剪取油菜幼苗的下胚轴，置于预培养基上，光照培养2-3天，预培养外植体。

转接活化表达抗逆线路的重组农杆菌菌株EHA-AcDwEm，离心收集菌株重悬至OD600＝1.0。将预培养的外植体浸泡于农杆菌菌液中90s，晾干后转移至共培养基上，暗培养2-3d。随后将生长良好的外植体转移至诱导培养基上培养。

选取愈伤组织长势良好的外植体转移到添加抗生物的筛选培养基上，光照培养45-50 d，在分化出芽。将分化出芽的愈伤组织转移到生根培养基，光照培养2周，待根系出现茎干长出4-5cm，转移至培养土中进行练苗，经驯化后移栽至温室，PCR检测阳性油菜苗。

三、实验结果

利用农杆菌介导的外植体共培养法，将抗逆功能线路AcDwEm转化油菜，经过侵染油菜外植体经过诱导培养、筛选培养、生根培养与练苗移植等步骤，经过PCR验证，最终得到表达抗逆功能线路的转基因油菜Bn-AcDwEm，可用于后续抗逆性能研究。

四、实验结论

通过农杆菌介导转化方法，最终获得转抗逆功能线路AcDwEm油菜Bn-AcDwEm 实施例3转抗逆功能线路AcDwEm油菜的抗逆性分析

一、实验材料

转基因油菜：Bn-AcDwEm

对照：非转基因野生型油菜

二、实验方法

分别以NaCl和聚乙二醇PEG-6000作为添加物质来模拟盐胁迫和干旱胁迫，采取浇灌的方式进行胁迫处理。

将获得的已鉴定为阳性的转基因油菜种子与野生型种子在MS固体培养中，待苗长出真叶后移栽到装有基质的塑料盆中，浇灌MS营养液待幼苗长出5-6片真叶进行逆境处理。

每天为植株浇灌等量的胁迫液，分别在胁迫处理的0,1,3,7,14,21d取样拍照，观测生长状态测定生理指标。

三、实验结果

生长状态观测结果显示：

盐胁迫和干旱胁迫处理前，转基因油菜Bn-AcDwEm与野生型油菜生长状态无差异，农艺性状未受影响。

15％重度干旱胁迫下7天时，野生型油菜开始枯黄落叶萎蔫的表型，转基因油菜Bn-AcDwEm生长速率变慢，但叶片与茎干生长未受明显影响；

干旱处理14天时，野生型油菜已经完全枯死，转基因油菜开始出现萎蔫。

高盐胁迫实验中，300mM NaCl胁迫处理7天，野生型油菜出现严重失水干枯的情况，转基因油菜Bn-AcDwEm部分叶片泛黄，生长状况显著好于野生型；

高盐处理14天，野生型油菜已经基本干枯死亡，转基因油菜Bn-AcDwEm仍存活，仅叶片出现卷曲，茎干萎蔫，生长变缓。

四、实验结论

逆功能线路AcDwEm在模式植物油菜中表达，显著提高了宿主植物耐盐性能和抗旱性能，具有重大育种应用潜力

实施例4农杆菌介导的转抗逆功能线路AcDwEm水稻的获得

一、实验材料

重组菌株EHA-AcDwEm：实施例1获得

水稻材料：水稻种子ZH11为本实验室保存。

二、实验方法

水稻种子去皮，用75％乙醇和0.1％的HgCl2浸泡消毒，均匀放置于植物组织培养基， 24℃组织培养室培养2周。用消毒手术剪取水稻愈伤组织，置于预培养基上，黑暗培养2周。

转接活化表达抗逆线路的重组农杆菌菌株EHA-AcDwEm，离心收集菌株重悬至OD600＝1.0。将预培养的外植体浸泡于农杆菌菌液中30分钟，晾干后转移至共培养基上，暗培养2-3d。随后转移至诱导培养基上培养。

选取愈伤组织转移到添加抗生物的筛选培养基上，暗培养2周，复筛一次暗培养2周，再 分化培养1周。将分化出芽的愈伤组织转移到生根培养基，待根系出现茎干长出4-5cm，转移至至温室，PCR检测阳性水稻苗。

三、实验结果

通过农杆菌介导愈伤组织共培养法，将抗逆功能线路AcDwEm转化水稻，经过侵染水稻愈伤组织经过诱导培养、抗性筛选培养、生根培养与建苗移植等步骤，经过PCR验证，最终得到表达抗逆功能线路的转基因水稻Os-AcDwEm，可用于后续抗逆性能研究。

四、实验结论

通过农杆菌介导转化方法，最终获得转抗逆功能线路AcDwEm水稻Os-AcDwEm

实施例5转抗逆功能线路AcDwEm水稻的抗逆性分析

一、实验材料

转基因水稻：Os-AcDwEm

对照：非转基因水稻

二、实验方法

转抗逆功能线路AcDwEm水稻Os-AcDwEm耐高温性能分析

将野生型水稻与阳性转基因水稻种子萌发出苗，进行高温处理。

将水稻种子在MS培养基中培养出苗。当水稻苗长到2叶1心时，大约12天左右进行胁迫处理，胁迫培养环境设置，45℃光照14小时，45℃黑暗条件10小时，处理7天，观测植株生长状态。

三、实验结果

生长状态观测结果显示，

无高温胁迫条件下，转基因水稻Os-AcDwEm出苗和生长与水稻野生型无差异。

高温胁迫处理7天，野生型水稻叶面枯黄卷曲，茎干萎蔫干枯，转基因水稻 Os-AcDwEm植株，生长几乎未受到影响。

四、实验结论

逆功能线路AcDwEm显著提高了宿主水稻的耐高温性能，具有重大育种应用潜力。

序列表

<110> 中国农业科学院生物技术研究所

<120> 抗逆基因线路AcDwEm及其提高作物耐盐抗旱耐高温的应用

<160> 7

<170> PatentIn version 3.1

<210> 1

<211> 10634

<212> DNA

<213> 人工序列

<400> 1

agctgagttg cttaatagag gaattgatgt agaagaaatc aggaagatta taactatgaa 60

aaaaagtact actgaatgat agattctcca cgttgcggga catggcgaac ttgatccact 120

tgtcgaagtg gggaacagtg tgctggttgc tggttttgag atgaaacttt atcatcttga 180

gagctgtgta gcgagttagg gtttcgttta tcgaagcagc atgcagtgta tcactatcat 240

caaaggaaag agaaggtata tcggaccata catgcctcca tcgtttttac aagacggaag 300

ttgtgatcga gagtttggtc ggaacaaata ataggatttg ttggaggatc tcatcaggca 360

ataagctgat taggtcaata ttccggcgat gacgaccacg atcgttacag tggataggaa 420

gactggaagt ggctgtggag ctctctcggg ccatcttact ttattaacct aacttgtgat 480

cttcttaatt agggtttaaa tcttaatttt agccgcatat tgttttctat atataataac 540

attctttcaa aatacatgtc aaacaattta tagcaaattt aattaactat tttttttaaa 600

aagtcttccc taataagtgc tcttagaaca ataaatcggc atttaaaaaa aaaaattggc 660

attttatttt tttcgttttt ttaaacttat tatatggtgt agtcgggcta tactggactt 720

ttgcaaaaat gtggttttta tattttatat tgtattaggt ttctgttaaa attaaatgag 780

aattttaatt aaaaagagaa attatttgtt aaaaaaaatc aggatggggt cctaattatt 840

atatgttttg attttctatg agaaagttgc accgtccatt gtttcttgaa aactattatc 900

tgactaaaag aacagaaaat gtaaagaaaa gacaaagaga cacagagacg actctgttaa 960

ataactctat agcagagtct ctcgagttaa atcaataaaa taaagacctg aaaacatata 1020

tttcttcgaa gcagtgtcta aaaccaatgt acaatttatg acaaaaggaa catgttattt 1080

tagtcgcata taattacaaa ataatcgcat gatttatcta agttggtctt tattaactct 1140

taacaaaaaa ataatataag aaaacagagt cagaatttaa aaaccactta attagtcctt 1200

caagaacaat tatcaaaacc ttaataatgt tttcatccaa taacatcctc gaagtctcct 1260

ctaaatcatt ggatccaacg aaattcatgt ttatctaaac taactcgaat aaagaaacga 1320

ttataataat tgcacactat gaaaaatatc agaagcgtca tagaaattgt cggctacctc 1380

catgcacgga accttcacga aacagttggt ccctcacaca cttcatcgcc acgctatacc 1440

acgtgtcaat tttacataca ccaaaacata tctactaatc atacctcttc acgtgtaaca 1500

aagtcccatt caacgtggca attacagacc ccaaaattat gaactaatca aacctcttca 1560

cgtgtcgcaa acttgtagaa cgttgaaacc ccccactcac acgaagtgta tatatcctct 1620

tcacaacaca aacataaaca ttacttcaaa caaagacttg aaagaactat ctttgttttc 1680

actcatatct tatctttatt aaatggcgat gtctttctca ggagctgttc tcactggtat 1740

ggcttcttct ttccacagcg gagccaagca gagcagcttc ggcgctgtca gagtcggcca 1800

gaaaactcag ttcgtcgtcg tttctcaacg caagaagtcg ttgatctacg ccttgacgtc 1860

cttgactctg cccggagggt ttggcagcgc gcctgtggcc aatttgtcga tgactttgga 1920

agtaaccaac cccaatcctc tgccgttgcg aatggctaat attgctgggg cgctcattat 1980

cgatggggcc gccgttggcg atgtgtcatt tcctaacgta gacatagcgg ctaggggggt 2040

atccacacaa agggcggatt tatctatacc tgtgacccta aatacagccg catcattctt 2100

gaaggttgcc cgtgggcagt tggttactta tagagttgat ggtggattta cttgatttct 2160

ccataataat gtgtgagtag ttcccagata agggaattag ggttcctata gggtttcgct 2220

catgtgttga gcatataaga aacccttagt atgtatttgt atttgtaaaa tacttctatc 2280

aataaaattt ctaattccta aaaccaaaat ccagtactaa aatccagatc aaagatacag 2340

tctcagaaga ccaaagggct ctttgccaat tcctctgtcc tcgataagag ctccaatacc 2400

agaaatgagt aaaaaaccaa ctccaatagt tcggattgta ctccatagct gttctttaaa 2460

gtgagtcctt tctgtggata tcgtatggat cggtgcacta gcagttccaa ggacaccatc 2520

ttttgttggt tttcctacgt ttctaaatgc cccaagacct ccaaaagtct cttcctccct 2580

tgcaacacca gcaataccta aaagaaaaac agcaaaacat ttcccaatat tatatagaga 2640

aatggctcag aagctgctaa tacaaatatg aaagcaagga cacaatccaa gtatccattc 2700

agattcaaac aaacagtctc tccctcctta cccacctctt tgcaatgttc gaaccagctc 2760

actttgatcg agcctatcaa ctttaaccaa tgccttaatg tattccgata acgcagaggc 2820

attcgcatgc aaagaaggtt ggctttcaaa cattctgata acagcctcag gatcatttct 2880

tcgaataagt tctctcagat gtgcaacctc attaacttct tctctatcac ggactctgcg 2940

agcaaagcta ccaacataac tcgattgaaa cctcgttcgc ggtaaagaag ctccaccacc 3000

acccacagct atagataaaa taaaaccaat ttacatcaaa aacacttcta aaacaacaag 3060

aacaactcat aaacggaatc agaatttacc tccagtaact cccactctag gaaaagagga 3120

gtaagctcta acaagtaagc ttcttaaact gctcaattcc ctttcatgac tcgaaacctg 3180

tcatcggtga agagaatcaa tacagatgaa gttaattact aatcccaaac tcaaaatgac 3240

actagacaac acatgaaaac attttgataa aagtgaatac ttttacccat tttgaatcaa 3300

aaaaattgaa actttttata ccaatttcaa aattaggtga cttgggtagt caaataaatc 3360

aaatgacaat atcacagaga caatctagaa tcctaaaaga caacattttg agggcagaga 3420

agaatttgag cttctagggt ttgaaaaata tcatctttag cttatgaatc acaaagatct 3480

tgataaaacc catcagaaat tattatctaa ttagatcaaa tccactacag tatcaaacca 3540

aagtcgaaac ctttttcgta ttaaaaattg gataaagggg aaaagagaaa aatgaaacaa 3600

aaaccttggt gatgatgcgc ctccaagcca tcaatcaaat ctctccttca cgatatctta 3660

aaaattggtg ttaatgtctg ataaatcgaa gttcctcgat ctatatcgga aacaaaagac 3720

ttcttcgttg tgtttgggga agaaccgttc taatcttatt ccctaaagtc ttaaaaacac 3780

taaattacac gtgagagacc tgtttggtta tcgggtgaga gaaaaattgt gcagcaggtg 3840

gagacacgca cgagatatgt aggtcgcctc ttaagtacat aaataccctt ggacataccc 3900

aataattcat tttagtaggc tttttctggc ggcccacatt aaaaagaagg acctagaaca 3960

taaattggca tcgttagaaa tgggcttaag taaaggccca tatgatatat atataaaaaa 4020

agagattcta gattagtaac gaagtttctg gaacattgtc ttgtcttgtc gccacgtgct 4080

cacataaatg tcaaagaagc ttcaatacag tgaaatgatc ttgtcttgtc tctagaacct 4140

tctcttctct ccccttataa tttcatttct ctctcctcca cgcctcaatc tctcaactca 4200

aaactcaaca ttttctgaag aaagtcgcaa actttaccca aaacccagtt tctaatttta 4260

gcaacaaaat caaaaatatc tacttttgtt tctcgaaagt tacgaaattc atacaatcta 4320

gcttatctct gagcttatgg atttgagata aacaacgaaa atggcagggg agaacttcgc 4380

tacgccgttc cacgggcacg tgggccgcgg cgccttcagc gacgtgtacg agcccgcgga 4440

ggacacgttt ctgcttttgg acgcgctgga ggcagcggct gccgaactgg caggagtgga 4500

aatatgcctg gaagtagggt cagggtctgg tgtagtatct gcattcctag cctctatgat 4560

aggccctcag gctttgtaca tgtgcactga tatcaaccct gaggcagcag cttgtaccct 4620

agagacagca cgctgtaaca aagttcacat tcaaccagtt attacagatt tggtcaaagg 4680

cttgctacca agattgaccg aaaaagttga tcttctggtg tttaatcccc cctatgtagt 4740

gactccacct caagaggtag gaagtcacgg aatagaggca gcttgggctg gtggcagaaa 4800

tggtcgggaa gtcatggaca ggttttttcc cctggttcca gatctccttt caccaagagg 4860

attattctat ttagttacca ttaaagaaaa caacccagaa gaaattttga aaataatgaa 4920

gacaaaaggt ctgcaaggaa ccactgcact ttccagacaa gcaggccaag aaactctttc 4980

agtcctcaag ttcaccaagt cttaggatcg ttcaaacatt tggcaataaa gtttcttaag 5040

attgaatcct gttgccggtc ttgcgatgat tatcatataa tttctgttga attacgttaa 5100

gcatgtaata attaacatgt aatgcatgac gttatttatg agatgggttt ttatgattag 5160

agtcccgcaa ttatacattt aatacgcgat agaaaacaaa atatagcgcg caaactagga 5220

taaattatcg cgcgcggtgt catctatgtt actagatcgc catagatgca attcaatcaa 5280

actgaaattt ctgcaagaat ctcaaacacg gagatctcaa agtttgaaag aaaatttatt 5340

tcttcgactc aaaacaaact tacgaaattt aggtagaact tatatacatt atattgtaat 5400

tttttgtaac aaaatgtttt tattattatt atagaatttt actggttaaa ttaaaaatga 5460

atagaaaagg tgaattaaga ggagagagga ggtaaacatt ttcttctatt ttttcatatt 5520

ttcaggataa attattgtaa aagtttacaa gatttccatt tgactagtgt aaatgaggaa 5580

tattctctag taagatcatt atttcatcta cttcttttat cttctaccag tagaggaata 5640

aacaatattt agctcctttg taaatacaaa ttaattttcg ttcttgacat cattcaattt 5700

taattttacg tataaaataa aagatcatac ctattagaac gattaaggag aaatacaatt 5760

cgaatgagaa ggatgtgccg tttgttataa taaacagcca cacgacgtaa acgtaaaatg 5820

accacatgat gggccaatag acatggaccg actactaata atagtaagtt acattttagg 5880

atggaataaa tatcataccg acatcagttt gaaagaaaag ggaaaaaaag aaaaaataaa 5940

taaaagatat actaccgaca tgagttccaa aaagcaaaaa aaaagatcaa gccgacacag 6000

acacgcgtag agagcaaaat gactttgacg tcacaccacg aaaacagacg cttcatacgt 6060

gtccctttat ctctctcagt ctctctataa acttagtgag accctcctct gttttactca 6120

caaatatgca aactagaaaa caatcatcag gaataaaggg tttgattact tctattggaa 6180

agaaaaaaat ctttggaaaa tggagaaaca gaggagagaa gaaagcagct ttcaacaacc 6240

tccatggatt cctcagacac ccatgaagcc attttcaccg atctgcccat acacggtgga 6300

ggatcaatat catagcagtc aattggagga aaggagattt gttgggaaca aggatatgag 6360

tggtcttgat cacttgtctt ttggggattt gcttgctcta gctaacactg catccctcat 6420

attctctggt cagactccaa tacctacaag aaacacagag gttatgcaaa aaggtactga 6480

agaagtggag agtttgagct cagtgagtaa caatgttgct gaacagatcc tcaagactcc 6540

tgaaaaacct aagaggaaga agcatcggcc aaaggttcgt agagaagcta aacccaagag 6600

ggagcctaaa ccacgagctc cgaggaagtc tgttgtcacc gatggtcaag aaagcaaaac 6660

accaaagagg aaatatgtgc ggaagaaggt tgaagtcagt aaggatcaag acgctactcc 6720

ggttgaatca tcagcagctg ttgaaacttc aactcgtcct aagaggctct gtagacgagt 6780

cttggatttt gaagccgaaa atggagaaaa ccagaccaac ggtgacatta gagaagcagg 6840

tgagatggaa tcagctcttc aagagaagca gttagattct gggaatcaag agttaaaaga 6900

ttgccttctt tcggctccta gcacgcccaa gagaaagcgc agccaaggta aaagaaaggg 6960

agttcaacca aagaaaaatg gcagtaatct agaagaagtc gatatttcga tggcgcaagc 7020

tgcaaagaga agacaaggac caacttgttg cgacatgaat ctatcaggga ttcagtatga 7080

tgagcaatgt gactaccaga aaatgcattg gttgtattcc ccaaacttgc aacagggagg 7140

gatgagatat gatgccattt gcagcaaagt attctctgga caacagcaca attatgtttc 7200

tgcctttcac gctacgtgct acagttccac atctcagctc agtgctaata gagtcctaac 7260

cgttgaagaa agacgagaag gtatctttca aggaaggcaa gagtctgagc taaatgttct 7320

ctcggataag atagacacgc cgatcaagaa gaaaacaaca ggccatgctc gattccggaa 7380

tttgtcttca atgaataaac ttgtggaagt tcctgagcat ttaacctcag gatattgtag 7440

caagccacag caaaataata agattcttgt tgatacgcgg gtgactgtga gcaaaaagaa 7500

gccaaccaag tctgagaaat cacaaaccaa acagaaaaat cttcttccga atctttgccg 7560

ttttccacct tcatttactg gtctttctcc agatgaactt tggaaacgac gtaactcgat 7620

cgaaacaatc agtgagctat tgcgtctatt agacatcaac agggagcatt ctgaaactgc 7680

tctcgttcct tacacaatga atagccagat tgtactcttt ggtggtggcg ctggagcaat 7740

tgtgcctgta actcctgtta aaaaaccacg cccacgacca aaggttgatc tagacgatga 7800

gacagacaga gtgtggaaac tgctattgga gaatattaat agcgaaggtg ttgacggatc 7860

agacgagcag aaggcgaaat ggtgggagga agaacgtaat gtgtttcgag gacgagctga 7920

ctcatttatt gcaaggatgc accttgtaca aggggatcga cgttttacgc cttggaaggg 7980

atccgtcgtg gattctgttg ttggagtatt tctcactcaa aatgtttcag accatctctc 8040

aagttcggct ttcatgtcgt tggcttccca gttccctgtc ccttttgtac cgagcagtaa 8100

ctttgacgct ggaacaagct cgatgccttc tattcaaata acgtacttgg actcagagga 8160

aacgatgtca agcccacccg atcacaatca cagttctgtt actttgaaaa atacacagcc 8220

tgatgaggag aaggattatg tacctagcaa tgaaacctcc agaagcagta gtgagattgc 8280

catctcagcc catgaatcag ttgacaaaac cacggattca aaggagtatg ttgattcaga 8340

tcgaaaaggc tcaagtgtag aggttgataa gacggatgag aagtgtcgtg tcctgaacct 8400

gtttccatct gaagattctg cacttacatg tcaacattcg atggtgtctg atgctcctca 8460

aaatacagag agagcaggat caagctcaga gatcgactta gaaggagagt atcgtacttc 8520

ctttatgaag ctcctacagg gggtacaagt ctctctagaa gattccaatc aagtatcacc 8580

aaatatgtct ccgggtgatt gtagctcaga aattaagggt ttccagtcaa tgaaagagcc 8640

cacaaaatcc tctgttgata gtagtgaacc tggttgttgc tctcagcaag atggggatgt 8700

tttgagttgt cagaaaccta ccttaaaaga aaaagggaaa aaggttttga aggaggaaaa 8760

aaaagcgttt gactgggatt gtttaagaag agaagcccaa gctagagcag gaattagaga 8820

aaaaacaaga agtacaatgg acaccgtgga ttggaaggca atacgagcag cagatgttaa 8880

ggaagttgct gaaacaatca agagtcgcgg gatgaaccat aaacttgcag aacgtataca 8940

gggcttcctt gatcgactgg taaatgacca tggaagtatc gatcttgaat ggttgagaga 9000

tgttccacca gataaagcaa aagaatatct tctgagcttt aacggattgg gactgaaaag 9060

tgtggagtgt gtgcggcttc taacacttca ccatcttgcc tttccagttg atacaaatgt 9120

tgggcgcata gccgtcagac ttggatgggt gccccttcag ccgctcccag agtcacttca 9180

gttgcatctt ctggaaatgt atcctatgct tgaatctatt caaaagtatc tttggccccg 9240

tctctgcaaa ctcgaccaaa aaacattgta tgagttgcac taccagatga ttacttttgg 9300

aaaggtcttt tgcacaaaga gcaaacctaa ttgcaatgca tgtccgatga aaggagaatg 9360

cagacatttt gccagtgcgt ttgcaagtgc aaggcttgct ttaccaagta cagagaaagg 9420

tatggggaca cctgataaaa accctttgcc tctacacctg ccagagccat tccagagaga 9480

gcaagggtct gaagtagtac agcactcaga accagcaaaa aaggtcacat gttgtgaacc 9540

aatcatcgaa gagcctgctt caccggagcc agaaaccgca gaagtatcaa tagctgacat 9600

agaggaggcg ttttttgagg atccagaaga aattcctacc atcaggctaa acatggatgc 9660

atttaccagt aacttgaaga agataatgga acacaacaag gaacttcaag acggaaacat 9720

gtccagcgct ttagttgcac ttactgctga aactgcttct cttccaatgc ctaagctcaa 9780

gaatatcagc cagttaagga cagaacaccg agtttacgaa cttccagacg agcatcctct 9840

tctagctcag ttggaaaaga gagaacctga tgatccatgt tcttatttgc ttgctatatg 9900

gacgccaggt gagacggctg attctattca accgtctgtt agtacgtgca tattccaagc 9960

aaatggtatg ctttgtgacg aggagacttg tttctcctgc aacagcatca aggagactag 10020

atctcaaatt gtgagaggga caattttgat tccttgtaga acagcgatga ggggtagttt 10080

tcctctaaat ggaacgtact ttcaagtaaa tgaggtgttt gcggatcatg catccagcct 10140

aaacccaatc aatgtcccaa gggaattgat atgggaatta cctcgaagaa cggtctattt 10200

tggtacctct gttcctacga tattcaaagg tttatcaact gagaagatac aggcttgctt 10260

ttggaaaggg tacgtatgtg tacgtggatt tgatcgaaag acgaggggac cgaagccttt 10320

gattgcaaga ttgcacttcc cggcgagcaa actgaaggga caacaagcta acctcgccta 10380

agatcgttca aacatttggc aataaagttt cttaagattg aatcctgttg ccggtcttgc 10440

gatgattatc atataatttc tgttgaatta cgttaagcat gtaataatta acatgtaatg 10500

catgacgtta tttatgagat gggtttttat gattagagtc ccgcaattat acatttaata 10560

cgcgatagaa aacaaaatat agcgcgcaaa ctaggataaa ttatcgcgcg cggtgtcatc 10620

tatgttacta gatc 10634

<210> 2

<211> 2360

<212> DNA

<213> 人工序列

<400> 2

agctgagttg cttaatagag gaattgatgt agaagaaatc aggaagatta taactatgaa 60

aaaaagtact actgaatgat agattctcca cgttgcggga catggcgaac ttgatccact 120

tgtcgaagtg gggaacagtg tgctggttgc tggttttgag atgaaacttt atcatcttga 180

gagctgtgta gcgagttagg gtttcgttta tcgaagcagc atgcagtgta tcactatcat 240

caaaggaaag agaaggtata tcggaccata catgcctcca tcgtttttac aagacggaag 300

ttgtgatcga gagtttggtc ggaacaaata ataggatttg ttggaggatc tcatcaggca 360

ataagctgat taggtcaata ttccggcgat gacgaccacg atcgttacag tggataggaa 420

gactggaagt ggctgtggag ctctctcggg ccatcttact ttattaacct aacttgtgat 480

cttcttaatt agggtttaaa tcttaatttt agccgcatat tgttttctat atataataac 540

attctttcaa aatacatgtc aaacaattta tagcaaattt aattaactat tttttttaaa 600

aagtcttccc taataagtgc tcttagaaca ataaatcggc atttaaaaaa aaaaattggc 660

attttatttt tttcgttttt ttaaacttat tatatggtgt agtcgggcta tactggactt 720

ttgcaaaaat gtggttttta tattttatat tgtattaggt ttctgttaaa attaaatgag 780

aattttaatt aaaaagagaa attatttgtt aaaaaaaatc aggatggggt cctaattatt 840

atatgttttg attttctatg agaaagttgc accgtccatt gtttcttgaa aactattatc 900

tgactaaaag aacagaaaat gtaaagaaaa gacaaagaga cacagagacg actctgttaa 960

ataactctat agcagagtct ctcgagttaa atcaataaaa taaagacctg aaaacatata 1020

tttcttcgaa gcagtgtcta aaaccaatgt acaatttatg acaaaaggaa catgttattt 1080

tagtcgcata taattacaaa ataatcgcat gatttatcta agttggtctt tattaactct 1140

taacaaaaaa ataatataag aaaacagagt cagaatttaa aaaccactta attagtcctt 1200

caagaacaat tatcaaaacc ttaataatgt tttcatccaa taacatcctc gaagtctcct 1260

ctaaatcatt ggatccaacg aaattcatgt ttatctaaac taactcgaat aaagaaacga 1320

ttataataat tgcacactat gaaaaatatc agaagcgtca tagaaattgt cggctacctc 1380

catgcacgga accttcacga aacagttggt ccctcacaca cttcatcgcc acgctatacc 1440

acgtgtcaat tttacataca ccaaaacata tctactaatc atacctcttc acgtgtaaca 1500

aagtcccatt caacgtggca attacagacc ccaaaattat gaactaatca aacctcttca 1560

cgtgtcgcaa acttgtagaa cgttgaaacc ccccactcac acgaagtgta tatatcctct 1620

tcacaacaca aacataaaca ttacttcaaa caaagacttg aaagaactat ctttgttttc 1680

actcatatct tatctttatt aaatggcgat gtctttctca ggagctgttc tcactggtat 1740

ggcttcttct ttccacagcg gagccaagca gagcagcttc ggcgctgtca gagtcggcca 1800

gaaaactcag ttcgtcgtcg tttctcaacg caagaagtcg ttgatctacg ccttgacgtc 1860

cttgactctg cccggagggt ttggcagcgc gcctgtggcc aatttgtcga tgactttgga 1920

agtaaccaac cccaatcctc tgccgttgcg aatggctaat attgctgggg cgctcattat 1980

cgatggggcc gccgttggcg atgtgtcatt tcctaacgta gacatagcgg ctaggggggt 2040

atccacacaa agggcggatt tatctatacc tgtgacccta aatacagccg catcattctt 2100

gaaggttgcc cgtgggcagt tggttactta tagagttgat ggtggattta cttgatttct 2160

ccataataat gtgtgagtag ttcccagata agggaattag ggttcctata gggtttcgct 2220

catgtgttga gcatataaga aacccttagt atgtatttgt atttgtaaaa tacttctatc 2280

aataaaattt ctaattccta aaaccaaaat ccagtactaa aatccagatc aaagatacag 2340

tctcagaaga ccaaagggct 2360

<210> 3

<211> 150

<212> PRT

<213> 人工序列

<400> 3

MET Ala MET Ser Phe Ser Gly Ala Val Leu Thr Gly MET Ala Ser Ser

1 5 10 15

Phe His Ser Gly Ala Lys Gln Ser Ser Phe Gly Ala Val Arg Val Gly

20 25 30

Gln Lys Thr Gln Phe Val Val Val Ser Gln Arg Lys Lys Ser Leu Ile

35 40 45

Tyr Ala Leu Thr Ser Leu Thr Leu Pro Gly Gly Phe Gly Ser Ala Pro

50 55 60

Val Ala Asn Leu Ser MET Thr Leu Glu Val Thr Asn Pro Asn Pro Leu

65 70 75 80

Pro Leu Arg MET Ala Asn Ile Ala Gly Ala Leu Ile Ile Asp Gly Ala

85 90 95

Ala Val Gly Asp Val Ser Phe Pro Asn Val Asp Ile Ala Ala Arg Gly

100 105 110

Val Ser Thr Gln Arg Ala Asp Leu Ser Ile Pro Val Thr Leu Asn Thr

115 120 125

Ala Ala Ser Phe Leu Lys Val Ala Arg Gly Gln Leu Val Thr Tyr Arg

145 150 155 160

Val Asp Gly Gly Phe Thr

165

<210> 4

<211> 2898

<212> DNA

<213> 人工序列

<400> 4

ctttgccaat tcctctgtcc tcgataagag ctccaatacc agaaatgagt aaaaaaccaa 60

ctccaatagt tcggattgta ctccatagct gttctttaaa gtgagtcctt tctgtggata 120

tcgtatggat cggtgcacta gcagttccaa ggacaccatc ttttgttggt tttcctacgt 180

ttctaaatgc cccaagacct ccaaaagtct cttcctccct tgcaacacca gcaataccta 240

aaagaaaaac agcaaaacat ttcccaatat tatatagaga aatggctcag aagctgctaa 300

tacaaatatg aaagcaagga cacaatccaa gtatccattc agattcaaac aaacagtctc 360

tccctcctta cccacctctt tgcaatgttc gaaccagctc actttgatcg agcctatcaa 420

ctttaaccaa tgccttaatg tattccgata acgcagaggc attcgcatgc aaagaaggtt 480

ggctttcaaa cattctgata acagcctcag gatcatttct tcgaataagt tctctcagat 540

gtgcaacctc attaacttct tctctatcac ggactctgcg agcaaagcta ccaacataac 600

tcgattgaaa cctcgttcgc ggtaaagaag ctccaccacc acccacagct atagataaaa 660

taaaaccaat ttacatcaaa aacacttcta aaacaacaag aacaactcat aaacggaatc 720

agaatttacc tccagtaact cccactctag gaaaagagga gtaagctcta acaagtaagc 780

ttcttaaact gctcaattcc ctttcatgac tcgaaacctg tcatcggtga agagaatcaa 840

tacagatgaa gttaattact aatcccaaac tcaaaatgac actagacaac acatgaaaac 900

attttgataa aagtgaatac ttttacccat tttgaatcaa aaaaattgaa actttttata 960

ccaatttcaa aattaggtga cttgggtagt caaataaatc aaatgacaat atcacagaga 1020

caatctagaa tcctaaaaga caacattttg agggcagaga agaatttgag cttctagggt 1080

ttgaaaaata tcatctttag cttatgaatc acaaagatct tgataaaacc catcagaaat 1140

tattatctaa ttagatcaaa tccactacag tatcaaacca aagtcgaaac ctttttcgta 1200

ttaaaaattg gataaagggg aaaagagaaa aatgaaacaa aaaccttggt gatgatgcgc 1260

ctccaagcca tcaatcaaat ctctccttca cgatatctta aaaattggtg ttaatgtctg 1320

ataaatcgaa gttcctcgat ctatatcgga aacaaaagac ttcttcgttg tgtttgggga 1380

agaaccgttc taatcttatt ccctaaagtc ttaaaaacac taaattacac gtgagagacc 1440

tgtttggtta tcgggtgaga gaaaaattgt gcagcaggtg gagacacgca cgagatatgt 1500

aggtcgcctc ttaagtacat aaataccctt ggacataccc aataattcat tttagtaggc 1560

tttttctggc ggcccacatt aaaaagaagg acctagaaca taaattggca tcgttagaaa 1620

tgggcttaag taaaggccca tatgatatat atataaaaaa agagattcta gattagtaac 1680

gaagtttctg gaacattgtc ttgtcttgtc gccacgtgct cacataaatg tcaaagaagc 1740

ttcaatacag tgaaatgatc ttgtcttgtc tctagaacct tctcttctct ccccttataa 1800

tttcatttct ctctcctcca cgcctcaatc tctcaactca aaactcaaca ttttctgaag 1860

aaagtcgcaa actttaccca aaacccagtt tctaatttta gcaacaaaat caaaaatatc 1920

tacttttgtt tctcgaaagt tacgaaattc atacaatcta gcttatctct gagcttatgg 1980

atttgagata aacaacgaaa atggcagggg agaacttcgc tacgccgttc cacgggcacg 2040

tgggccgcgg cgccttcagc gacgtgtacg agcccgcgga ggacacgttt ctgcttttgg 2100

acgcgctgga ggcagcggct gccgaactgg caggagtgga aatatgcctg gaagtagggt 2160

cagggtctgg tgtagtatct gcattcctag cctctatgat aggccctcag gctttgtaca 2220

tgtgcactga tatcaaccct gaggcagcag cttgtaccct agagacagca cgctgtaaca 2280

aagttcacat tcaaccagtt attacagatt tggtcaaagg cttgctacca agattgaccg 2340

aaaaagttga tcttctggtg tttaatcccc cctatgtagt gactccacct caagaggtag 2400

gaagtcacgg aatagaggca gcttgggctg gtggcagaaa tggtcgggaa gtcatggaca 2460

ggttttttcc cctggttcca gatctccttt caccaagagg attattctat ttagttacca 2520

ttaaagaaaa caacccagaa gaaattttga aaataatgaa gacaaaaggt ctgcaaggaa 2580

ccactgcact ttccagacaa gcaggccaag aaactctttc agtcctcaag ttcaccaagt 2640

cttaggatcg ttcaaacatt tggcaataaa gtttcttaag attgaatcct gttgccggtc 2700

ttgcgatgat tatcatataa tttctgttga attacgttaa gcatgtaata attaacatgt 2760

aatgcatgac gttatttatg agatgggttt ttatgattag agtcccgcaa ttatacattt 2820

aatacgcgat agaaaacaaa atatagcgcg caaactagga taaattatcg cgcgcggtgt 2880

catctatgtt actagatc 2898

<210> 5

<211> 214

<212> PRT

<213> 人工序列

<400> 5

MET Ala Gly Glu Asn Phe Ala Thr Pro Phe His Gly His Val Gly Arg

1 5 10 15

Gly Ala Phe Ser Asp Val Tyr Glu Pro Ala Glu Asp Thr Phe Leu Leu

20 25 30

Leu Asp Ala Leu Glu Ala Ala Ala Ala Glu Leu Ala Gly Val Glu Ile

35 40 45

Cys Leu Glu Val Gly Ser Gly Ser Gly Val Val Ser Ala Phe Leu Ala

50 55 60

Ser MET Ile Gly Pro Gln Ala Leu Tyr MET Cys Thr Asp Ile Asn Pro

65 70 75 80

Glu Ala Ala Ala Cys Thr Leu Glu Thr Ala Arg Cys Asn Lys Val His

85 90 95

Ile Gln Pro Val Ile Thr Asp Leu Val Lys Gly Leu Leu Pro Arg Leu

100 105 110

Thr Glu Lys Val Asp Leu Leu Val Phe Asn Pro Pro Tyr Val Val Thr

115 120 125

Pro Pro Gln Glu Val Gly Ser His Gly Ile Glu Ala Ala Trp Ala Gly

130 135 140

Gly Arg Asn Gly Arg Glu Val MET Asp Arg Phe Phe Pro Leu Val Pro

145 150 155 160

Asp Leu Leu Ser Pro Arg Gly Leu Phe Tyr Leu Val Thr Ile Lys Glu

165 170 175

Asn Asn Pro Glu Glu Ile Leu Lys Ile MET Lys Thr Lys Gly Leu Gln

180 185 190

Gly Thr Thr Ala Leu Ser Arg Gln Ala Gly Gln Glu Thr Leu Ser Val

195 200 205

Leu Lys Phe Thr Lys Ser

210

<210> 6

<211> 5376

<212> DNA

<213> 人工序列

<400> 6

gccatagatg caattcaatc aaactgaaat ttctgcaaga atctcaaaca cggagatctc 60

aaagtttgaa agaaaattta tttcttcgac tcaaaacaaa cttacgaaat ttaggtagaa 120

cttatataca ttatattgta attttttgta acaaaatgtt tttattatta ttatagaatt 180

ttactggtta aattaaaaat gaatagaaaa ggtgaattaa gaggagagag gaggtaaaca 240

ttttcttcta ttttttcata ttttcaggat aaattattgt aaaagtttac aagatttcca 300

tttgactagt gtaaatgagg aatattctct agtaagatca ttatttcatc tacttctttt 360

atcttctacc agtagaggaa taaacaatat ttagctcctt tgtaaataca aattaatttt 420

cgttcttgac atcattcaat tttaatttta cgtataaaat aaaagatcat acctattaga 480

acgattaagg agaaatacaa ttcgaatgag aaggatgtgc cgtttgttat aataaacagc 540

cacacgacgt aaacgtaaaa tgaccacatg atgggccaat agacatggac cgactactaa 600

taatagtaag ttacatttta ggatggaata aatatcatac cgacatcagt ttgaaagaaa 660

agggaaaaaa agaaaaaata aataaaagat atactaccga catgagttcc aaaaagcaaa 720

aaaaaagatc aagccgacac agacacgcgt agagagcaaa atgactttga cgtcacacca 780

cgaaaacaga cgcttcatac gtgtcccttt atctctctca gtctctctat aaacttagtg 840

agaccctcct ctgttttact cacaaatatg caaactagaa aacaatcatc aggaataaag 900

ggtttgatta cttctattgg aaagaaaaaa atctttggaa aatggagaaa cagaggagag 960

aagaaagcag ctttcaacaa cctccatgga ttcctcagac acccatgaag ccattttcac 1020

cgatctgccc atacacggtg gaggatcaat atcatagcag tcaattggag gaaaggagat 1080

ttgttgggaa caaggatatg agtggtcttg atcacttgtc ttttggggat ttgcttgctc 1140

tagctaacac tgcatccctc atattctctg gtcagactcc aatacctaca agaaacacag 1200

aggttatgca aaaaggtact gaagaagtgg agagtttgag ctcagtgagt aacaatgttg 1260

ctgaacagat cctcaagact cctgaaaaac ctaagaggaa gaagcatcgg ccaaaggttc 1320

gtagagaagc taaacccaag agggagccta aaccacgagc tccgaggaag tctgttgtca 1380

ccgatggtca agaaagcaaa acaccaaaga ggaaatatgt gcggaagaag gttgaagtca 1440

gtaaggatca agacgctact ccggttgaat catcagcagc tgttgaaact tcaactcgtc 1500

ctaagaggct ctgtagacga gtcttggatt ttgaagccga aaatggagaa aaccagacca 1560

acggtgacat tagagaagca ggtgagatgg aatcagctct tcaagagaag cagttagatt 1620

ctgggaatca agagttaaaa gattgccttc tttcggctcc tagcacgccc aagagaaagc 1680

gcagccaagg taaaagaaag ggagttcaac caaagaaaaa tggcagtaat ctagaagaag 1740

tcgatatttc gatggcgcaa gctgcaaaga gaagacaagg accaacttgt tgcgacatga 1800

atctatcagg gattcagtat gatgagcaat gtgactacca gaaaatgcat tggttgtatt 1860

ccccaaactt gcaacaggga gggatgagat atgatgccat ttgcagcaaa gtattctctg 1920

gacaacagca caattatgtt tctgcctttc acgctacgtg ctacagttcc acatctcagc 1980

tcagtgctaa tagagtccta accgttgaag aaagacgaga aggtatcttt caaggaaggc 2040

aagagtctga gctaaatgtt ctctcggata agatagacac gccgatcaag aagaaaacaa 2100

caggccatgc tcgattccgg aatttgtctt caatgaataa acttgtggaa gttcctgagc 2160

atttaacctc aggatattgt agcaagccac agcaaaataa taagattctt gttgatacgc 2220

gggtgactgt gagcaaaaag aagccaacca agtctgagaa atcacaaacc aaacagaaaa 2280

atcttcttcc gaatctttgc cgttttccac cttcatttac tggtctttct ccagatgaac 2340

tttggaaacg acgtaactcg atcgaaacaa tcagtgagct attgcgtcta ttagacatca 2400

acagggagca ttctgaaact gctctcgttc cttacacaat gaatagccag attgtactct 2460

ttggtggtgg cgctggagca attgtgcctg taactcctgt taaaaaacca cgcccacgac 2520

caaaggttga tctagacgat gagacagaca gagtgtggaa actgctattg gagaatatta 2580

atagcgaagg tgttgacgga tcagacgagc agaaggcgaa atggtgggag gaagaacgta 2640

atgtgtttcg aggacgagct gactcattta ttgcaaggat gcaccttgta caaggggatc 2700

gacgttttac gccttggaag ggatccgtcg tggattctgt tgttggagta tttctcactc 2760

aaaatgtttc agaccatctc tcaagttcgg ctttcatgtc gttggcttcc cagttccctg 2820

tcccttttgt accgagcagt aactttgacg ctggaacaag ctcgatgcct tctattcaaa 2880

taacgtactt ggactcagag gaaacgatgt caagcccacc cgatcacaat cacagttctg 2940

ttactttgaa aaatacacag cctgatgagg agaaggatta tgtacctagc aatgaaacct 3000

ccagaagcag tagtgagatt gccatctcag cccatgaatc agttgacaaa accacggatt 3060

caaaggagta tgttgattca gatcgaaaag gctcaagtgt agaggttgat aagacggatg 3120

agaagtgtcg tgtcctgaac ctgtttccat ctgaagattc tgcacttaca tgtcaacatt 3180

cgatggtgtc tgatgctcct caaaatacag agagagcagg atcaagctca gagatcgact 3240

tagaaggaga gtatcgtact tcctttatga agctcctaca gggggtacaa gtctctctag 3300

aagattccaa tcaagtatca ccaaatatgt ctccgggtga ttgtagctca gaaattaagg 3360

gtttccagtc aatgaaagag cccacaaaat cctctgttga tagtagtgaa cctggttgtt 3420

gctctcagca agatggggat gttttgagtt gtcagaaacc taccttaaaa gaaaaaggga 3480

aaaaggtttt gaaggaggaa aaaaaagcgt ttgactggga ttgtttaaga agagaagccc 3540

aagctagagc aggaattaga gaaaaaacaa gaagtacaat ggacaccgtg gattggaagg 3600

caatacgagc agcagatgtt aaggaagttg ctgaaacaat caagagtcgc gggatgaacc 3660

ataaacttgc agaacgtata cagggcttcc ttgatcgact ggtaaatgac catggaagta 3720

tcgatcttga atggttgaga gatgttccac cagataaagc aaaagaatat cttctgagct 3780

ttaacggatt gggactgaaa agtgtggagt gtgtgcggct tctaacactt caccatcttg 3840

cctttccagt tgatacaaat gttgggcgca tagccgtcag acttggatgg gtgccccttc 3900

agccgctccc agagtcactt cagttgcatc ttctggaaat gtatcctatg cttgaatcta 3960

ttcaaaagta tctttggccc cgtctctgca aactcgacca aaaaacattg tatgagttgc 4020

actaccagat gattactttt ggaaaggtct tttgcacaaa gagcaaacct aattgcaatg 4080

catgtccgat gaaaggagaa tgcagacatt ttgccagtgc gtttgcaagt gcaaggcttg 4140

ctttaccaag tacagagaaa ggtatgggga cacctgataa aaaccctttg cctctacacc 4200

tgccagagcc attccagaga gagcaagggt ctgaagtagt acagcactca gaaccagcaa 4260

aaaaggtcac atgttgtgaa ccaatcatcg aagagcctgc ttcaccggag ccagaaaccg 4320

cagaagtatc aatagctgac atagaggagg cgttttttga ggatccagaa gaaattccta 4380

ccatcaggct aaacatggat gcatttacca gtaacttgaa gaagataatg gaacacaaca 4440

aggaacttca agacggaaac atgtccagcg ctttagttgc acttactgct gaaactgctt 4500

ctcttccaat gcctaagctc aagaatatca gccagttaag gacagaacac cgagtttacg 4560

aacttccaga cgagcatcct cttctagctc agttggaaaa gagagaacct gatgatccat 4620

gttcttattt gcttgctata tggacgccag gtgagacggc tgattctatt caaccgtctg 4680

ttagtacgtg catattccaa gcaaatggta tgctttgtga cgaggagact tgtttctcct 4740

gcaacagcat caaggagact agatctcaaa ttgtgagagg gacaattttg attccttgta 4800

gaacagcgat gaggggtagt tttcctctaa atggaacgta ctttcaagta aatgaggtgt 4860

ttgcggatca tgcatccagc ctaaacccaa tcaatgtccc aagggaattg atatgggaat 4920

tacctcgaag aacggtctat tttggtacct ctgttcctac gatattcaaa ggtttatcaa 4980

ctgagaagat acaggcttgc ttttggaaag ggtacgtatg tgtacgtgga tttgatcgaa 5040

agacgagggg accgaagcct ttgattgcaa gattgcactt cccggcgagc aaactgaagg 5100

gacaacaagc taacctcgcc taagatcgtt caaacatttg gcaataaagt ttcttaagat 5160

tgaatcctgt tgccggtctt gcgatgatta tcatataatt tctgttgaat tacgttaagc 5220

atgtaataat taacatgtaa tgcatgacgt tatttatgag atgggttttt atgattagag 5280

tcccgcaatt atacatttaa tacgcgatag aaaacaaaat atagcgcgca aactaggata 5340

aattatcgcg cgcggtgtca tctatgttac tagatc 5376

<210> 7

<211> 1393

<212> PRT

<213> 人工序列

<400> 7

MET Glu Lys Gln Arg Arg Glu Glu Ser Ser Phe Gln Gln Pro Pro Trp

1 5 10 15

Ile Pro Gln Thr Pro MET Lys Pro Phe Ser Pro Ile Cys Pro Tyr Thr

20 25 30

Val Glu Asp Gln Tyr His Ser Ser Gln Leu Glu Glu Arg Arg Phe Val

35 40 45

Gly Asn Lys Asp MET Ser Gly Leu Asp His Leu Ser Phe Gly Asp Leu

50 55 60

Leu Ala Leu Ala Asn Thr Ala Ser Leu Ile Phe Ser Gly Gln Thr Pro

65 70 75 80

Ile Pro Thr Arg Asn Thr Glu Val MET Gln Lys Gly Thr Glu Glu Val

85 90 95

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

100 105 110

Thr Pro Glu Lys Pro Lys Arg Lys Lys His Arg Pro Lys Val Arg Arg

115 120 125

Glu Ala Lys Pro Lys Arg Glu Pro Lys Pro Arg Ala Pro Arg Lys Ser

130 135 140

Val Val Thr Asp Gly Gln Glu Ser Lys Thr Pro Lys Arg Lys Tyr Val

145 150 155 160

Arg Lys Lys Val Glu Val Ser Lys Asp Gln Asp Ala Thr Pro Val Glu

165 170 175

Ser Ser Ala Ala Val Glu Thr Ser Thr Arg Pro Lys Arg Leu Cys Arg

180 185 190

Arg Val Leu Asp Phe Glu Ala Glu Asn Gly Glu Asn Gln Thr Asn Gly

195 200 205

Asp Ile Arg Glu Ala Gly Glu MET Glu Ser Ala Leu Gln Glu Lys Gln

210 215 220

Leu Asp Ser Gly Asn Gln Glu Leu Lys Asp Cys Leu Leu Ser Ala Pro

225 230 235 240

Ser Thr Pro Lys Arg Lys Arg Ser Gln Gly Lys Arg Lys Gly Val Gln

245 250 255

Pro Lys Lys Asn Gly Ser Asn Leu Glu Glu Val Asp Ile Ser MET Ala

260 265 270

Gln Ala Ala Lys Arg Arg Gln Gly Pro Thr Cys Cys Asp MET Asn Leu

275 280 285

Ser Gly Ile Gln Tyr Asp Glu Gln Cys Asp Tyr Gln Lys MET His Trp

290 295 300

Leu Tyr Ser Pro Asn Leu Gln Gln Gly Gly MET Arg Tyr Asp Ala Ile

305 310 315 320

Cys Ser Lys Val Phe Ser Gly Gln Gln His Asn Tyr Val Ser Ala Phe

325 330 335

His Ala Thr Cys Tyr Ser Ser Thr Ser Gln Leu Ser Ala Asn Arg Val

340 345 350

Leu Thr Val Glu Glu Arg Arg Glu Gly Ile Phe Gln Gly Arg Gln Glu

355 360 365

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

370 375 380

Lys Thr Thr Gly His Ala Arg Phe Arg Asn Leu Ser Ser MET Asn Lys

385 390 395 400

Leu Val Glu Val Pro Glu His Leu Thr Ser Gly Tyr Cys Ser Lys Pro

405 410 415

Gln Gln Asn Asn Lys Ile Leu Val Asp Thr Arg Val Thr Val Ser Lys

420 425 430

Lys Lys Pro Thr Lys Ser Glu Lys Ser Gln Thr Lys Gln Lys Asn Leu

435 440 445

Leu Pro Asn Leu Cys Arg Phe Pro Pro Ser Phe Thr Gly Leu Ser Pro

450 455 460

Asp Glu Leu Trp Lys Arg Arg Asn Ser Ile Glu Thr Ile Ser Glu Leu

465 470 475 480

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

485 490 495

Pro Tyr Thr MET Asn Ser Gln Ile Val Leu Phe Gly Gly Gly Ala Gly

500 505 510

Ala Ile Val Pro Val Thr Pro Val Lys Lys Pro Arg Pro Arg Pro Lys

515 520 525

Val Asp Leu Asp Asp Glu Thr Asp Arg Val Trp Lys Leu Leu Leu Glu

530 535 540

Asn Ile Asn Ser Glu Gly Val Asp Gly Ser Asp Glu Gln Lys Ala Lys

545 550 555 560

Trp Trp Glu Glu Glu Arg Asn Val Phe Arg Gly Arg Ala Asp Ser Phe

565 570 575

Ile Ala Arg MET His Leu Val Gln Gly Asp Arg Arg Phe Thr Pro Trp

580 585 590

Lys Gly Ser Val Val Asp Ser Val Val Gly Val Phe Leu Thr Gln Asn

595 600 605

Val Ser Asp His Leu Ser Ser Ser Ala Phe MET Ser Leu Ala Ser Gln

610 615 620

Phe Pro Val Pro Phe Val Pro Ser Ser Asn Phe Asp Ala Gly Thr Ser

625 630 635 640

Ser MET Pro Ser Ile Gln Ile Thr Tyr Leu Asp Ser Glu Glu Thr MET

645 650 655

Ser Ser Pro Pro Asp His Asn His Ser Ser Val Thr Leu Lys Asn Thr

660 665 670

Gln Pro Asp Glu Glu Lys Asp Tyr Val Pro Ser Asn Glu Thr Ser Arg

675 680 685

Ser Ser Ser Glu Ile Ala Ile Ser Ala His Glu Ser Val Asp Lys Thr

690 695 700

Thr Asp Ser Lys Glu Tyr Val Asp Ser Asp Arg Lys Gly Ser Ser Val

705 710 715 720

Glu Val Asp Lys Thr Asp Glu Lys Cys Arg Val Leu Asn Leu Phe Pro

725 730 735

Ser Glu Asp Ser Ala Leu Thr Cys Gln His Ser MET Val Ser Asp Ala

740 745 750

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

755 760 765

Gly Glu Tyr Arg Thr Ser Phe MET Lys Leu Leu Gln Gly Val Gln Val

770 775 780

Ser Leu Glu Asp Ser Asn Gln Val Ser Pro Asn MET Ser Pro Gly Asp

785 790 795 800

Cys Ser Ser Glu Ile Lys Gly Phe Gln Ser MET Lys Glu Pro Thr Lys

805 810 815

Ser Ser Val Asp Ser Ser Glu Pro Gly Cys Cys Ser Gln Gln Asp Gly

820 825 830

Asp Val Leu Ser Cys Gln Lys Pro Thr Leu Lys Glu Lys Gly Lys Lys

835 840 845

Val Leu Lys Glu Glu Lys Lys Ala Phe Asp Trp Asp Cys Leu Arg Arg

850 855 860

Glu Ala Gln Ala Arg Ala Gly Ile Arg Glu Lys Thr Arg Ser Thr MET

865 870 875 880

Asp Thr Val Asp Trp Lys Ala Ile Arg Ala Ala Asp Val Lys Glu Val

885 890 895

Ala Glu Thr Ile Lys Ser Arg Gly MET Asn His Lys Leu Ala Glu Arg

900 905 910

Ile Gln Gly Phe Leu Asp Arg Leu Val Asn Asp His Gly Ser Ile Asp

915 920 925

Leu Glu Trp Leu Arg Asp Val Pro Pro Asp Lys Ala Lys Glu Tyr Leu

930 935 940

Leu Ser Phe Asn Gly Leu Gly Leu Lys Ser Val Glu Cys Val Arg Leu

945 950 955 960

Leu Thr Leu His His Leu Ala Phe Pro Val Asp Thr Asn Val Gly Arg

965 970 975

Ile Ala Val Arg Leu Gly Trp Val Pro Leu Gln Pro Leu Pro Glu Ser

980 985 990

Leu Gln Leu His Leu Leu Glu MET Tyr Pro MET Leu Glu Ser Ile Gln

995 1000 1005

Lys Tyr Leu Trp Pro Arg Leu Cys Lys Leu Asp Gln Lys Thr Leu Tyr

1010 1015 1020

Glu Leu His Tyr Gln MET Ile Thr Phe Gly Lys Val Phe Cys Thr Lys

1025 1030 1035 1040

Ser Lys Pro Asn Cys Asn Ala Cys Pro MET Lys Gly Glu Cys Arg His

1045 1050 1055

Phe Ala Ser Ala Phe Ala Ser Ala Arg Leu Ala Leu Pro Ser Thr Glu

1060 1065 1070

Lys Gly MET Gly Thr Pro Asp Lys Asn Pro Leu Pro Leu His Leu Pro

1075 1080 1085

Glu Pro Phe Gln Arg Glu Gln Gly Ser Glu Val Val Gln His Ser Glu

1090 1095 1100

Pro Ala Lys Lys Val Thr Cys Cys Glu Pro Ile Ile Glu Glu Pro Ala

1105 1110 1115 1120

Ser Pro Glu Pro Glu Thr Ala Glu Val Ser Ile Ala Asp Ile Glu Glu

1125 1130 1135

Ala Phe Phe Glu Asp Pro Glu Glu Ile Pro Thr Ile Arg Leu Asn MET

1140 1145 1150

Asp Ala Phe Thr Ser Asn Leu Lys Lys Ile MET Glu His Asn Lys Glu

1155 1160 1165

Leu Gln Asp Gly Asn MET Ser Ser Ala Leu Val Ala Leu Thr Ala Glu

1170 1175 1180

Thr Ala Ser Leu Pro MET Pro Lys Leu Lys Asn Ile Ser Gln Leu Arg

1185 1190 1195 1200

Thr Glu His Arg Val Tyr Glu Leu Pro Asp Glu His Pro Leu Leu Ala

1205 1210 1215

Gln Leu Glu Lys Arg Glu Pro Asp Asp Pro Cys Ser Tyr Leu Leu Ala

1220 1225 1230

Ile Trp Thr Pro Gly Glu Thr Ala Asp Ser Ile Gln Pro Ser Val Ser

1235 1240 1245

Thr Cys Ile Phe Gln Ala Asn Gly MET Leu Cys Asp Glu Glu Thr Cys

1250 1255 1260

Phe Ser Cys Asn Ser Ile Lys Glu Thr Arg Ser Gln Ile Val Arg Gly

1265 1270 1275 1280

Thr Ile Leu Ile Pro Cys Arg Thr Ala MET Arg Gly Ser Phe Pro Leu

1285 1290 1295

Asn Gly Thr Tyr Phe Gln Val Asn Glu Val Phe Ala Asp His Ala Ser

1300 1305 1310

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

1315 1320 1325

Arg Arg Thr Val Tyr Phe Gly Thr Ser Val Pro Thr Ile Phe Lys Gly

1330 1335 1340

Leu Ser Thr Glu Lys Ile Gln Ala Cys Phe Trp Lys Gly Tyr Val Cys

1345 1350 1355 1360

Val Arg Gly Phe Asp Arg Lys Thr Arg Gly Pro Lys Pro Leu Ile Ala

1365 1370 1375

Arg Leu His Phe Pro Ala Ser Lys Leu Lys Gly Gln Gln Ala Asn Leu

1380 1385 1390

Ala

Claims (2)

1.SEQ ID NO:1所示核苷酸序列的基因在提高农作物抗逆功能中的应用，所述的应用，是在农作物品种选育时提高细胞抗干旱，耐高盐和耐高温能力。

2.含有SEQ ID NO:1所示序列的基因的质粒在增强农作物抗干旱，耐高盐和耐高温能力方面的应用。

Images