CN114836429B - PhCLV3和PhCLV1基因在调控矮牵牛植株观赏性中的应用 - Google Patents

Abstract

本发明公开了PhCLV3和PhCLV1基因在调控矮牵牛植株观赏性中的应用。本发明发现利用基因编辑技术对PhCLV3和PhCLV1基因进行敲除，功能丧失的单突变体表现花瓣数目增加，两个基因功能丧失的双突变体表型更加剧烈，呈现出稀缺型头状花蕾、花瓣重瓣、花瓣面积增大、开花密度增大或株高变矮，大大提高了矮牵牛作为花卉植物的观赏价值，说明PhCLV3和PhCLV1基因在矮牵牛生长和发育过程中具有重要调控作用。因此，本发明在矮牵牛花卉性状改良方面具有较好的应用前景。

Description

PhCLV3和PhCLV1基因在调控矮牵牛植株观赏性中的应用

技术领域

本发明属于生物技术领域，尤其涉及PhCLV3和PhCLV1基因在调控矮牵牛植株观赏性中的应用。

背景技术

矮牵牛原产自南美洲，是茄科中最为常见的花卉植物。在适宜的条件下，矮牵牛一年四季都能开花，因其颜色和花器官形态的多样性成为世界上广受人们欢迎的园艺花卉之一。在美国，矮牵牛的种植和消费居草本花卉植物之首，且多个品种获得花坛植物奖项。在日本，矮牵牛是最受欢迎的花卉之一。我国矮牵牛品种引进较晚，主要兴起于20世纪80年代，当今商业化种植的矮牵牛是科学家在近两个世纪内经常规育种培育出来的，由P.axillaris N和P.inflata S6亲本杂交产生的一系列近交种质。矮牵牛作为花坛后起之秀，市场需求量越来越大；矮牵牛常规育种往往需要较长年限，大大限制了性状改良的进程(Bombarely et al.,2016,Dai et al；2004,W Kevin Lutke,2006)。

在高等被子植物包括矮牵牛中，CLAVATA3(CLV3)/ESR(EMBRYO SURROUNDINGREGION,CLE)是广泛存在且保守的小肽家族。由CLV-WUS负反馈通路核心成员对植物茎尖干细胞的调控决定了植物地上部分花器官数目、果实大小和株型(Betsuyaku et al.,2011；Goad et al.,2017)。现阶段关于CLV-WUS在矮牵牛生长发育过程中发挥的功能仍未报道。此外，随着人们对矮牵牛花色及花瓣重瓣等研究的不断深入，已有个别矮牵牛株型相关基因EVERGREEN被鉴定(Castel et al.,2010；Rebocho et al.,2008)，但其应用前景还有待探究。从基因出发，利用基因工程手段来提高花卉植物的观赏性，已成为花卉性状改良的一项重要内容。

发明内容

本发明的目的在于提供一种PhCLV3和PhCLV1基因在调控矮牵牛植株观赏性中的应用。克服现有技术的不足，利用基因编辑技术，发现使矮牵牛花蕾呈现稀缺型头状花蕾、花瓣重叠、花瓣面积增大、开花密度增大或株高变矮的两个基因PhCLV3和PhCLV1，大大提高了矮牵牛作为花卉植物的观赏性。

本发明提供了如下(1)-(6)中任一种物质在调控矮牵牛植株观赏性中的应用；

(1)矮牵牛PhCLV3和PhCLV1基因起始密码子上游序列；

(2)矮牵牛PhCLV3和PhCLV1基因终止密码子下游序列；

(3)矮牵牛PhCLV3和PhCLV1基因组序列；

(4)矮牵牛PhCLV3和PhCLV1基因的编码序列；

(5)矮牵牛PhCLV3和PhCLV1基因编码的蛋白氨基酸序列；

(6)含有矮牵牛PhCLV3和PhCLV1基因的重组载体、表达盒或重组工程菌；

PhCLV3基因起始密码子上游5000bp序列如SEQ ID NO.1所示，

PhCLV1基因起始密码子上游5000bp序列如SEQ ID NO.6所示，

PhCLV3基因终止密码子下游579bp序列如SEQ ID NO.2所示，

PhCLV1基因终止密码子下游983bp序列如SEQ ID NO.7所示，

PhCLV3基因组序列如SEQ ID NO.3所示，PhCLV1基因组序列如SEQ ID NO.8所示，

PhCLV3基因的编码序列如SEQ ID NO.4所示，PhCLV1基因的编码序列如SEQ IDNO.9所示，

PhCLV3基因编码的蛋白氨基酸序列如SEQ ID NO.5所示，PhCLV1基因编码的蛋白氨基酸序列如SEQ ID NO.10所示。

所述调控矮牵牛植株观赏性主要为调控矮牵牛头状花序、重叠花瓣、花瓣面积增大、开花密度增大或株高变矮。

本发明构建包含PhCLV3和PhCLV1基因靶点的重组质粒，靶点设计在CRISPR P2.0网站进行，构建U6p-Target-gRNA表达盒，利用边切边连反应体系连接进pM双元载体。构建重组载体时，可以采用pM载体作为双元载体(pM-Cas9-sgRNA植物多个sgRNA基因编辑CRISPR-Cas9质粒载体)，构建方式是：将矮牵牛PhCLV3和PhCLV1的靶点序列扩增、连接到中间载体，通过边切边连的方式重组到最终双元载体。使用重组载体时，在其转录起始核苷酸前可加上一种增强型启动子；为了便于对基因编辑植物进行鉴定和筛选，对表达载体进行卡那霉素标记物等抗性基因的加工。

将构建成功的双元载体转化进入GV3101根癌农杆菌，阳性克隆进行摇菌侵染矮牵牛真叶外植体。经过一系列诱导生芽，生根培养，待生根苗长至瓶口处时，取叶片提取DNA进行编辑鉴定并移栽，对获得的纯合突变体进行表型观察和统计分析。

与现有技术相比，本发明具有如下有益效果：

本发明发现利用基因编辑技术，将PhCLV3和PhCLV1基因进行敲除，两个基因功能丧失的单突变体的花瓣增加，双突变体表型更加剧烈，花蕾呈现头状花蕾、花重瓣、花瓣面积增大、开花密度大或株高变矮，大大提高了矮牵牛作为花卉植物的观赏价值，说明PhCLV3和PhCLV1基因在矮牵牛生长和发育过程中具有重要调控作用。因此，本发明在矮牵牛花卉性状改良方面具有较好的应用前景。

附图说明：

图1为CRISPR/Cas9靶向PhCLV3和PhCLV1基因的靶点及突变体基因型鉴定结果示意图。

图2为基因编辑矮牵牛花瓣的表型，和野生型相比，phclv3和phclv1单突变体花瓣数目增加，phclv3 phclv1双突变体的花瓣增多，表型强于任一单突。

图3为phclv3 phclv1双突变体，花蕾呈现稀缺型头状花蕾，和野生型相比，同一花期开花密度增大，株高变矮。

图4为基因编辑矮牵牛的表型统计，和野生型相比，phclv1单突变体的花瓣和萼片无显著性差异，phclv3单突变体的花瓣和萼片显著性增加，并且phclv3 phclv1双突变体的花瓣和萼片数目，花蕾和花瓣的直径都呈现极显著性增加，开花密度极显著性增大，花瓣表现出重瓣、花面积增大(****,P≤0.0001,双尾T检验)。

具体实施方式：

下述实施例中所使用的实验方法如无特殊说明，均为常规方法。下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。

实施例1：矮牵牛靶向PhCLV3及PhCLV1基因靶点的制备

根据PhCLV3及PhCLV1基因序列，在两个基因的编码区域选择20bp为靶点，定向敲除两个基因，使用CRISPR-P v2.0在线网络工具(http://cbi.hzau.edu.cn/CRISPR2/)选择sgRNA靶点。首先使用p_U6p载体作为模板扩增获得p_U6p-Target-gRNA表达盒，使用包含靶点和Bsal1酶切位点的PhCLV3-g1-F等靶点引物和公用引物U-R来扩增gRNA片段(引物序列如下)。扩增的sgRNA_U6的片段经第一次Goleden Gate反应体系进行边切边连反应连接到p51、p61等系列中间载体中，经p-F/R引物鉴定阳性克隆；条带正确的中间载体质粒经第二次Goleden Gate边切边连反应体系连接入pM最终载体(图1)，克隆转化DH5a感受态细胞，使用pM-F/R引物进行菌落PCR筛选阳性克隆；将测序正确的质粒转到GV3101根癌农杆菌菌株中。

引物序列如下：

PhCLV3-g1-F(如SEQ ID NO.11)：

TGTGGTCTCAATTGAAGTTACCAGAAGAAATGCTGTTTTAGAGCTAGAAATAGCAAG

PhCLV3-g2-F(如SEQ ID NO.12)：

TGTGGTCTCAATTGCTCAAAATGCATTTCACGAAGTTTTAGAGCTAGAAATAGCAAG

PhCLV3-g3-F(如SEQ ID NO.13)：

TGTGGTCTCAATTGCCTTTCCATAAGCAGCCCATGTTTTAGAGCTAGAAATAGCAAG

PhCLV3-g4-F(如SEQ ID NO.14)：

TGTGGTCTCAATTGTGAATTGGATCAGGACCAGCGTTTTAGAGCTAGAAATAGCAAG

PhCLV1-g1-F(如SEQ ID NO.15)：

TGTGGTCTCAATTGTATGAAACTTAAAGAATCCAGTTTTAGAGCTAGAAATAGCAAG

PhCLV1-g2-F(如SEQ ID NO.16)：

TGTGGTCTCAATTGGGTACCATACCACCAGAAATGTTTTAGAGCTAGAAATAGCAAG

U-R(如SEQ ID NO.17)：

TGTGGTCTCAACCGTAATGCCAACTTTGTAC

p-F(如SEQ ID NO.18)：TCCTGTCAAACACTGATAG

p-R(如SEQ ID NO.19)：TAATGTACTGGGGTGGATGCAG

pM-F(如SEQ ID NO.20)：ATAAGCCCATCAGGGAGCAG

pM-R(如SEQ ID NO.21)：CGGATAAACCTTTTCACGCC

具体步骤为：

1.矮牵牛遗传转化采用叶盘法，主要参考文献(Deroles et al.,1996)。详细步骤如下：(1)剪取矮牵牛幼嫩叶片，流水冲洗叶片表面，用75％酒精消30s，无菌水冲洗3-4次，10％次氯酸钠消毒10min，无菌水冲洗3-4次；(2)去除叶片的中脉和边缘部分，将叶片剪成1.0cm×1.0cm，摆放于PhD1培养基上，将外植体远轴端朝下，19℃黑暗培养2d；(3)将农杆菌接种到3mL含有相关抗性的LB液体培养基中过夜，次日按照1:100的比例接种，摇OD值至0.6-0.8，5000g,10min收集菌体，在超净台里用MSO液体重悬；(4)将(2)的叶片倒入(3)的MSO重悬液中，手动轻轻摇晃10-15min，进行侵染；(5)外植体侵染结束倒出菌液，将叶片再次摆入PhD1培养皿中，19℃黑暗培养2d；(6)将叶片转入Ph2Z的培养基，每两周换一次培养基直至生芽，待新生芽长至1-2cm高可切除外植体及愈伤组织，转入PhMSSV生根培养基中，直至生根；(7)待生根苗生长至瓶口处时，取叶片提取DNA进行编辑鉴定并移栽。

2.基因编辑苗子DNA的提取，具体步骤如下：(1)取适量植物叶片于2mL离心管中，每管中放入一粒3mm钢珠，液氮充分冷冻后打样；(2)向含有打碎样品的离心管中加入2％CTAB缓冲液500μL，轻轻颠倒混匀；(3)放入65℃烘箱裂解1h后取出，冷却至常温；(4)加入等体积的氯仿/异戊醇(24:1)，颠倒混匀；(5)室温12000g，离心10min；(6)取上清于新的1.5mL的离心管中，加入等体积异丙醇后混匀，室温放置10min；(7)室温12000g，离心10min；(8)去上清，加1mL 70％酒精，重复洗涤两次；(9)倒掉酒精后室温晾干或烘箱烘干；(10)加入适量ddH₂O充分溶解，分光光度计检测DNA的浓度和质量。

3.DNA提取完成，对基因编辑苗进行PCR鉴定。

4.和野生型相比，phclv3和phclv1功能丧失的单突变体以及双突变体花瓣增多，并且对突变体的花瓣和萼片数目、及双突变体的花苞及花瓣的直径进行了统计。

如图1所示，DNA水平上鉴定发现PhCLV3及PhCLV1分别发生了碱基的删除和插入，导致二者功能的丧失。对突变体进行表型观察，和野生型相比，phclv3和phclv1单突变体矮牵牛花瓣增多(图2)，phclv3 phclv1双突变体呈现稀缺型头状花蕾、花瓣重瓣、开花密度大、株高变矮，表型强于二者的任一单突变体(图3)。对花器官进一步统计发现，与野生型相比，phclv1单突变体的花瓣和萼片无显著性差异，phclv3单突变体的花瓣和萼片显著性增加，并且phclv3 phclv1双突变体的花瓣和萼片数目，花蕾及花瓣的直径都呈现极显著性增加，矮牵牛花瓣表现出重瓣且面积增大(****,P≤0.0001，双尾T检验)(图4)，phclv3和phclv1单突变体到双突变体表型渐次增强，大大提高了矮牵牛作为花卉植物的观赏价值，为矮牵牛性状改良扩充了基因资源。

序列表

<110> 中国科学院遗传与发育生物学研究所

<120> PhCLV3 和PhCLV1基因在调控矮牵牛植株观赏性中的应用

<160> 10

<170> SIPOSequenceListing 1.0

<210> 1

<211> 5000

<212> DNA

<213> PhCLV3基因起始密码子上游()

<400> 1

caaaatagtt ggccatttag ccaatctcaa ttggtgtaaa atagttggcc atttaggaaa 60

ataagacaaa attaatgttg cttttccttt aatacccttg catagttaat gacaacgtga 120

acaacttcat ttcaactcct aataaagagc aacatagtta aattacacta caagaagtac 180

attttttcaa ttttcttaat aactgtgcaa aagccaaaat gaccaactat tttggaccgg 240

aaggagtaat tttcacgttt taagttattt catgccctaa gttgtacaaa tacgcgtatg 300

acctgtcggg aaggtgaaga acaccgaaat taaaaaaata attaaaaaac aattaaattc 360

ggatttaata aaaataaaaa aaacttacct cgaggttgat ttgtgctcca atagcaccta 420

gtaatgattc cccactagtt atatgcttaa gaactacaaa aaattgagta aaaaagttag 480

ggtttaagta ttagaaaata attaaaataa ccctaaaact aagaatttac cttgaatttc 540

ttgttatatg tagtgtttag gagggtagaa tgtgtttgat ttttgatttt gggggtgggt 600

tatggtggtt tttgggtata gaggtttggg gggggggggt gtttagagag aattttagtg 660

tttggggaag aagatagaaa tggggaaagg ggggttctgc tgaaacttaa aacgggtaga 720

tcgaccgacc tttcgaccga aattcggtcg aaaggtacaa aatttttttt tgttcgaccg 780

atttgtattt cgaccgattt tcggtcgaac agttgtgact atgttcactg ttcgactgaa 840

aatcggttag tggttcgacc gattttcggt cgatagatgg tagtcgaaaa aacctgtatt 900

tttagtagtg agctattccc tgcatttaat ttatgtgacg taaattttga gtcagtactt 960

aattacgttt aaaaaagaaa tgaaagatta ttattagacg tgaaatggaa agttaaggtt 1020

ataatattgt tttcaaatat agaaaaatat ttagaacaga ctaataataa aatagtgtca 1080

tatcaatcga aatgagtaat tcttttataa agcacgtagg gatattagtg tattgtgcat 1140

ttatttggta cattacagta cattaattca aaaagctttt cttattagtt tattagaaaa 1200

agaatgattt atttctaaat gtagaaataa tttgattcca aatcgtccat tttaattgcc 1260

caaaagacta atttgaatat tttaaatgga atagaagccg gagtgttctc atgccaccat 1320

tcagctacgg tcccaggatt aaaatgttgt attacagcca tgtatctcag aagttatgaa 1380

taaactattt ttttgcaaaa aagttcacct agtgccctga atagacatgg tcttacctaa 1440

ttgcccctac ttacaaatat tttaattaaa tgccctccac atcatactcc ccctatgaaa 1500

ggactatttt ggcccccgac ctatttaatt catgaacatg tttccttctc tctttcccca 1560

ttattggcct ccttctttct tctttgtgtg ctgaaaattt gggatttttt attctctctt 1620

tttcgtatat ttgttctatt attttttcgt tatattgtaa tttttatttc gcgtaatcat 1680

ttatctgcat ttattttttc gtattatttt tttatactta ttgtttatat atttatctgt 1740

tagttttttc gattcgatct atgattgttg aattgagttt gaggttcctc tggatttttt 1800

tcttgtgttt gcttgattcc agtgatttga aggtttttat cgcctatggt tattagatat 1860

tcatgttttg catagagttt aaaatcttga ccatggccaa gagttgcata aactttctct 1920

gcaagttgct tattgcagta gagtttaaag tcttggctat tgtaaatact tgcagagatg 1980

ttccgtgtaa gttgtcgctt ccaccagaac ttagagtctt ggctattgcc aagagttgca 2040

gagactttgt ctccaacttg ttcactgtaa tagaggtaga agtgttggaa attgctaata 2100

ctcttagaga ttattagtgt aaggtctccc ttgcagcaga atttaaagtc ttggctattg 2160

ccaagagttg tagagacttt ctctgcaagt tttgcactgc aatataggta aaagtctttg 2220

gcaattgtca atacttgcag agactatttg tacaagtttc tccttccact agagtttaaa 2280

gtcttggcaa atgtcaatac ttgcagatac tatttgtaca agttgcccct tccacttgag 2340

tttaaagtgt tggctattag caatagttgt agagactttt tctgtagttt actcttttca 2400

tcaaacatgt gttcagtagt tgtcattgta tgtttgtatg tttgtatatg gtcttttcat 2460

tattttttca ttgttttgtt attatgtgtt gtcattgttt tttttaaaaa tttacaagta 2520

atgaatattg aatcttcaat ctcctgatcc tgatcatgta catttagata tggtggatga 2580

ttttataagt cagccaagat cttctaatga attccaaggc aataatgtga tgtgggcgat 2640

gaattttcaa attttcgttt gttatctctg ccgcaagatg atctgtgtag tagtccaaat 2700

gaggctttta atactacaaa agagttagag aaagttgttg agtagttttt aattacatat 2760

atttcgtatt taccgtgttt tttatgttta tgtaaacaag tacacttgga tgtttgatat 2820

ttttgttatt ttaaagactg aagttgcttt gaatcagatg aacggaaagt gttggcaatt 2880

gcaggacttc aagacatttt agttgttgtg gggcgaacac atttagatgg aaagtcttgc 2940

caatgggcaa gacttgaatt ctaattaatt aattccaact ctaatatttt agaattaatt 3000

taaaattgat aaataattca ttgagggttg tgctggtaat gttgttgcca agacttggtc 3060

tcaattttgt tgtcaatatt attgcctatg gcaagacatc aaggcaagtt aactatttct 3120

aatattaatg tcgataaata agttttggta ggaaatatct tgtcctttgc caagacttta 3180

cgtcaagtta aattgaattg gttatctgta tgagcgtaaa gtcttggcta ttgccaatag 3240

ttcagtttat tgataatatt aactttccaa tcacaattga ttttaaaaat attcataatc 3300

aataattgtc cttccttttg atgcaacacc atgctatggt cagcttcatt cacaaagaag 3360

cgggattttc tgacatgtca gttcttttaa taacttctac tatgaagcag gattttctga 3420

aattctcata attcttcagc agatacatgc aagtgataag ttcttgatca tttgttacca 3480

tcatgcctct gcacctttct agattcgtgt caaaccttat ctttgcttca ctttcatcag 3540

tgttgatggt tagagcatta caaatttctt tttcaaatac gtggtagctt ataccatctt 3600

ggacaagtat caattttata tcatggttga tgtatttgct gtttgcatct catttcccat 3660

tataagctat ataaatgtag cgagttgcca tagtgttaat cctgcacata aacaagacga 3720

tatgcttaga atttcgtact caaatcgctt tgatagaagc aggatcatgg gtaaaagtga 3780

tgccctttgt gggctttaga tcttgcgaag aagactctcc taggttccta ttgcaccaga 3840

gcagtaactc ttcgcaatag ccaagacttg cagagacttt gtctgcaagc ccccattgca 3900

acaggtctta aagtcttggc aataggcaag aagtctctcc taggtgctta tcgtacccga 3960

gttgtaagtc ttgacatgac ttgaagagac tttgtctgca aggacctcat tgcaacaggg 4020

ctttaagtct tggcaagagg gaagaagtgt ttataaagcc cagagacttt gcctgcaagt 4080

cccccattac aacagatctt aaagttttgg caataagtaa gaagtctcta caagcctccc 4140

attgcagtag agtttaaagt cttggctatt accaatactt tagagatttt cagtataagt 4200

ttctctttgc agcagaactt aaagtcttgg ctattgtcga aacttgaaga gattttctcc 4260

tgaagttggc cactgcaaga gggcttaaag tcttggcaat agccaagaag tctctccaag 4320

gtgcccattg caccagagct taaactcttg gctataagtc aagggttgca gagactttct 4380

cttcaagttg gcacttgcaa caggccatag agtcttggca ataggcaaga agtgtatgta 4440

gggtgctgat tgcaccagag cttaaactct tggcaatagt tgcagagaac tttacccttg 4500

caacaggact taaaggaatt aatcgaactg ttagtaaaat tcaatagaaa tttgaagaaa 4560

tcttacgctg aatctttgct tgaacggagc aatctgtata aggaatgggt tttagggttt 4620

ctgtgatcgt gtgactggtg aaaaggaacg ggagggactg ggcgtgttgt tttattggtt 4680

tttagggggt attttggtcc caaaaaggtt ttattaaact aatggagagc atttttgcta 4740

aaccattgtt attgagggta ttttggcaaa ccattgcttc cggtagcata tttttgcaaa 4800

tcaaccattt tttattcata acaacatttt aaacacgtac ccataatttt ttttcgaact 4860

gattagaagg cctctcagat aacctattta cttgtaattg agtaaaatca cttggaacat 4920

taagagcaac acaatcttgc taaaaaagaa cttcattcat catgtgtggt tctgtactga 4980

tataagtttc aagcaatcgt 5000

<210> 2

<211> 579

<212> DNA

<213> PhCLV3基因终止密码子下游()

<400> 2

aattatgcag tatcgatttt gtttattagc tctatatatt tccaagtttt tgctaaaggg 60

tacctggttt cgtctctctt tttgctgttt ttggcccagc agaacaaaaa atgaagcaaa 120

aatatgtaaa tgggcggcta agtgttaacc acttggaata gaagtttatg cctgacaata 180

tgtatatagt gtccattttg gtcctaagta ataataacaa tgaacatagt tttttgctat 240

cacaagtgaa tgctctcatt gtcattaatg cagaaaagct tgtttttgga gctaaagggt 300

tagctgtata ttgtttctta gctgataaaa gaaatgaaaa caggaaataa gggacaaaat 360

cagtcgtaaa gatgatgctt tctgccctag ttgagggaga aattttttct gtcactggtt 420

atataagttg cgcagaaatg taaattatca accacattca tataattcat ttacatcatc 480

ttcaacatag ccagacactc ttagctcaac tgacaaattc tcaagcattg cataaatctg 540

ctctgagtgc atatgtgatc tatctccagc cacaaattc 579

<210> 3

<211> 1893

<212> DNA

<213> PhCLV3基因组序列()

<400> 3

atggacatta cgcccagcat ttcttctggt aacttcaaaa attccgaaaa tgtctcatcg 60

tcatatatat tccactcaca atatattggc gcaccttgac tagtagatga gcgtggatac 120

ttaccaaaaa ttaaaatctt tacccgtgcg ggatcaacat tcattttctt aaacaaaata 180

tcaacaaact tatgatacaa aatagaagtt ggcactttaa aattactttt aggaggtaag 240

ctatacctga ttgagttgtt ttcatctaac acttcacccc ccccccctcc aataaaatga 300

tacatttact ttggacatga cgagaagaaa agaaatattg tttattgcag ataagaaatg 360

aggctcggtg gtggtggtac atatgagatg tgaggctaga gttaaataga gtacaaaata 420

tatggaagtt ggcagaagtt ttgccaactt atgtaacggc aaaacttatg ccgactaaca 480

tttttataaa atataacaag acggtgttta tccttctgtc ttattaagtc ggtggaatag 540

atgtcgactt atttaaacac tatgccgatt aacattttta taaaagataa caagtcggcg 600

tttattctac cgacttattt aaacatatgt tctgttactc catttgattg cttcgtctgt 660

caaatgctat aagtcggtat tagaagtgcc gataaactca taattgtaac attttagttg 720

accaacctat atatgttgct gaactacaaa aaaaatccca tttcggcggt ggattcctca 780

tgtagaccac aaattataaa attcttataa tttctgtcta attctctgta agtcaaacaa 840

tgacttataa attgaacaga tagaatacat gtttagctta tggtaaaaaa gtttcttttt 900

tatagatttg tattcagtcc aatactatca tacaaactga agcgattgga gtaacgttac 960

attgactatt cttttatcct caaaatgaat gagctaactg agttttcgga aagaagttag 1020

gagaagttac tcattattaa cttcctgcaa gcgataattt tgtgtttaat gaaacagata 1080

atttacttta agcatacaaa aatgtactaa tgatttggag gaagtttaat accaccaagg 1140

gttagcatat gagatgagct cacctaatga tggtcctatc ctattctatt gcctcggcat 1200

gtagggtcta aaactacaca ctgtcctgtc ataaaagtat atgatagata aaaatggctg 1260

catggcttat cattttcttt tgctctataa attagcttta acttcacttc ctcaaccttc 1320

aaaaacatct tgttccattt gtgttttatt aagattaaga acttgcgtct agcagcattt 1380

ccctttcgct tctgaaatgt cttgcactgc caagtgtttc acaatctttg tcttgctttg 1440

ctttttggtt atgcaagagt cacatggtct ctatctctat ctctttctca catttatgtt 1500

tgttttttgt gctgtttgtg atgatgtttt attttgatgt agggtgcact agtagctcaa 1560

aatgcatttc acgaaaggct gttgcttcaa tgagacttct aaacaggaag gtttgaacat 1620

tgcctatatg actgcctata gtgttttcat ctttttgctc atactatctt ttattctctt 1680

ctaatttcca tgcctacata tttcttaaac caatgtgcaa tgcaggtttt aggaagccaa 1740

tgggctgctt atggaaaggg cttacaatat ggaaactaca ataataaagg gaagaatgag 1800

aagtttgctg attgggagct tagaggagtt ccagctggtc ctgatccaat tcaccatcat 1860

ggtggcagtc ctaagaaacc aaagactcca tga 1893

<210> 4

<211> 264

<212> DNA

<213> PhCLV3基因的编码序列()

<400> 4

atggacatta cgcccagcat ttcttctggg tgcactagta gctcaaaatg catttcacga 60

aaggctgttg cttcaatgag acttctaaac aggaaggttt taggaagcca atgggctgct 120

tatggaaagg gcttacaata tggaaactac aataataaag ggaagaatga gaagtttgct 180

gattgggagc ttagaggagt tccagctggt cctgatccaa ttcaccatca tggtggcagt 240

cctaagaaac caaagactcc atga 264

<210> 5

<211> 87

<212> PRT

<213> PhCLV3基因编码的蛋白序列()

<400> 5

Met Asp Ile Thr Pro Ser Ile Ser Ser Gly Cys Thr Ser Ser Ser Lys

1 5 10 15

Cys Ile Ser Arg Lys Ala Val Ala Ser Met Arg Leu Leu Asn Arg Lys

20 25 30

Val Leu Gly Ser Gln Trp Ala Ala Tyr Gly Lys Gly Leu Gln Tyr Gly

35 40 45

Asn Tyr Asn Asn Lys Gly Lys Asn Glu Lys Phe Ala Asp Trp Glu Leu

50 55 60

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

65 70 75 80

Pro Lys Lys Pro Lys Thr Pro

85

<210> 6

<211> 5000

<212> DNA

<213> PhCLV1基因起始密码子上游()

<400> 6

aaaggtaata tcacaactaa agaataataa aaaatatttt tttttaaaaa aaaaaagaaa 60

gaaagaaaaa ataagaagtt tttaaataaa attaaatgaa atagaattga aaagaaaatt 120

ataaatgaaa aaaaaaaaaa aaaagtagaa ataccatgta attacacagt gtaattacct 180

ccaattctca gctcccccct aagaattgaa gagtgtaatt gcaccctctc aattacactc 240

aattccatgt tgaccaagta attacttggt cagacaaaca agccaaactg tgtaattaca 300

ctcaattaca cccaaatcca attacatggt ggcttttcaa acaagcccta agtaattact 360

ccctctgttt caaaaaggat gaacctatta ctatttaaag ggtcaaacaa gattatgttt 420

gaccacatgt cttacaaata ttttctaact attttgaatt gttaactatt gtgacttata 480

gtatttttca tgtagtttct aaatatgtaa attttatttc aaaaaattta aaaattcgat 540

gtgcaaattc atggtcaaaa ttagtcaatt tgaccctcgt actccgcaaa ggttcatcct 600

ttttgaaacg gagggagtag ttttgaggat taccccgttt aatatgattt taataaaata 660

ccctccaatt ggtctgtaga caactgttca agttatcaat ttgcaagtat ttttttttag 720

aaagagtcaa caagcaagtg ttgtttaggc aacacttcat attatctgaa agaatacggc 780

aatcaacatt ttctctgcga aaatgtgaaa ttcaaaggta agtattgtct agtcgtggca 840

aatgggaggg ttggatcgga tttggatgga tcaaaaatgg attgagaaaa attggttggg 900

ttgtaatcca cccatattaa tataggtaaa aaatgggtcg gataaatctg aattgaataa 960

aaaatggatt ggataaaata tgggtaaaat atgatttgga taacccaccc attggggact 1020

tcccttagtg ttattatctt acataaacag agactatttg ttggtaaata tggatgattg 1080

agttctatga aaatgggtac tataatttta tatactctgc atccttatct taatgaatta 1140

ctaggcattc ttactatgta acaacaatta ttttatttac attttaaaaa tatctttgtt 1200

ctttaatttc ctcgagtatc tttgttctta tcagtttagg tatttatgta catactaaca 1260

attcaaattt tgttctatat tacttctatg tatattactt ctatgtataa aacaatacta 1320

ttttgtactt cagactaaat tattttagtg atatacagtg gttgttttgt gagaaaaaag 1380

tcaaataaaa aaggttggca attaaaaggg caaatagtga accgactaag ctttacacat 1440

ggaacaaaaa aattaaaata aaaaattcaa ccggctacta gctacagtta aataaaaagc 1500

tgcagttagc cggtatactt taaaaaccca gttaccaaat cttatacata gtaaatgaat 1560

tctgcaatat gatgtctcct tcacaatcca tattttaccc gtttgcaacc catatcccgc 1620

ccagttaatg gagggtctaa tatgaactga accgactaag ctttacacat tttaaaatga 1680

gcgggttcaa tgtgggttta atgtgggtaa aataggtggg ttaatgaaat ttgggtttaa 1740

attgccacct ctagtattat ctaaaaaaag aggaattaaa ggcaaatatt gcctcctagg 1800

cagtacttat ggagaaaagg agggccgagt gctgcccatt caacattttc tctagaaaat 1860

tgtgagaatc aaggggcaag tgttgcccta aaaaaggagt caaagacaaa tattttttct 1920

tagacagtac ttaatgaacg tcattataag atttatagga atatttccgt ctgaaattga 1980

ttcattaaac taagcgaaga atacttttgc gaaaccacgg gaaaaaaatc aaaccattgc 2040

ctatggtgta atagttctac aagtcagtca tttgcttaag ggccgcatta attgaggccc 2100

acataaaaat gtttagtaat ttattatagg gcatcttaca aaagtatata aacaaacatt 2160

aatatcacac aatttttaaa atatagtaat atttctttta caaaaacata ataaatattt 2220

ataaaaagat tggataaaac cctagaactc cctcttacaa ccctctgcca acctcccagc 2280

cgcccccaag acttccctag ctgtgccgat caattttttt tcattttttt gtatattatt 2340

tgtatatata tacttgtatg actaatgtat ttctcttgaa tagacgttca atacatcaga 2400

aatacaatga atatacaatg ttgaggtttt atacatgtgg tgtatgtgag tgtatatact 2460

ttgtatgtcc attgtatata catgacatac aacaaactta taatgggtat acacttggaa 2520

tacatttccc cacaatcaac aaccacaaaa aatacaccat agccaatata tatccgccat 2580

agccacaaaa gaaactcaaa gaaaaaatgg aagtagcagc gccagcgacc ggagaagagc 2640

agccaaggca ttgctctatg ctggacaaga accttcgaaa cttgtatgtc gccaatggag 2700

caaagtagac ctgtcggtga tgagcacaac tgagatgatg gaacgacgag cgatggagat 2760

ggtgtggtga ttggaaacga tgacatgtca gtggtgacag cgccgccgaa gaaagtgggt 2820

gaggggtagt gagaagagag ggaagggaat taatgtttaa tagcaatgtt tttgtattta 2880

tgcaatctgt attacatttc gatctcaagc aagttcatat tggactatat tactgtccat 2940

attgctccat taaaacgtgt attaatttga tataaaagaa agattatttt tccttacaaa 3000

ctcaacattt tcctagcata ttgattatgg aggtcataag atattttgtt catttgctaa 3060

atatggatgg aaaaaacctt tgttctttat ttagcatgtt aaatagtaag ttatattatt 3120

agtgtggtgg gaagaatttt gaaacttcta ctaaaatgta gtataatatt tgtgtgactt 3180

taaatttttt aaattaaaga taaaacaatg agttaaactt aatttagttc aaaatataaa 3240

agtgtgtcaa tcttttcgta tatactacta acaaagaaac tgtatcacac tattttgcct 3300

ctcatatcga caatattcca ataaagaaat aatatatgtg ctttcaataa actatagtta 3360

aaaaagaaac atatctcatt gaaatgtgtc attggaatat acgactagta gattaattca 3420

gtttaatttt aaaaataaga aaactcataa attttcacac tatttgctct cctgacaaac 3480

tcagaagcat caatcatcta atgacgttaa agtcctttgt ttacacaatg acagaagccg 3540

ccagatattt acactcttgc tcctccagcc atttcatccc ttgcgtttaa agtccactgc 3600

taaaatcatt tactttagac aaaatgatgt tgtggctagt caattatcaa aattttaagt 3660

ttatgagttt tgaattttcc atcaaactct ttatttgtct tatttgttat cttaatgtta 3720

gcatgtctga attcacgatc aaaatgaaac agtttaactc tcgaaatttg aacagaatgc 3780

agggcccctc ctataaggca agtaaagcca ccgctttagg cccgtatttt ttggggccct 3840

atttttgtta tacatataat agattatact accttcattt tttaatgcac gttatttttt 3900

ttctttttat ttttgtcatt ataagtgtca tcttaagaaa tcaatataaa tttaattatt 3960

attttcttat ctttcccatt caattaaaga tataggaaat gaataattgt acttaagttt 4020

gatattcgag attggtaaat caggacaatg tagtaagtta tataatccat ccatttcagt 4080

ttatatgaac ttgtttgaca tgagaaatgt ttaataaaga aaataagact ttttaaactt 4140

gtggtctaaa ctaagtcatt gatatttgtg tggatataaa taatttattt tggtaacatt 4200

ggtaaatttg gggttaagtt gtttctaagt ttagaaatat gtcttttttt taacggacta 4260

aaaaataatt aagacgacgt gcaaaaaagg aagtaaaatg gacgtttgta atgagactga 4320

ggtagtatgt aagttaaaat agacgacatc ttaagtaaaa aagtaaaata catggcaagt 4380

tatattaaat tgaacaaatc ttatctaaaa tctaatatca acaatctctc aagtgagatt 4440

aaatggattg tttatattat caattgagaa agactcatca aaagaaatcg attaaaaaaa 4500

ttattaataa ctttgcattt caaaaaacta aaaaaaatag aattcaaagt tatatttatt 4560

gttttttatt aaaaaaatta agacccctca ttaaagtttt gtttcaagcc acaaaattcg 4620

tcgggctgcc accgacagaa tcacataaat ttagacggag agtgtaatat ttataaatgt 4680

taaatgaatc tctaatacta cacttcattt catatatcac taatgatgta cacttaacat 4740

gaaccaaatt accaaagcta gcaggttcac tatgacttta gagctaattg agtacataaa 4800

aatgattaaa aaggttcatt aaaaaataat tataaccact tccctcctcg tatcttgctc 4860

atatatattc aaaggcaaaa gtttagtaag tggtaacaga acttttgatt ctcctaccac 4920

tgaatactta tacttcaaat agcctaataa acctatccta aaattcgtat aaaccaaaaa 4980

acaaagctaa gcattgtaac 5000

<210> 7

<211> 983

<212> DNA

<213> PhCLV1基因终止密码子下游()

<400> 7

agccacttgc aaggatcagt gtctggttga gaactcgtag agttaagggc tgcgagcaca 60

aatctatgca agtatagatg tattttgtgt attagagtgt tttctttttt gtgccatttg 120

tgtttcgttt gcttattgtt aagttgtatt tggcaaaatg tataaaaggg gcaagaagaa 180

aaaaggaatg accatgagaa caaacaaatg aaaacatggc aatgagttgc tacttgctat 240

taataacatg atctttgatt aggactttgt aaaggtgaaa tttgtttcta taatggaagt 300

atttgatata ttcaatgttg cttcgctatg ctcgtgatct atctagaaaa ttttctgaaa 360

caacgggcga tatctaattg tcaaataatc agccattttc atcatatgca aatttgtgtg 420

acttgctgat acttatgttt tgaaattacc gtcgatatct ttttagtagt gaatttctta 480

tcctaaatca agttttttac attatttaag ttactcatat ttaaaaaagt aaatgtaact 540

actcatattt aactagaact aaggacactc agtgcactca ggggtcgttt ggttcccctc 600

attaaacatt aatacctgcg caaaactctg cgttaattta gcgcagcgtt tgattggttt 660

tctgtgaaaa tttaataccc acataaaatt agtgcaacgt ttggttgcgt ttttgtatat 720

tccgcattat taatatccgt attattaatg cgggaatcta tgtattattt tatgcggggt 780

agaaggtggt ttaataatat gggtattaat aatgcaggta ttaaaattta aaatgacaaa 840

attaacccct gtaataataa ggatatttta aaattttatt taaggaataa ataaatattt 900

taaataataa tattttttgt taatttgtaa tttatcagtg attcaacaaa taatcctacg 960

ttataatata tgcgtggcta ata 983

<210> 8

<211> 3942

<212> DNA

<213> PhCLV1基因组序列()

<400> 8

atgtcactcc caaagatgtt cactgcttgt cttctccttg tccaaacttt catcttcttt 60

tcatacacca ttaatgcaaa caatgatctt gaaacactta tgaaacttaa agaatccatg 120

gttggtcctg aaagttctgg tttaaaagat tggaacaaca caaattattg ttcattttct 180

ggtgttacat gtaacaatgt ttcacgtgtg atatctttga acatttctaa tgttccatta 240

tttggtacca taccaccaga aattggttat ttagacaaac ttgttaacct tactatattt 300

ggagataatc ttactggtac acttcctttg gagatgtcca aacttgttaa tgttaagtac 360

attaatcttt catacaatgg tttttcaggt ccttttccta gagaaatctt gttgggatta 420

atagaacttg aaacatttga tgtgtataac aataatttca ctggtgagct tcctactgaa 480

tttgtgaagc ttaagaagtt aaagacatta caccttggag gaaattattt tcatggagag 540

atacctgaag cttattctta tattgaaagt ttaaaatggt taggcttgga cggtaattca 600

cttactggaa aaattcctaa gagtttggct ttgcttccaa atcttgaaga gttaaggttg 660

ggttattata atagttatga aggaggtatt ccacctgagt ttggttctat tactactctt 720

aagcttcttg atcttggaag ttgtaatctt gatggtgaaa ttcctgcaag tcttggaaat 780

ttgaagaagt tgcattctct gtttctccaa atgaatagac ttacaggtaa gggcagacct 840

atacttttga atactttttt gttttataat agtagtattg tgttaagata tatgctggtc 900

cgactaatcc ggagtcgtgt aggaccccgg atttttccat tcatagggct tgaaaccgag 960

acctttgttg atgggaaggg tggtggaatt agtgattgct tttggatatt tatttattgc 1020

atagatatat atgtgagaaa tcttgagaac ttttgtgaat tgtaagtttt gaagttgtaa 1080

ttttgatgaa cttagaacct ttaacgcgtt aagtttaaaa ttctgaatcc gtttctgctt 1140

tacaggttac atacctccgg aactctctgg tttagagagc ttgatgtctt tcgacctgtc 1200

cattaaccaa ctcattggag aaatacctga gagttttgtg aagttgaaga aatttacatt 1260

gataaatttg ttcagaaaca atctgcatgg tccaattccc ccttttattg gtgaccttcc 1320

aaatcttgaa gtgttgcaaa tttgggacaa taattttacc ctccaattgc ctgaaaatct 1380

cggtcgtaac ggtaggctaa agctgcttga tgttgctact aatcatttca ctggaagaat 1440

accttctgat ttgtgtaaag gggggaagtt aaagactttg attcttatgg agaattattt 1500

ctttggctca attcctgaaa aacttggtga atgtacatcg ttgactcgaa ttcgtgtaag 1560

gaagaattac ctcaatggta ctattccagc tggttttttc aggtttcctt cattggatat 1620

gcttgagctt gacaataact atttcaccgg gcagctgcca acggagatta atgcgaataa 1680

tctcacgagt cttagacttt ctaacaactg gatcactgga aagattcctc catcaattgg 1740

taacttgatg aacctagtga ctttgtcact tgacatgaac agattttctg gtgacattcc 1800

tcaagaagtt gcaagtttaa agaaacttgt gaccatcaac ttgagtggca acaacttaac 1860

aggtgaaata ccaagttcaa ttgctcaatg ttctgaacta acgtcaattg acttgagcag 1920

aaaccaatta gttggtgaag ttccaaaaga gatctccaaa ttaaacagct tgaatgcgct 1980

caacttgtca cggaaccaac tcaatggtgc catccctgga gaaattggag tgatggatgg 2040

tttaacagtt ttggatcttt cttacaatga tctttctggc agaagaccga tcaatggaca 2100

gttaaagttc ttcagcgaca ggtcttttgc aggaaatcct aatctctgct cgcctcgtgc 2160

tacttactgt ccgtcagctt ccaagtcagc agaagttccc cacaagaatc attctgggaa 2220

gtttacaact acacaattgg tgattacaat aatcatcttg gtaactgttg cattgctgct 2280

agctgttaca tgggtgatca tcaagaagga aaagttcaga agatcaaaac tttggaagct 2340

aacagcattc cagagacttg atttcagagc tgaggatgtt ttggaatgtt taaaagacga 2400

gaacataatt ggtaaaggtg gagctggtgt agtgtatcga gggtcgatgc ctaatggcat 2460

tgatgttgca ataaaaaaac ttgttggccg aggaaccgga caccatgatc acggtttctc 2520

agctgaaatc caaacactag gaagaatcag gcacaggaac attgtacgac tactagggta 2580

tgtctcgaac aaagacacaa atttgttgtt gtacgaatac atgtcgaatg gtagcttagg 2640

ggaaacgtta catggtgcaa aaggtgctca tttgaggtgg gagacgaggt accgtattgc 2700

tgtggaagct gctaagggat tgagctattt gcaccatgat tgttcacctt ccattattca 2760

tagggatgtt aagtccaata atattctgct cgattcagat tatgaagctc atgttgctga 2820

ttttggccta gccaaattct ttcaggatgc aggtgcatct gaatgcatgt cttctattgc 2880

tggctcatat ggttatattg caccaggtaa ttatctgcca tccttcttat tcttttcatc 2940

ctctttttat tacataattt gcagaacttg aattaaggaa tacaaaaaat caagatccta 3000

ttgaaaaaga atgaactaag gaaaatagag ttatgaaagt gttattgaat ttggtttttc 3060

taatcctttc cattatagtt cagtgttttg ctgatttttt tgtggtcaag ttttactgat 3120

ttatcataaa tagccatgat taatatgtag tggaggagac aacctctact agaccacttt 3180

gtagaacaat tacttcatct atttgtttaa aaatactctt caatatatta tgaatatatg 3240

caatatgttg acatcgttta aaaagaacaa aaaattatat tttgggcagg aagtgaactt 3300

taaaaccagt agagtctcaa aaacttgcaa cccggtttgt gacaaggagc tgataccact 3360

attacaatgt tttgttagta gactaattat actaaaagta caagcttctt ctttttacaa 3420

agtacaaact gaaagaaata atggcaaact tttggaattt actgaactgg ggttaaaaga 3480

gtgatgaggg taaagttact ttccaataca agagctgtta aaacattaaa tgctgtcatt 3540

aatgttgttc tgaactatat ttttgttgga tatgaaacag agtacgcata cacgttgaaa 3600

gttgaccaga aaagtgatgt gtacagtttt ggagttgtac tattggaact aatcacaggt 3660

cacaagccag ttggtgaatt tggtgatggt gtagatattg taagatgggt aaataagaca 3720

atttctgaat tatctcagcc atcagatgct gcttcagttt tagcagttgt tgactcaagg 3780

cttattagtt accctcttgc aagtgttgtc aatttgttca agattgctat gatgtgtgtt 3840

gaagaagaga gttgtgctag gcctagtatg agggaagttg ttcacatgct cacaaatcct 3900

cctcagtcta ctactactac cccgaccctc ctcgcgcttt ga 3942

<210> 9

<211> 2949

<212> DNA

<213> PhCLV1基因的编码序列()

<400> 9

atgtcactcc caaagatgtt cactgcttgt cttctccttg tccaaacttt catcttcttt 60

tcatacacca ttaatgcaaa caatgatctt gaaacactta tgaaacttaa agaatccatg 120

gttggtcctg aaagttctgg tttaaaagat tggaacaaca caaattattg ttcattttct 180

ggtgttacat gtaacaatgt ttcacgtgtg atatctttga acatttctaa tgttccatta 240

tttggtacca taccaccaga aattggttat ttagacaaac ttgttaacct tactatattt 300

ggagataatc ttactggtac acttcctttg gagatgtcca aacttgttaa tgttaagtac 360

attaatcttt catacaatgg tttttcaggt ccttttccta gagaaatctt gttgggatta 420

atagaacttg aaacatttga tgtgtataac aataatttca ctggtgagct tcctactgaa 480

tttgtgaagc ttaagaagtt aaagacatta caccttggag gaaattattt tcatggagag 540

atacctgaag cttattctta tattgaaagt ttaaaatggt taggcttgga cggtaattca 600

cttactggaa aaattcctaa gagtttggct ttgcttccaa atcttgaaga gttaaggttg 660

ggttattata atagttatga aggaggtatt ccacctgagt ttggttctat tactactctt 720

aagcttcttg atcttggaag ttgtaatctt gatggtgaaa ttcctgcaag tcttggaaat 780

ttgaagaagt tgcattctct gtttctccaa atgaatagac ttacaggtta catacctccg 840

gaactctctg gtttagagag cttgatgtct ttcgacctgt ccattaacca actcattgga 900

gaaatacctg agagttttgt gaagttgaag aaatttacat tgataaattt gttcagaaac 960

aatctgcatg gtccaattcc cccttttatt ggtgaccttc caaatcttga agtgttgcaa 1020

atttgggaca ataattttac cctccaattg cctgaaaatc tcggtcgtaa cggtaggcta 1080

aagctgcttg atgttgctac taatcatttc actggaagaa taccttctga tttgtgtaaa 1140

ggggggaagt taaagacttt gattcttatg gagaattatt tctttggctc aattcctgaa 1200

aaacttggtg aatgtacatc gttgactcga attcgtgtaa ggaagaatta cctcaatggt 1260

actattccag ctggtttttt caggtttcct tcattggata tgcttgagct tgacaataac 1320

tatttcaccg ggcagctgcc aacggagatt aatgcgaata atctcacgag tcttagactt 1380

tctaacaact ggatcactgg aaagattcct ccatcaattg gtaacttgat gaacctagtg 1440

actttgtcac ttgacatgaa cagattttct ggtgacattc ctcaagaagt tgcaagttta 1500

aagaaacttg tgaccatcaa cttgagtggc aacaacttaa caggtgaaat accaagttca 1560

attgctcaat gttctgaact aacgtcaatt gacttgagca gaaaccaatt agttggtgaa 1620

gttccaaaag agatctccaa attaaacagc ttgaatgcgc tcaacttgtc acggaaccaa 1680

ctcaatggtg ccatccctgg agaaattgga gtgatggatg gtttaacagt tttggatctt 1740

tcttacaatg atctttctgg cagaagaccg atcaatggac agttaaagtt cttcagcgac 1800

aggtcttttg caggaaatcc taatctctgc tcgcctcgtg ctacttactg tccgtcagct 1860

tccaagtcag cagaagttcc ccacaagaat cattctggga agtttacaac tacacaattg 1920

gtgattacaa taatcatctt ggtaactgtt gcattgctgc tagctgttac atgggtgatc 1980

atcaagaagg aaaagttcag aagatcaaaa ctttggaagc taacagcatt ccagagactt 2040

gatttcagag ctgaggatgt tttggaatgt ttaaaagacg agaacataat tggtaaaggt 2100

ggagctggtg tagtgtatcg agggtcgatg cctaatggca ttgatgttgc aataaaaaaa 2160

cttgttggcc gaggaaccgg acaccatgat cacggtttct cagctgaaat ccaaacacta 2220

ggaagaatca ggcacaggaa cattgtacga ctactagggt atgtctcgaa caaagacaca 2280

aatttgttgt tgtacgaata catgtcgaat ggtagcttag gggaaacgtt acatggtgca 2340

aaaggtgctc atttgaggtg ggagacgagg taccgtattg ctgtggaagc tgctaaggga 2400

ttgagctatt tgcaccatga ttgttcacct tccattattc atagggatgt taagtccaat 2460

aatattctgc tcgattcaga ttatgaagct catgttgctg attttggcct agccaaattc 2520

tttcaggatg caggtgcatc tgaatgcatg tcttctattg ctggctcata tggttatatt 2580

gcaccagagt acgcatacac gttgaaagtt gaccagaaaa gtgatgtgta cagttttgga 2640

gttgtactat tggaactaat cacaggtcac aagccagttg gtgaatttgg tgatggtgta 2700

gatattgtaa gatgggtaaa taagacaatt tctgaattat ctcagccatc agatgctgct 2760

tcagttttag cagttgttga ctcaaggctt attagttacc ctcttgcaag tgttgtcaat 2820

ttgttcaaga ttgctatgat gtgtgttgaa gaagagagtt gtgctaggcc tagtatgagg 2880

gaagttgttc acatgctcac aaatcctcct cagtctacta ctactacccc gaccctcctc 2940

gcgctttga 2949

<210> 10

<211> 982

<212> PRT

<213> PhCLV1基因编码的蛋白序列()

<400> 10

Met Ser Leu Pro Lys Met Phe Thr Ala Cys Leu Leu Leu Val Gln Thr

1 5 10 15

Phe Ile Phe Phe Ser Tyr Thr Ile Asn Ala Asn Asn Asp Leu Glu Thr

20 25 30

Leu Met Lys Leu Lys Glu Ser Met Val Gly Pro Glu Ser Ser Gly Leu

35 40 45

Lys Asp Trp Asn Asn Thr Asn Tyr Cys Ser Phe Ser Gly Val Thr Cys

50 55 60

Asn Asn Val Ser Arg Val Ile Ser Leu Asn Ile Ser Asn Val Pro Leu

65 70 75 80

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

85 90 95

Leu Thr Ile Phe Gly Asp Asn Leu Thr Gly Thr Leu Pro Leu Glu Met

100 105 110

Ser Lys Leu Val Asn Val Lys Tyr Ile Asn Leu Ser Tyr Asn Gly Phe

115 120 125

Ser Gly Pro Phe Pro Arg Glu Ile Leu Leu Gly Leu Ile Glu Leu Glu

130 135 140

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

145 150 155 160

Phe Val Lys Leu Lys Lys Leu Lys Thr Leu His Leu Gly Gly Asn Tyr

165 170 175

Phe His Gly Glu Ile Pro Glu Ala Tyr Ser Tyr Ile Glu Ser Leu Lys

180 185 190

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

195 200 205

Leu Ala Leu Leu Pro Asn Leu Glu Glu Leu Arg Leu Gly Tyr Tyr Asn

210 215 220

Ser Tyr Glu Gly Gly Ile Pro Pro Glu Phe Gly Ser Ile Thr Thr Leu

225 230 235 240

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

245 250 255

Ser Leu Gly Asn Leu Lys Lys Leu His Ser Leu Phe Leu Gln Met Asn

260 265 270

Arg Leu Thr Gly Tyr Ile Pro Pro Glu Leu Ser Gly Leu Glu Ser Leu

275 280 285

Met Ser Phe Asp Leu Ser Ile Asn Gln Leu Ile Gly Glu Ile Pro Glu

290 295 300

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

305 310 315 320

Asn Leu His Gly Pro Ile Pro Pro Phe Ile Gly Asp Leu Pro Asn Leu

325 330 335

Glu Val Leu Gln Ile Trp Asp Asn Asn Phe Thr Leu Gln Leu Pro Glu

340 345 350

Asn Leu Gly Arg Asn Gly Arg Leu Lys Leu Leu Asp Val Ala Thr Asn

355 360 365

His Phe Thr Gly Arg Ile Pro Ser Asp Leu Cys Lys Gly Gly Lys Leu

370 375 380

Lys Thr Leu Ile Leu Met Glu Asn Tyr Phe Phe Gly Ser Ile Pro Glu

385 390 395 400

Lys Leu Gly Glu Cys Thr Ser Leu Thr Arg Ile Arg Val Arg Lys Asn

405 410 415

Tyr Leu Asn Gly Thr Ile Pro Ala Gly Phe Phe Arg Phe Pro Ser Leu

420 425 430

Asp Met Leu Glu Leu Asp Asn Asn Tyr Phe Thr Gly Gln Leu Pro Thr

435 440 445

Glu Ile Asn Ala Asn Asn Leu Thr Ser Leu Arg Leu Ser Asn Asn Trp

450 455 460

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

465 470 475 480

Thr Leu Ser Leu Asp Met Asn Arg Phe Ser Gly Asp Ile Pro Gln Glu

485 490 495

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

500 505 510

Leu Thr Gly Glu Ile Pro Ser Ser Ile Ala Gln Cys Ser Glu Leu Thr

515 520 525

Ser Ile Asp Leu Ser Arg Asn Gln Leu Val Gly Glu Val Pro Lys Glu

530 535 540

Ile Ser Lys Leu Asn Ser Leu Asn Ala Leu Asn Leu Ser Arg Asn Gln

545 550 555 560

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

565 570 575

Val Leu Asp Leu Ser Tyr Asn Asp Leu Ser Gly Arg Arg Pro Ile Asn

580 585 590

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

595 600 605

Leu Cys Ser Pro Arg Ala Thr Tyr Cys Pro Ser Ala Ser Lys Ser Ala

610 615 620

Glu Val Pro His Lys Asn His Ser Gly Lys Phe Thr Thr Thr Gln Leu

625 630 635 640

Val Ile Thr Ile Ile Ile Leu Val Thr Val Ala Leu Leu Leu Ala Val

645 650 655

Thr Trp Val Ile Ile Lys Lys Glu Lys Phe Arg Arg Ser Lys Leu Trp

660 665 670

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

675 680 685

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

690 695 700

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

705 710 715 720

Leu Val Gly Arg Gly Thr Gly His His Asp His Gly Phe Ser Ala Glu

725 730 735

Ile Gln Thr Leu Gly Arg Ile Arg His Arg Asn Ile Val Arg Leu Leu

740 745 750

Gly Tyr Val Ser Asn Lys Asp Thr Asn Leu Leu Leu Tyr Glu Tyr Met

755 760 765

Ser Asn Gly Ser Leu Gly Glu Thr Leu His Gly Ala Lys Gly Ala His

770 775 780

Leu Arg Trp Glu Thr Arg Tyr Arg Ile Ala Val Glu Ala Ala Lys Gly

785 790 795 800

Leu Ser Tyr Leu His His Asp Cys Ser Pro Ser Ile Ile His Arg Asp

805 810 815

Val Lys Ser Asn Asn Ile Leu Leu Asp Ser Asp Tyr Glu Ala His Val

820 825 830

Ala Asp Phe Gly Leu Ala Lys Phe Phe Gln Asp Ala Gly Ala Ser Glu

835 840 845

Cys Met Ser Ser Ile Ala Gly Ser Tyr Gly Tyr Ile Ala Pro Glu Tyr

850 855 860

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

865 870 875 880

Val Val Leu Leu Glu Leu Ile Thr Gly His Lys Pro Val Gly Glu Phe

885 890 895

Gly Asp Gly Val Asp Ile Val Arg Trp Val Asn Lys Thr Ile Ser Glu

900 905 910

Leu Ser Gln Pro Ser Asp Ala Ala Ser Val Leu Ala Val Val Asp Ser

915 920 925

Arg Leu Ile Ser Tyr Pro Leu Ala Ser Val Val Asn Leu Phe Lys Ile

930 935 940

Ala Met Met Cys Val Glu Glu Glu Ser Cys Ala Arg Pro Ser Met Arg

945 950 955 960

Glu Val Val His Met Leu Thr Asn Pro Pro Gln Ser Thr Thr Thr Thr

965 970 975

Pro Thr Leu Leu Ala Leu

980

Claims (1)

1.敲除PhCLV3和/或PhCLV1基因在调控矮牵牛植株观赏性中的应用；

所述PhCLV3基因起始密码子上游5000 bp的序列如SEQ ID NO.1所示，所述PhCLV1基因起始密码子上游5000 bp的序列如SEQ ID NO.6所示；

所述PhCLV3基因终止密码子下游579 bp的序列如SEQ ID NO.2所示，所述PhCLV1基因终止密码子下游983 bp的序列如SEQ ID NO.7所示；

所述PhCLV3基因组序列如SEQ ID NO.3所示，所述PhCLV1基因组序列如SEQ ID NO.8所示；

所述PhCLV3基因的编码序列如SEQ ID NO.4所示，所述PhCLV1基因的编码序列如SEQID NO.9所示；

所述PhCLV3基因编码的蛋白序列如SEQ ID NO.5所示，所述PhCLV1基因编码的蛋白序列如SEQ ID NO.10所示；

所述调控矮牵牛植株观赏性为调控矮牵牛稀缺头状花蕾、重叠花瓣、花瓣面积增大、开花密度增大或株高变矮。

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