CN104193814B

CN104193814B - 青虾卵黄蛋白原Vg基因、编码蛋白及应用

Info

Publication number: CN104193814B
Application number: CN201410456699.2A
Authority: CN
Inventors: 白鸿坤; 傅洪拓; 吴滟; 乔慧; 李法君; 张文宜; 龚永生; 颜跃弟; 金舒博
Original assignee: Freshwater Fisheries Research Center of Chinese Academy of Fishery Sciences
Current assignee: Freshwater Fisheries Research Center of Chinese Academy of Fishery Sciences
Priority date: 2014-09-09
Filing date: 2014-09-09
Publication date: 2017-09-05
Anticipated expiration: 2034-09-09
Also published as: CN104193814A

Abstract

本发明公开一种青虾卵黄蛋白原Vg基因、编码蛋白及应用。利用本发明提供的引物组从青虾肝胰腺中克隆到了一种Vg基因全长cDNA序列，该基因dsRNA注入雌性青虾围心腔后可以沉默卵巢和肝脏内的卵黄蛋白原基因的表达，使青虾卵巢发育显著延缓。本发明为解决青虾性早熟现象，培育优良品种提供新的思路和有效手段。

Description

青虾卵黄蛋白原Vg基因、编码蛋白及应用

技术领域

本发明公开一种青虾卵黄蛋白原Vg基因、编码蛋白及应用，涉及生物技术和发育调控领域，具体涉及青虾卵黄蛋白原基因的克隆，还涉及利用该基因dsRNA来调控青虾卵巢的发育速度。

背景技术

青虾，学名日本沼虾(Macrobrachium nipponense)，俗称河虾，隶属十足目（Decapoda）、长臂虾科（Palaemonidae）、沼虾属（Macrobrachium），广泛分布于我国各地水域，生长快、适应性强、养殖经济效益高，是我国重要的淡水养殖虾类。据2012年《中国渔业年鉴》记载，全国养殖青虾年产量超过23万吨，年产值超过150亿元，是目前淡水养殖业中最有发展前途的品种之一。近年来,随着其自然资源的锐减和养殖规模的扩大,养殖的青虾出现了生产性能下降和种质资源退化的情况,其中虾个体小型化和“性早熟”的现象尤为突出。“性早熟”即雌、雄性个体性腺提前发育，在生产中小虾性腺过早成熟，产卵导致个体变小、池塘养殖密度过大、生长规格不齐、近亲交配等一系列问题，最终商品虾的规格降低,繁殖性能下降,严重影响了青虾的品质和产值，成为青虾养殖业发展的一大瓶颈。为实现青虾产业的可持续发展，寻找解决雌性青虾“性早熟”的解决方法变得刻不容缓。

卵黄蛋白原(Vitellogenin,Vg)是一种普遍存在于卵生非哺乳动物血液中的蛋白,是几乎所有卵生动物中卵黄蛋白(vitellin,Vn)的前体。卵黄蛋白是所有卵生的脊椎动物和无脊椎动物雌性卵黄的主要成分，在卵黄发生期迅速积累。它被认为是由卵巢外组织合成，分泌释放到血液中，通过血液运输到***，以卵黄蛋白的形式积累和储存，是胚胎和幼体早期发育主要的营养来源。卵黄蛋白和卵黄蛋白原含有许多脂肪成分,可为正在发育的胚胎提供氨基酸、脂肪、碳水化合物、维生素、磷和硫等营养和功能性物质。

RNAi（RNA interference, RNAi）是一种广泛存在于真核生物中的高效的序列特异性基因剔除技术。RNAi是由不同来源和长度的双链 RNA（double-stranded RNA, dsRNA）前体所诱发的，可以有效地抑制靶基因RNA 的翻译。近年开始在甲壳动物基因功能研究中使用。目前，甲壳类关于Vg基因的沉默表达尚未见报道。

发明内容

本发明要解决的技术问题是提供一种青虾卵黄蛋白原Vg基因、编码蛋白及应用，该基因用于调控雌性青虾卵巢发育速度，为解决青虾性早熟提供理论基础。

技术方案：

一种青虾卵黄蛋白原，其氨基酸序列如SEQ ID NO.2所示。

编码上述青虾卵黄蛋白原的青虾卵黄蛋白原Vg基因。

所述的青虾卵黄蛋白原Vg基因，其核苷酸序列如SEQ ID NO.1中的第35位～第7645位所示。

所述的青虾卵黄蛋白原Vg基因所合成的dsRNA。

所述的青虾卵黄蛋白原Vg基因在延缓雌性青虾卵巢发育中的应用。

所述的应用，包括如下步骤：对青虾DNA样本中的青虾卵黄蛋白原Vg基因进行PCR扩增，将扩增产物进行体外转录合成dsRNA，再将dsRNA以注射到青虾的围心腔内。

所述的应用，PCR扩增中使用的上下游引物的序列如SEQ ID NO.3～4所示。

所述的应用，注射步骤中的注射剂量是4μg/g。

有益效果

本发明提供的青虾卵黄蛋白原Vg基因可以制备得到dsRNA，通过注射的方式注入青虾围心腔内，可以有效地延缓青虾卵巢成熟的时间，证明该基因的表达对雌性卵巢发育有非常重要的影响。

附图说明

图1是外源性注射Vg-dsRNA对雌性青虾卵巢GSI性腺发育指数影响的示意图。

注： 1d，3d, 5d, 7d, 9d, 11d, 13d, 15d，17d分别为注射后1，3，5，7，9，13，15和17天(样本量N≥ 3)。

图2是外源性注射Vg-dsRNA对雌性青虾内源Vg基因的表达量影响的示意图。

注： 1d, 3d, 5d, 7d, 9d,11d, 13d, 15d，17d分别为注射后1，3，5，7，9，13，15和17天(样本量N≥ 3)。对照组注射相同剂量的DEPC水。

图3是在肝脏中外源性注射Vg-dsRNA对雌性青虾内源Vg基因的表达量的影响的示意图。

注： 1d，3d, 5d, 7d, 9d, 11d, 13d, 15d，17d分别为注射后1，3，5，7，9，13，15和17天(样本量N≥3)。对照组注射相同剂量的DEPC水。

具体实施方式

下面通过具体实施方式对本发明作进一步详细说明。但本领域技术人员将会理解，下列实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体技术或条件者，按照本领域内的文献所描述的技术或条件(例如参考J. 萨姆布鲁克等著，黄培堂等译的《分子克隆实验指南》，第三版，科学出版社)或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。

本文使用的近似语在整个说明书和权利要求书中可用于修饰任何数量表述，其可在不导致其相关的基本功能发生变化的条件下准许进行改变。因此，由诸如“约”的术语修饰的值并不局限于所指定的精确值。在至少一些情况下，近似语可与用于测量该值的仪器的精度相对应。除非上下文或语句中另有指出，否则范围界限可以进行组合和/或互换，并且这种范围被确定为且包括本文中所包括的所有子范围。除了在操作实施例中或其他地方中指明之外，说明书和权利要求书中所使用的所有表示成分的量、反应条件等等的数字或表达在所有情况下都应被理解为受到词语“约”的修饰。

本研究的目的就是通过用Vg-dsRNA 注入青虾体内，研究该基因在dsRNA 刺激下的表达变化以及对卵巢发育的影响，阐释 dsRNA 对青虾卵巢发育的调节机制，为利用Vg-dsRNA干扰技术抗性早熟的实用化提供新思路和参考依据。

实施例1：青虾Vg基因全长cDNA的获得

总RNA提取：选取3～4支成熟雌虾的肝脏，迅速放入盛有液氮的预冷研钵中，迅速的研磨。总RNA的提取使用Takara公司的RNAiso Plus提取试剂结合传统的酚仿抽提法进行，提取方法参照使用说明书。经过琼脂糖凝胶电泳检测RNA的质量，分光光度计分析该样品OD260/280 比值在1.8～2.0 之间，并测定RNA的浓度。

cDNA第一链合成：参照Takara M-MLV反转录试剂盒进行第一链cDNA的合成。

青虾Vg基因全长cDNA序列的克隆：

根据已获得的Vg基因全长cDNA序列(罗氏沼虾Macrobrachium rosenbergii,BAB69831；日本仿长额虾Pandalopsis japonica, ACU51164; 高背长额虾Pandalus hypsinotus BAD11098;)的编码区进行比对，依据比对结果，在保守区域设计引物，利用引物组一（SEQ ID NO:5～SEQ ID NO:14）和上述反转录的cDNA为模板进行中间片段的克隆。

更具体地，上述的引物组的组成如下：

正向引物VGAF1的核苷酸序列如SEQ ID NO.5所示；

正向引物VGBF2的核苷酸序列如SEQ ID NO.6所示；

正向引物VGCF3的核苷酸序列如SEQ ID NO.7所示；

正向引物VGDF4的核苷酸序列如SEQ ID NO.8所示；

正向引物VGEF5的核苷酸序列如SEQ ID NO.9所示;

反向引物VGAR1的核苷酸序列如SEQ ID NO.10所示；

反向引物VGBR2的核苷酸序列如SEQ ID NO.11所示；

反向引物VGCR3的核苷酸序列如SEQ ID NO.12所示；

反向引物VGDR4的核苷酸序列如SEQ ID NO.13所示；

反向引物VGER5的核苷酸序列如SEQ ID NO.14所示。

进行PCR扩增反应体系如下：

PCR反应程序为：94℃预变性3min，然后进入下列循环：94℃ 30s，55℃ 30s，72℃30s，30个循环，最后72℃延伸10min，4℃保存。1.5%琼脂糖凝胶电泳检测。

利用上海生工生物工程有限公司(Sangon)的柱式DNA 胶回收试剂盒进行目的片段的回收，将产物连接到pMD18-T载体（Takara），并转化入大肠杆菌DH5α，进行蓝白斑筛选，挑取单克隆白斑扩增培养后，将***目的片段的阳性克隆送至上海铂尚生物公司进行测序分析。将得到的几段产物进行拼接，得到青虾Vg基因的中间片段。

根据Vg基因cDNA中间片段序列，又设计特异性正向引物3VGF1（SEQ ID NO.15）、3VGF2（SEQ ID NO.16）；和反向引物5VGR1（SEQ ID NO.17）、5VGR2（SEQ ID NO.18）分别进行cDNA 3’和5’快速扩增，操作步骤按TaKaRa 3’-RACE和TaKaRa5’-RACE试剂盒说明书进行。按上述克隆的步骤进行随后的切胶、回收、转化、克隆及测序最终得到Vg 基因的3’和5’端的序列。将中间片段和3’和5’端的测序结果进行比对，拼接得到青虾Vg基因全长cDNA序列。经过序列比对显示，青虾Vg基因和其它甲壳类的同源性在70%以上。其核苷酸序列如SEQ IDNO.1中的第35位～第7645位所示（第1～34和第7646～7800位是3’和5’端的非编码区），其编码蛋白的氨基酸序列如SEQ ID NO.2所示。

实施例2：青虾Vg基因dsRNA的合成

以SEQ ID NO.1核苷酸序列为依据，在开放阅读框以内采用NCBI在线dsRNA引物设计软件（http://www.flyrnai.org/cgi-bin/RNAi_find_primers.pl）设计制备双链RNA 的引物(SEQ ID NO.3- SEQ ID NO.4)，根据Transcript AidTM T7 High YieldTranscription kit (Fermentas, Inc., USA)试剂盒说明进行体外转录合成dsRNA。将制备好的dsRNA溶解在DEPC 水中，测定其浓度并在1.5%琼脂糖凝胶上检测其纯度。将上述dsRNA溶液以4μg/g的剂量注射到青虾的围心腔内。对照组注射相同体积的DEPC 水，实验所用的雌虾都处于卵巢发育初期（刚排完卵）。在注射后的第1、3、5、7、9、11、13、15和17天收集样品(N≥3)，并将组织保存在RNA 保存液中（Takara）。最后提取各样品的总RNA并反转成cDNA，采用Real Time PCR检测Vg基因相对于对照组的表达量变化，以此来计算Vg基因干扰的效率。性腺指数是评价动物性腺发育状况的重要指标。根据公式GSI=性腺湿重/体重湿重×100%，计算两组雌性青虾的GSI性腺发育指数（Gonad Somatic Index，GSI)。

实验结果

图1所示，为对照组的GSI性腺发育指数和注射组的性腺发育指数对比的示意图，可以看出Vg-dsRNA 注射显著延缓了青虾卵巢的发育在第11天对照组卵巢发育已处于成熟期实验组仍处于卵黄积累时期。

如图2所示，在卵巢中，相对于对照组，注射后第1天，青虾Vg基因的表达量显著下降了76%，在第5天已经下降了92%，随着对照组卵巢的发育，Vg 基因在卵巢中呈现了先升高后降低的表达规律。然而在对照组这一规律明显被延后，在第15天时处理组处于卵黄发生的高峰期，而对照组卵巢已完成一轮发育周期，已经排空。

如图3所示，在肝脏中，相对于对照组，注射后第1天，青虾Vg基因的表达量显著下降了74%，在第5天已经下降了97%，随着对照组卵巢的发育，Vg 基因在肝脏中同样呈现了先升高后降低的表达规律。根据表达规律来看对照组的发育明显被延缓。结果证明外源注射Vg-dsRNA ,可以有效地延缓性腺发育，这一研究结果为解决青虾性早熟这一难题提供了理论基础，也为后续将该方法应用到生产实践提供指导意义。

SEQUENCE LISTING

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catcagagaa tgggtacaac tcaactttga agttagcgga gatctgaggt atcatgaatg 3420

gtgcagaata cctcttccac aaagactgcg caaattcgaa actaaagtag cacttagaaa 3480

ttggcacatc atttctttca tgagaaagga aggtgaatcc caatacaagt caactctcaa 3540

gattggtcaa cgaggaaatg aaaagattga tataattggc aaccatgtca tggaaggaag 3600

ttcatacaga gatatgacct tcaaatctaa catagaagct aaaattggaa atgccagata 3660

caagaaaaac tttgttttat acaaccaaga aagtaaaatg ggagttgaat ggcaagtggc 3720

ttcccaagaa ggtggaaagg ttattgaatt agaaatgatg ctaattcggt cgggagagac 3780

atctcaagtc aaatttttgc ttgatattcc cgagcgcatg aagaaagtcc tgctcgaagc 3840

ctctgcagca ggccaaggaa gctctcagta ccaagtcaga gcaatggcca agtatggaga 3900

aagtcctatt ttccatgtag aaggaccagt caccgcaatg ctctcatcta aaaacactca 3960

cctcaagacc gaattgagga ctgttttact tcgcagccaa ccctacacag tcacaacctc 4020

tcttgtaatg atgcatggca aacaagccgt tgtctttgaa ctaaaagaca gagaagaaca 4080

tctcattact gtgcaatgga atatggcgac tcgagatgga caggaaaccc atctggattt 4140

taaaatgctc attccatcca tgatagaaaa gacaatgaat gccattgtca gtgagaaagt 4200

ctttcacttg agtgtcaacc agctgatagg accaaagagc tcttccccac tgagaaggaa 4260

aggtttcttg gatgttgatt tcgaaggcaa gagagcaaac gtcgaatttg cttgggacgc 4320

tgatcgtaac ccaaataaga aaattaaggc tgaagtcaaa atggttaatc cactttcaac 4380

tctacgagac tgtgttgtcc agggtaactg ggcctactta gagagacaac atcaattcaa 4440

ggctgaactg aaactcagtg acatccgctc ttggtttgtt ggaaggaaca gcttgatgct 4500

agaggtaaca aaccccaatc aacaaatgta caaaatgaat tctatcttaa tggtggagaa 4560

aaagggatca ggaccaaaag taggggcaga cataaccttc aggacacctg aaaacagaga 4620

gtataagtgg aattccgaaa gttctcttga gtgccgcgaa ggactacaga acgtgaggct 4680

gatgaccaaa gcagatcttg tttggccaga aggtcgacaa tctatagtaa acttagaagc 4740

aatgcatcgc tggacaccta gccaaaggga agctgaatta aagattgaag tgacctgccc 4800

atcactccca caaccaatca agaccaaact tcagctgaat aatcaacagg gtgaatacag 4860

taccaagtgg gtcattgaaa tgggctttcc agtaaatggt gcaatgtaca aactaaaatt 4920

gtctcctgag catggagtac aagggttcga agtagaactt aacctagaag aagtacttaa 4980

gctcctgaag gcgatagatc acctcatatc ttctagctct ttgtccatgt ctgatagccc 5040

tcggcgtctg ctctttatcg tgctcactac aaacatcatg accaccattc ttattccctg 5100

ttgcctgatg tcaccctctc gtacaatgga aggaggatgt ggatgcaata cttgcacagg 5160

cccaagttgt gctgactgcc attgcagctt cgttcctcat aagggtttaa gtgattccaa 5220

atatgagatt gccttccgcc gatctcattc cgattggagt ggagaatcca gatatgaagg 5280

tcgtgtcagc catccaggac tagagaggga ccgtcgaatt gatattcagc ttagaagaga 5340

ggaacagaag gtcacaggaa gtgttgagct cgatattttc ctcagaccag aagataagat 5400

cactggaaag ttagaatcac tcatttatgc tcgaaacact gtcatcgttg aagcaaaact 5460

tgaaggaaag gtactgaagg atcaccccaa ggtcattgta cgtggagcct atggtccaag 5520

caatgttgga tttgatatta aattgcacaa gactcactct tctcctacat ccttcaggat 5580

gtcgggaaga tatgagaaga ggactgccag aaatgccgct gcttccttca tcatagagaa 5640

tgaacatcaa aaaaccattg acatttctat agcacttcat cctcatcaga gtccttggtg 5700

ttatggagta cgagctgaag ctaaggcaaa gtcgtccatc attggaactt acgacatcta 5760

ttctgagctt tgcaagccag gatttgtatc attgaccaca aagaaacatg gaagtgacaa 5820

gatgtacata actaaactcg gtatagaagg aatgaagaac atcgaagcca gtattttgga 5880

agctaaccca caaaccatgg agaaaagacc acttggcatg gctcgtatgg tgctaacttc 5940

tcccacaatg atgaagctgg aaactaagta tgaaggtgat aagctacatg aaatcaagag 6000

tactgtacgc gatatgtggt ggaggcttgt ttcatctgca ggtaacggga tggattcttt 6060

gtgcagtgaa gttttacaag agggctctgg atcaccttct ggccacatgt cttcggtatg 6120

gcgggaaatt aagaatgaag cctcaagaat ctattctgat ttggaaaacg atttagtcat 6180

tcctaaatat gagaagctca gggaatggct tcatggtggc attctctcta acattgccga 6240

gggatgttca aaggtttggt ctcattatgc tcacttccaa agcagtttat catcatctgt 6300

atccaacatg ataagaacta ttcgagagga attccctgga ttaactagag tagtcactga 6360

agttgtaatg ggaactgctc gtggcctgca aactggtgaa atgccggagg tgttccgtcg 6420

atggtggaac gatttccttg agagttcttt ctgcagggct atagcatctg actttgaatc 6480

actctggagt gaacaccaag aggagtacca gggattgcag cagatctgga ggaaaatcaa 6540

gaacactctt gcaaaggacg ttaacagaca gagacgtaat cttatgcact acaagaaacc 6600

caggcatctt gttaactgga tcgtccacga catgaacttt gaacgaatgg ttttcaaggg 6660

tgtagataag gttatcaaga atgttgtgca gaaagctctc ttcgtacctt ttcagatcga 6720

tggtaaccac ttccagcttc agttacctat tcgtcgacca gtgcactcac ttcctcaagc 6780

tttgtcttat gtatcaatga atccaactcc agttgttgac cgtgcattat ggtggtttga 6840

agctcttatg cccactccag tcgataacat tgtatgggct tattataaat tccttcctcg 6900

ccatgcacgt tatcttctgc caccctacaa tcacacagcc atggttgttg atggatcaga 6960

gatcctcacc tttgatggag tagttctgcg agtacctcat tctccctgca aagtcttgct 7020

tgcacagtac aagactcact ccttgattat gcaaaatcag cagcctggcc atttgccaca 7080

cttcattttg aaggctgctg gagctacagt ggaggtcaag cctgacttcg ttgttaccgt 7140

caatggaaat ccagtaagtg ggcccaggga agtgcaaggt gaagtcgtaa tcatgaaaga 7200

aaatgagaag atcaaggtca ggactccatt cataacactc cgtgtctaca aagacagccg 7260

caccgcttca gtcgaggtgt ctggctggac atttggtaga atagctggtc ttttgggcat 7320

ctatgatggg gaaaaggcaa ctgataggtt gacacctcag ggtacaaggg catctaatct 7380

tcaagagctg gtcaagtcct ggcaagagaa tcctcagtgc gagactcctc acgtagcccc 7440

catgagtcct atgcaggttc cagtggtcca tatgctccac tgccaaaccc tcttcggagt 7500

tcggtctcgc tgcaacccag tcgtccatcc agggccattc aggaagatgt gcttcgccag 7560

caagaacgcc tgtcacgttg ctaaggccta tcgggccatg tgcgagacga agggcattaa 7620

ggagaccttc cctctgggat gctaatgacc aaacctatcc atggaagtca ttgtgtaaat 7680

gagtgactcc aagcctagtg acctcattgt tctagttttt aagaaatgca acgaaaatgt 7740

acaaaaaata ataaatttct agatcgataa taaatatgag atgcataaca agaaaaaaaa 7800

<210> 2

<211> 2536

<212> PRT

<213> vg-prt

<400> 2

Met Thr Ser Leu Ala Leu Leu Leu Leu Ala Ile Ala Ser Phe Ser Pro

1 5 10 15

Ala Pro Trp Pro Ser Gly Thr Asn Leu Cys Ser Lys Glu Cys Pro Val

20 25 30

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

35 40 45

Ser Tyr Thr Gly Lys Ser Arg Ile His Leu Lys Asp Val Glu Gly Ala

50 55 60

Asn Val Asp Met Glu Trp Asn Ser Gln Val Glu Leu Ser Trp Leu Ser

65 70 75 80

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

85 90 95

Ser Gly Ser Ala Glu Ala Arg Phe Leu Glu Arg Tyr Pro Leu Val Val

100 105 110

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

115 120 125

Asp Thr Trp Ser Ile Asn Leu Lys Lys Gly Ile Ala Ser Ala Phe Gln

130 135 140

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

145 150 155 160

Glu Thr Asp Ile Ile Gly Asn Cys Ser Thr Val Tyr Glu Val Gln His

165 170 175

Glu Gly Glu Lys Val Val Val Thr Lys Leu Lys Asn His Arg Phe Cys

180 185 190

Gln Asp His Tyr Ala Asn Arg Ala Glu Thr Pro Lys Ala Trp Met Lys

195 200 205

Ala Pro Leu Pro Met Glu Glu Ser Tyr Ser Glu Cys Lys Gln Glu Ile

210 215 220

Thr Asn Gly Ile Tyr Thr Ser Ile Thr Cys Lys Asp Lys Asn Val Ile

225 230 235 240

Lys Pro Ala Tyr Gly Ser Tyr Lys Tyr Val Glu Ala His Gln Glu Ser

245 250 255

Val Leu Arg Phe Gln Ser Glu Thr Asp Gln Ile Pro Pro Ser Val Ser

260 265 270

Gln Leu Pro Ser Arg Phe Ile Arg Lys Thr Leu Arg Tyr Asp Gln His

275 280 285

Thr Leu Lys Lys Asp Pro Ser Met Ala Ala Lys Leu Asp Glu Met Leu

290 295 300

Lys Gln Val Cys Glu Lys Val Lys His Gly Val His Glu His Ala Ala

305 310 315 320

Ser Gln Phe Ala Gln Ala Leu His Phe Leu Arg Arg Val Pro Glu Glu

325 330 335

Ala Ile Pro Gln Thr Leu Glu Lys Ile Arg Gly Gly Gln Ile Cys Glu

340 345 350

Gln Arg Gln Lys Met Glu Ser Met Phe Leu Asp Gly Leu Ala Phe Val

355 360 365

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

370 375 380

Lys Ala Thr Gly Gly Arg Ala Ala Leu Tyr Ala Ala Ser Met Tyr Phe

385 390 395 400

Met Pro Arg Pro Cys Ile His Ser Ile Glu Ala Leu Lys Pro Leu Phe

405 410 415

Glu Asn Phe Gln Arg Phe Pro Arg Thr Thr Leu Ala Gly Ala Thr Met

420 425 430

Val His Thr Tyr Cys Arg Gln Asn Pro Lys Cys Gln Glu Lys Ala Pro

435 440 445

Val Arg Gln Leu Ala Glu Thr Leu Ser Ser Lys Val Gly Gln Met Cys

450 455 460

Thr Ala Ser Pro Asn Glu Glu Thr Arg Glu His Ala Leu Ala Leu Leu

465 470 475 480

Lys Ser Leu Gly Asn Met Gly Val Met Asn Ser Glu Ile Ala Arg Pro

485 490 495

Ile Leu Gln Cys Ile Glu Asn Pro Glu Ala Asp Lys Gly Ile Arg Arg

500 505 510

Ala Ala Thr Gln Thr Phe Arg Asn Val His Cys His Gln Glu Ile His

515 520 525

Pro Ile Met Lys Gln Leu Ile Asn Val Ala Leu Asp His Arg Lys Arg

530 535 540

Thr Glu Val Arg Ile Gly Ser Tyr Leu Ala Ala Ile Lys Cys Ala Lys

545 550 555 560

His Glu Glu Leu His Lys Ile Thr Glu Lys Ile Ala Lys Glu Glu Asn

565 570 575

Thr Gln Val Arg Ser Phe Ile Leu Ser His Leu His Asn Ile Lys Glu

580 585 590

Ser Thr Val Pro Tyr Lys Ala Asn Leu Lys Ser Arg Leu Glu Ser Ile

595 600 605

Val Leu Pro Ser Asn Phe Thr Lys Asp Trp Arg Lys His Ser Arg Asn

610 615 620

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

625 630 635 640

Glu Ser Asn Val Ile Tyr Ala Pro Gly Ser Tyr Val Pro Arg Ser Val

645 650 655

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

660 665 670

Glu Ile Gly Ala Arg Phe Glu Gly Leu Glu Thr Ile Ile Glu Glu Leu

675 680 685

Phe Gly Pro His Ser Tyr Phe Lys Arg Thr Pro Ala Arg Gln Ile Trp

690 695 700

Glu Asp Leu Lys Glu Thr Leu Ser Lys Ile Ser Glu Arg Leu Glu Gly

705 710 715 720

Ser Phe Arg Gln Arg Arg Ser Ile Asp Leu Ser Gln Ile Ser His Leu

725 730 735

Phe Asp Lys Leu Tyr Gly Asn Arg His Met Gln Lys Ala Asp Ile Tyr

740 745 750

Ala Arg Ile Asn Asn Gln Glu Met Ala Phe Gly Ser Trp Glu Gly Asn

755 760 765

Met Lys Asn Ile Lys Ile Asp Glu Leu Ile Asn Asn Leu Phe Asp Lys

770 775 780

Phe Asp Asn Val Ile Asn Arg Ala Ala Thr Thr Asn Ile Asp Thr Val

785 790 795 800

Arg Thr Ala Gln Phe Tyr Met Asn Asn His Leu Pro Thr Ile Gln Gly

805 810 815

Leu Pro Ile Lys Ile Lys Leu Glu Gly Thr Ala Val Ala Gly Ile Lys

820 825 830

Met Glu Thr His Val Arg Gly Leu Thr Ser Gly Thr Pro Gly Val Val

835 840 845

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

850 855 860

Asp Cys His Ile Val Arg Ala Gly Ile Lys Met Lys Asn His Ile Ala

865 870 875 880

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

885 890 895

Phe Glu Val Glu Leu Glu Met Pro Glu Lys Met Glu Leu Leu Lys Ala

900 905 910

Gln Ser Glu Thr Tyr Leu Met Lys Arg Val Lys Gly Gln Gln Glu Thr

915 920 925

Lys Val His Pro Ser Ser Val Gln Ser Thr Arg Phe Gln Thr Gln Ser

930 935 940

Cys Leu Thr Lys Met Glu Pro Val Leu Gly Leu Lys Leu Cys Tyr Asp

945 950 955 960

Val Asn Met Pro Asn Ile Phe Arg Ser Gln Gly Leu Pro Leu Gly Pro

965 970 975

Pro Ala Ile Phe Lys Val Val Leu Glu Lys Ala Glu Ser Gly Met Arg

980 985 990

Gly Phe Leu Val Lys Gly Arg Glu Glu His Val Gly Gly Lys Lys Val

995 1000 1005

Ile Lys Val Glu Leu Glu Thr Pro Gly Thr Ser Ser Pro Lys Lys

1010 1015 1020

Ala Thr Ala Ile Ile Thr Ser Ala Asn Glu Gly Glu Glu Lys Lys

1025 1030 1035

Ile Ser Ala Thr Phe Glu Cys Glu Arg Leu Gly Gly Ile Ser Ile

1040 1045 1050

His Met Met Arg Lys Trp Thr Gln Ser Glu Lys Gln Val Glu Met

1055 1060 1065

Ile Ala Tyr Phe Ser Glu Ser Arg Gln Tyr Asn Pro Ser Thr Lys

1070 1075 1080

Gly Met Glu Ala Lys Phe Leu Trp Asn Gly Glu Gly Glu Glu Val

1085 1090 1095

Lys Leu Asp Met Ala Leu Asn Thr Leu Ala Ala Ile Arg Glu Trp

1100 1105 1110

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

1115 1120 1125

Trp Cys Arg Ile Pro Leu Pro Gln Arg Leu Arg Lys Phe Glu Thr

1130 1135 1140

Lys Val Ala Leu Arg Asn Trp His Ile Ile Ser Phe Met Arg Lys

1145 1150 1155

Glu Gly Glu Ser Gln Tyr Lys Ser Thr Leu Lys Ile Gly Gln Arg

1160 1165 1170

Gly Asn Glu Lys Ile Asp Ile Ile Gly Asn His Val Met Glu Gly

1175 1180 1185

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

1190 1195 1200

Ile Gly Asn Ala Arg Tyr Lys Lys Asn Phe Val Leu Tyr Asn Gln

1205 1210 1215

Glu Ser Lys Met Gly Val Glu Trp Gln Val Ala Ser Gln Glu Gly

1220 1225 1230

Gly Lys Val Ile Glu Leu Glu Met Met Leu Ile Arg Ser Gly Glu

1235 1240 1245

Thr Ser Gln Val Lys Phe Leu Leu Asp Ile Pro Glu Arg Met Lys

1250 1255 1260

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

1265 1270 1275

Tyr Gln Val Arg Ala Met Ala Lys Tyr Gly Glu Ser Pro Ile Phe

1280 1285 1290

His Val Glu Gly Pro Val Thr Ala Met Leu Ser Ser Lys Asn Thr

1295 1300 1305

His Leu Lys Thr Glu Leu Arg Thr Val Leu Leu Arg Ser Gln Pro

1310 1315 1320

Tyr Thr Val Thr Thr Ser Leu Val Met Met His Gly Lys Gln Ala

1325 1330 1335

Val Val Phe Glu Leu Lys Asp Arg Glu Glu His Leu Ile Thr Val

1340 1345 1350

Gln Trp Asn Met Ala Thr Arg Asp Gly Gln Glu Thr His Leu Asp

1355 1360 1365

Phe Lys Met Leu Ile Pro Ser Met Ile Glu Lys Thr Met Asn Ala

1370 1375 1380

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

1385 1390 1395

Gly Pro Lys Ser Ser Ser Pro Leu Arg Arg Lys Gly Phe Leu Asp

1400 1405 1410

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

1415 1420 1425

Ala Asp Arg Asn Pro Asn Lys Lys Ile Lys Ala Glu Val Lys Met

1430 1435 1440

Val Asn Pro Leu Ser Thr Leu Arg Asp Cys Val Val Gln Gly Asn

1445 1450 1455

Trp Ala Tyr Leu Glu Arg Gln His Gln Phe Lys Ala Glu Leu Lys

1460 1465 1470

Leu Ser Asp Ile Arg Ser Trp Phe Val Gly Arg Asn Ser Leu Met

1475 1480 1485

Leu Glu Val Thr Asn Pro Asn Gln Gln Met Tyr Lys Met Asn Ser

1490 1495 1500

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

1505 1510 1515

Asp Ile Thr Phe Arg Thr Pro Glu Asn Arg Glu Tyr Lys Trp Asn

1520 1525 1530

Ser Glu Ser Ser Leu Glu Cys Arg Glu Gly Leu Gln Asn Val Arg

1535 1540 1545

Leu Met Thr Lys Ala Asp Leu Val Trp Pro Glu Gly Arg Gln Ser

1550 1555 1560

Ile Val Asn Leu Glu Ala Met His Arg Trp Thr Pro Ser Gln Arg

1565 1570 1575

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

1580 1585 1590

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

1595 1600 1605

Ser Thr Lys Trp Val Ile Glu Met Gly Phe Pro Val Asn Gly Ala

1610 1615 1620

Met Tyr Lys Leu Lys Leu Ser Pro Glu His Gly Val Gln Gly Phe

1625 1630 1635

Glu Val Glu Leu Asn Leu Glu Glu Val Leu Lys Leu Leu Lys Ala

1640 1645 1650

Ile Asp His Leu Ile Ser Ser Ser Ser Leu Ser Met Ser Asp Ser

1655 1660 1665

Pro Arg Arg Leu Leu Phe Ile Val Leu Thr Thr Asn Ile Met Thr

1670 1675 1680

Thr Ile Leu Ile Pro Cys Cys Leu Met Ser Pro Ser Arg Thr Met

1685 1690 1695

Glu Gly Gly Cys Gly Cys Asn Thr Cys Thr Gly Pro Ser Cys Ala

1700 1705 1710

Asp Cys His Cys Ser Phe Val Pro His Lys Gly Leu Ser Asp Ser

1715 1720 1725

Lys Tyr Glu Ile Ala Phe Arg Arg Ser His Ser Asp Trp Ser Gly

1730 1735 1740

Glu Ser Arg Tyr Glu Gly Arg Val Ser His Pro Gly Leu Glu Arg

1745 1750 1755

Asp Arg Arg Ile Asp Ile Gln Leu Arg Arg Glu Glu Gln Lys Val

1760 1765 1770

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

1775 1780 1785

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

1790 1795 1800

Ile Val Glu Ala Lys Leu Glu Gly Lys Val Leu Lys Asp His Pro

1805 1810 1815

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

1820 1825 1830

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

1835 1840 1845

Met Ser Gly Arg Tyr Glu Lys Arg Thr Ala Arg Asn Ala Ala Ala

1850 1855 1860

Ser Phe Ile Ile Glu Asn Glu His Gln Lys Thr Ile Asp Ile Ser

1865 1870 1875

Ile Ala Leu His Pro His Gln Ser Pro Trp Cys Tyr Gly Val Arg

1880 1885 1890

Ala Glu Ala Lys Ala Lys Ser Ser Ile Ile Gly Thr Tyr Asp Ile

1895 1900 1905

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

1910 1915 1920

Lys His Gly Ser Asp Lys Met Tyr Ile Thr Lys Leu Gly Ile Glu

1925 1930 1935

Gly Met Lys Asn Ile Glu Ala Ser Ile Leu Glu Ala Asn Pro Gln

1940 1945 1950

Thr Met Glu Lys Arg Pro Leu Gly Met Ala Arg Met Val Leu Thr

1955 1960 1965

Ser Pro Thr Met Met Lys Leu Glu Thr Lys Tyr Glu Gly Asp Lys

1970 1975 1980

Leu His Glu Ile Lys Ser Thr Val Arg Asp Met Trp Trp Arg Leu

1985 1990 1995

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

2000 2005 2010

Leu Gln Glu Gly Ser Gly Ser Pro Ser Gly His Met Ser Ser Val

2015 2020 2025

Trp Arg Glu Ile Lys Asn Glu Ala Ser Arg Ile Tyr Ser Asp Leu

2030 2035 2040

Glu Asn Asp Leu Val Ile Pro Lys Tyr Glu Lys Leu Arg Glu Trp

2045 2050 2055

Leu His Gly Gly Ile Leu Ser Asn Ile Ala Glu Gly Cys Ser Lys

2060 2065 2070

Val Trp Ser His Tyr Ala His Phe Gln Ser Ser Leu Ser Ser Ser

2075 2080 2085

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

2090 2095 2100

Thr Arg Val Val Thr Glu Val Val Met Gly Thr Ala Arg Gly Leu

2105 2110 2115

Gln Thr Gly Glu Met Pro Glu Val Phe Arg Arg Trp Trp Asn Asp

2120 2125 2130

Phe Leu Glu Ser Ser Phe Cys Arg Ala Ile Ala Ser Asp Phe Glu

2135 2140 2145

Ser Leu Trp Ser Glu His Gln Glu Glu Tyr Gln Gly Leu Gln Gln

2150 2155 2160

Ile Trp Arg Lys Ile Lys Asn Thr Leu Ala Lys Asp Val Asn Arg

2165 2170 2175

Gln Arg Arg Asn Leu Met His Tyr Lys Lys Pro Arg His Leu Val

2180 2185 2190

Asn Trp Ile Val His Asp Met Asn Phe Glu Arg Met Val Phe Lys

2195 2200 2205

Gly Val Asp Lys Val Ile Lys Asn Val Val Gln Lys Ala Leu Phe

2210 2215 2220

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

2225 2230 2235

Ile Arg Arg Pro Val His Ser Leu Pro Gln Ala Leu Ser Tyr Val

2240 2245 2250

Ser Met Asn Pro Thr Pro Val Val Asp Arg Ala Leu Trp Trp Phe

2255 2260 2265

Glu Ala Leu Met Pro Thr Pro Val Asp Asn Ile Val Trp Ala Tyr

2270 2275 2280

Tyr Lys Phe Leu Pro Arg His Ala Arg Tyr Leu Leu Pro Pro Tyr

2285 2290 2295

Asn His Thr Ala Met Val Val Asp Gly Ser Glu Ile Leu Thr Phe

2300 2305 2310

Asp Gly Val Val Leu Arg Val Pro His Ser Pro Cys Lys Val Leu

2315 2320 2325

Leu Ala Gln Tyr Lys Thr His Ser Leu Ile Met Gln Asn Gln Gln

2330 2335 2340

Pro Gly His Leu Pro His Phe Ile Leu Lys Ala Ala Gly Ala Thr

2345 2350 2355

Val Glu Val Lys Pro Asp Phe Val Val Thr Val Asn Gly Asn Pro

2360 2365 2370

Val Ser Gly Pro Arg Glu Val Gln Gly Glu Val Val Ile Met Lys

2375 2380 2385

Glu Asn Glu Lys Ile Lys Val Arg Thr Pro Phe Ile Thr Leu Arg

2390 2395 2400

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

2405 2410 2415

Thr Phe Gly Arg Ile Ala Gly Leu Leu Gly Ile Tyr Asp Gly Glu

2420 2425 2430

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

2435 2440 2445

Leu Gln Glu Leu Val Lys Ser Trp Gln Glu Asn Pro Gln Cys Glu

2450 2455 2460

Thr Pro His Val Ala Pro Met Ser Pro Met Gln Val Pro Val Val

2465 2470 2475

His Met Leu His Cys Gln Thr Leu Phe Gly Val Arg Ser Arg Cys

2480 2485 2490

Asn Pro Val Val His Pro Gly Pro Phe Arg Lys Met Cys Phe Ala

2495 2500 2505

Ser Lys Asn Ala Cys His Val Ala Lys Ala Tyr Arg Ala Met Cys

2510 2515 2520

Glu Thr Lys Gly Ile Lys Glu Thr Phe Pro Leu Gly Cys

2525 2530 2535

<210> 3

<211> 39

<212> DNA

<213> 人工序列

<400> 3

taatacgact cactataggt gccaagaaaa agctcctgt 39

<210> 4

<211> 39

<212> DNA

<213> 人工序列

<400> 4

taatacgact cactataggg ccaaaggttg gtgcatagt 39

<210> 5

<211> 22

<212> DNA

<213> 人工序列

<400> 5

tctggcgaca gcctcagctg gt 22

<210> 6

<211> 22

<212> DNA

<213> 人工序列

<400> 6

ctggagcagt caaggttatg gt 22

<210> 7

<211> 22

<212> DNA

<213> 人工序列

<400> 7

gccagagaaa atggagttgg tg 22

<210> 8

<211> 22

<212> DNA

<213> 人工序列

<400> 8

gtcaggcgaa acatcacaag tc 22

<210> 9

<211> 23

<212> DNA

<213> 人工序列

<400> 9

ctgttgcttg atgtcaccct ctc 23

<210> 10

<211> 23

<212> DNA

<213> 人工序列

<400> 10

ttgatgacag tgaacgttcc tga 23

<210> 11

<211> 22

<212> DNA

<213> 人工序列

<400> 11

tacagagcac acgattccag ac 22

<210> 12

<211> 22

<212> DNA

<213> 人工序列

<400> 12

ttggtactga gagcttcctt gg 22

<210> 13

<211> 22

<212> DNA

<213> 人工序列

<400> 13

cctgtgacct tctgttcctc tc 22

<210> 14

<211> 22

<212> DNA

<213> 人工序列

<400> 14

accgtgcatt atggtggctt ga 22

<210> 15

<211> 23

<212> DNA

<213> 人工序列

<400> 15

tcttgttaac tggatcgtcc acg 23

<210> 16

<211> 23

<212> DNA

<213> 人工序列

<400> 16

aagctctctt cgtacctgtt cag 23

<210> 17

<211> 24

<212> DNA

<213> 人工序列

<400> 17

attacaggtg tgcagagttc cctc 24

<210> 18

<211> 22

<212> DNA

<213> 人工序列

<400> 18

aagtacccta cctgaaccac ct 22

Claims

1.青虾卵黄蛋白原Vg基因在延缓雌性青虾卵巢发育中的应用；所述的青虾卵黄蛋白原Vg基因的核苷酸序列如SEQ ID NO.1中的第35位～第7645位所示；包括如下步骤：对青虾DNA样本中的青虾卵黄蛋白原Vg基因进行PCR扩增，将扩增产物进行体外转录合成dsRNA，再将dsRNA以注射到青虾的围心腔内；PCR扩增中使用的上下游引物的序列如SEQ ID NO.3～4所示；所述的注射步骤中的注射剂量是4μg/g。