CN116267799B - 一种抗鲫疱疹病毒银鲫的创制方法 - Google Patents
一种抗鲫疱疹病毒银鲫的创制方法 Download PDFInfo
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Abstract
本发明涉及鱼类遗传育种技术领域，尤其涉及一种抗鲫疱疹病毒银鲫的创制方法。向银鲫受精卵注射含有ptpn6‑A和ptpn6‑B各自两个特异敲除靶位点的sgRNA与Cas9mRNA混合物，获得F0代嵌合体，F0代银鲫饲养至性成熟后经过雌核生殖获得F1代，筛选F1代中产生ptpn6‑A一个或2个等位基因提前终止突变的个体再次进行雌核生殖获得F2代，F2代中ptpn6‑A的1个等位基因突变型的银鲫，即为所需抗鲫疱疹病毒银鲫。本发明能够获得抵抗鲫疱疹病毒能力提高的银鲫，为银鲫抗病良种选育提供基础，也为其它养殖鱼类抗病育种提供重要参考价值。
Description
技术领域
本发明涉及鱼类抗病育种技术领域，尤其涉及一种抗鲫疱疹病毒银鲫的创制方法。
技术背景
非受体型蛋白酪氨酸磷酸酶6(protein tyrosine phosphatase nonreceptortype6，ptpn6)参与多条免疫相关信号通路，包括适应性免疫TCR、BCR信号通路，以及天然免疫JAK-STAT、MAPK、RLR等信号通路。有少数研究报道了ptpn6在细菌攻击后的表达变化及其在免疫反应中的作用。在鼠伤寒沙门氏菌或海鱼分枝杆菌感染期间，敲降ptpn6基因的斑马鱼胚胎中先天性免疫反应基因如ifnφ1、il1b、il8、tnfa和tnfb被细菌过度诱导。ptpn6基因在尼罗罗非鱼中的表达水平经过无乳链球菌感染后显著上调，可能涉及B细胞受体(Bcell receptor，BCR)信号通路。
银鲫(Carassius gibelio)是我国最重要的淡水养殖鱼类之一。银鲫是含有两套三倍体染色体组的双三倍体(AAABBB)，能通过单性雌核生殖进行繁殖。随着异育银鲫“中科3号”(A+系)和“中科5号”(F系)等几个优良品种的大规模推广养殖，银鲫已成为我国主要的淡水养殖品种，但近年来高密度养殖导致银鲫爆发许多疾病问题并造成重大经济损失。危害比较严重的就是由鲫疱疹病毒(CaHV)引发的鲫急性鳃出血症，培育抗病力强的银鲫新品种是鲫鱼产业的重大需求。
鲫疱疹病毒(CaHV)感染异育银鲫“中科3号”致死率可达100％，感染异育银鲫“中科5号”也有较高的致死率。
专利CN115486411A公开了一种抗疱疹病毒雌核生殖银鲫新品系的创制方法，通过向雌核生殖异育银鲫中整入一套有性生殖双二倍体鲫基因组，获得倍性升高同时恢复有性生殖能力的合成双四倍体雄鱼，然后利用其有性生殖能力与双二倍体日本白鲫回交，从而创制了一个倍性恢复并且同时整合有银鲫、红鲫和日本白鲫基因组的新双三倍体群体。该专利利用日本白鲫具有显著的抗鲫疱疹病毒(CaHV)的能力，获得具有抗病能力的新品种，但该方法引入了新的基因，创造新的品种。
有必要提出一种不依靠引入外源基因创制具有显著的抗鲫疱疹病毒(CaHV)能力的银鲫的方法。
发明内容
为了解决现有技术中存在的问题，本发明提供了一种抗鲫疱疹病毒银鲫的创制方法。
本发明采用了以下技术方案：
一种抗鲫疱疹病毒银鲫的创制方法，利用CRISPR/Cas9基因编辑技术，向银鲫受精卵注射含有ptpn6-A和ptpn6-B各自两个特异敲除靶位点的sgRNA与Cas9 mRNA混合物获得F0代嵌合体，F0代银鲫饲养至性成熟后经过雌核生殖获得F1代，筛选F1代中产生ptpn6-A一个或2个等位基因提前终止突变的个体再次进行雌核生殖获得F2代，F2代中ptpn6-A的1个等位基因突变型的银鲫，即为所需抗鲫疱疹病毒银鲫；所述ptpn6-A的两个靶位点序列如SEQ ID NO:1和SEQ ID NO:2所示，ptpn6-B的两个靶位点序列如SEQ ID NO:3和SEQ IDNO:4所示。
优选的，包括以下步骤：
S1.获取浓度为1000～1500ng/μL的ptpn6-A和ptpn6-B各自两个特异敲除靶位点的ptpn6-A-sgRNA1、ptpn6-A-sgRNA2、ptpn6-B-sgRNA1、ptpn6-B-sgRNA2，制备浓度为1500～2000ng/μL的Cas9 mRNA，备用；
S2.分别取S1中的ptpn6-A-sgRNA1、ptpn6-A-sgRNA2、ptpn6-B-sgRNA1、ptpn6-B-sgRNA2和Cas9 mRNA各1μL混合，用于注射受精卵，5μL混合物注射500～2000个银鲫受精卵；
S3.显微注射完成后待受精卵孵化，获得F0代嵌合体，F0代养至性成熟后经过雌核生殖获得F1代；
S4.取F1代中产生ptpn6-A一个或2个等位基因提前终止突变的银鲫，再次通过雌核生殖方式获得F2代，F2代中出现的ptpn6-A 1个等位基因突变型的银鲫，即为所需抗鲫疱疹病毒银鲫。
优选的，所述受精卵由银鲫成熟的卵子与兴国红鲤雄鱼的精子混合受精得到。
优选的，所述步骤S3、S4中的雌核生殖的具体操作为：将银鲫成熟的卵子与兴国红鲤雄鱼的精子混合受精并孵化，产生银鲫全雌性子代。
优选的，所述步骤S4中，筛选F1代中产生ptpn6-A一个或2个等位基因提前终止突变的银鲫的方法为：分别单独提取F1代成鱼鳍条基因组DNA，将每尾成鱼鳍条的基因组DNA作为模板，同时分别用ptpn6-A靶位点检测引物和ptpn6-B靶位点检测引物进行PCR扩增，扩增的PCR产物经Sanger测序获得突变序列，将突变序列与野生型序列进行比较，从而确定并选择所需的等位基因的突变型银鲫。
优选的，所述ptpn6-A和ptpn6-B的sgRNA的制备方法为：分别合成ptpn6-A和ptpn6-B各自两个特异靶位点引物，以所述靶位点引物和pUC19-gRNA质粒为模板，进行PCR扩增得到ptpn6-A-sgRNA1、ptpn6-A-sgRNA2、ptpn6-B-sgRNA1和ptpn6-B-sgRNA2片段，再对所述四个片段进行纯化及回收，得到最终所需的ptpn6-A-sgRNA1、ptpn6-A-sgRNA2、ptpn6-B-sgRNA1和ptpn6-B-sgRNA2；
所述ptpn6-A的两个特异靶位点引物的正向引物序列如SEQ ID NO:5或SEQ IDNO:6所示，所述ptpn6-B的两个特异靶位点引物的正向引物序列如SEQ IDNO:7或SEQ IDNO:8所示，ptpn6-A和ptpn6-B的特异靶位点引物的反向引物序列如SEQ ID NO:9所示。
优选的，所述纯化及回收通过体外转录和氯化锂沉淀法进行。
本发明的有益效果在于：
由ptpn6基因编码的含SH2结构域的蛋白酪氨酸磷酸酶1(Src homology region2domain-containing phosphatase 1，SHP1)是PTPs家族的一员，它能催化由蛋白酪氨酸激酶(PTKs)磷酸化的蛋白质中酪氨酸残基的去磷酸化。Ptpn6参与多条免疫相关信号通路，包括适应性免疫TCR、BCR信号通路，以及天然免疫JAK-STAT、MAPK、RLR等信号通路。
银鲫ptpn6-A和ptpn6-B负调控IFN(干扰素)反应主要通过两种机制：ptpn6-A和ptpn6-B都能抑制TBK1对MITA的磷酸化，以及ptpn6-A能够通过自噬途径降解MITA，ptpn6-A和ptpn6-B已经发生亚功能化，且在RLR介导的IFN应答过程中，相对于ptpn6-B，ptpn6-A起主导作用，为培育具有更高抗病能力的银鲫提供靶基因。本发明通过CRISPR/Cas9基因编辑技术，将银鲫的IFN负调控等免疫相关基因进行编辑，使银鲫抗病能力提高，为鱼类精准抗病育种奠定基础。
相比于二倍体，银鲫是双三倍体(AAABBB)，每个基因包括两个部分同源基因和六个等位基因，多倍体的基因编辑比二倍体更具挑战性。
本发明优化了双三倍体银鲫在体基因功能研究的方法，根据ptpn6-A和ptpn6-B在第四个外显子的不同序列，分别设计ptpn6-A和ptpn6-B特异的敲除靶位点。利用基因编辑技术，将高浓度的四个特异靶位点sgRNA与Cas9 mRNA共同注射到银鲫受精卵中以破坏每个部分同源基因和其等位基因的功能。
向银鲫受精卵注射含有ptpn6-A和ptpn6-B各自两个特异敲除靶位点的sgRNA与Cas9 mRNA混合物获得F0代嵌合体，F0代银鲫饲养至性成熟后经过雌核生殖获得F1代，F1代中ptpn6-A一个或两个等位基因提前终止的突变个体能正常性成熟，将其继续雌核生殖产生F2代，发现F2代中出现ptpn6-A 1个等位基因突变型的银鲫，具有较高的抵抗鲫疱疹病毒能力，其抗病能力相较野生型提高约10％。
本发明方法能够获得抵抗鲫疱疹病毒能力提高的银鲫，同时没有引入外源基因，避免了基因污染的风险，为银鲫抗病良种选育提供基础，也为其它养殖鱼类抗病育种提供重要参考价值。
附图说明
图1为银鲫ptpn6-A和ptpn6-B各自两个靶位点设计示意图，外显子和内含子分别用矩形框和粗线表示，ptpn6-A和ptpn6-B差异序列用加粗表示；
图2为银鲫野生型(WT-F)及两个ptpn6-A敲除突变克隆系感染CaHV后的存活率。
图3为实施例2中野生型银鲫和试验鱼的Sanger测序峰图；图中a部分为野生型和ptpn6-A突变体银鲫的ptpn6-A-sgRNA1靶位点测序峰图，下划线为靶位点；b部分为野生型和ptpn6-A突变体银鲫的ptpn6-A-sgRNA2靶位点测序峰图，下划线为靶位点；c部分为野生型和ptpn6-B突变体银鲫的ptpn6-B-sgRNA1靶位点测序峰图，下划线为靶位点；d部分为野生型和ptpn6-B突变体银鲫的ptpn6-B-sgRNA2靶位点测序峰图，下划线为靶位点。
具体实施方式
为了便于理解，下面结合实施例对本发明的技术方案做出更为具体的说明：
实施例1
1银鲫ptpn6-A和ptpn6-B靶位点设计
根据ptpn6-A和ptpn6-B在第四外显子的不同序列，分别设计ptpn6-A和ptpn6-B特异的两个敲除靶位点。如图1所示，靶位点分别对应的CDS序列为171-191bp和249-269bp，对应蛋白序列57-64和83-90氨基酸位置，位于C-SH2和PTPc结构域之前。且ptpn6-A和ptpn6-B各自的两个靶位点位置均是对应的，只是存在单核苷酸多态性(single nucleotidepolymorphism，SNP)差异。
其中ptpn6-A靶位点序列为：GGGGATTATTACGATCTGT(SEQ ID NO:1)和GGAACTCTACAGGATAGAGA(SEQ ID NO:2)，ptpn6-B靶位点序列为：GGTGATTATTATGACCTGTA(SEQ ID NO:3)和GGAACCCTACAGGATAAAGA(SEQ ID NO:4)。
2合成ptpn6-A和ptpn6-B特异靶位点引物如下：
ptpn6-A的特异靶位点引物的正向引物序列：
ptpn6-A-sgRNA1-F:5′-GTAATACGACTCACTATAGGGGATTATTACGATCTGTAGTTTTAGAGCTAGAAATAGC-3′(SEQ ID NO:5)
ptpn6-A-sgRNA2-F:5′-GTAATACGACTCACTATAGGAACTCTACAGGATAGAGAGTTTTAGAGCTAGAAATAGC-3′(SEQ ID NO:6)
ptpn6-B的特异靶位点引物的正向引物序列：
ptpn6-B-sgRNA1-F:5′-GTAATACGACTCACTATAGGTGATTATTATGACCTGTAGTTTTAGAGCTAGAAATAGC-3′(SEQ ID NO:7)
ptpn6-B-sgRNA2-F:5′-GTAATACGACTCACTATAGGAACCCTACAGGATAAAGAGTTTTAGAGCTAGAAATAGC-3′(SEQ ID NO:8)
ptpn6-A和ptpn6-B的特异靶点引物的反向引物序列(sgRNA-R)：
5′-AAAAGCACCGACTCGGTGCC-3′(SEQ ID NO:9)。
3.制备ptpn6-A和ptpn6-B sgRNA
以上述靶位点引物(ptpn6-A-sgRNA1-F和sgRNA-R)或(ptpn6-A-sgRNA2-F和sgRNA-R)以及(ptpn6-B-sgRNA1-F和sgRNA-R)或(ptpn6-B-sgRNA2-F和sgRNA-R)和pUC19-gRNA质粒为模板，用FastPfu Fly DNA Polymerase(Transgen)的高保真酶进行PCR扩增。
使用TranscriptAid T7 High Yield Transcription Kit(Thermo FishScientific)分别对ptpn6-A-sgRNA1、ptpn6-A-sgRNA2片段和ptpn6-B-sgRNA1、ptpn6-B-sgRNA2片段进行sgRNA的体外转录，并利用氯化锂沉淀法对sgRNA的体外转录产物纯化回收。
测定其浓度后取100～200ng的sgRNA溶液通过琼脂糖凝胶检测RNA的质量，其余溶液分装并冻存于-80℃冰箱，备用。
4制备Cas9 mRNA
酶切zebrafish-codon-optimized Cas9质粒后通过琼脂糖凝胶分析酶切产物，确认质粒线性化后，使用Gel Extraction Kit(Omega)进行切胶纯化回收线性化Cas9质粒。使用Mmessage mMACHINETM T7(Thermo Fish Scientific)对线性化zebrafish-codon-optimized Cas9质粒进行Cas9 mRNA的体外转录，并利用氯化锂沉淀法对Cas9 mRNA的体外转录产物纯化回收，最终得到Cas9 mRNA。测定其浓度后后取100～200ng的Cas9 mRNA溶液通过琼脂糖凝胶检测RNA的质量，其余Cas9 mRNA溶液分装冻存于-80℃冰箱，备用。
5受精卵显微注射
将1μL ptpn6-A-sgRNA1(1000～1500ng/μL)、1μL ptpn6-A-sgRNA2(1000～1500ng/μL)、1μL ptpn6-B-sgRNA1(1000～1500ng/μL)、1μLptpn6-B-sgRNA2(1000～1500ng/μL)和1μL Cas9 mRNA(1500～2000ng/μL)在冰上混合均匀后转移到显微注射针头进行显微注射，5μL混合物可注射500-2000个银鲫受精卵。
将200～500粒异育银鲫F系受精卵平铺到10cm无菌培养皿中，采用氮气加压的PLI-100定量显微注射仪(Harvard)将混合液注射到1细胞期的银鲫胚胎动物极中，每次注射量为2nL；显微注射完成后将受精卵转移至水温22～23℃孵化缸中孵化，获得F0代。
6抗鲫疱疹病毒银鲫的创制
将F0代个体鱼苗饲养到成年，经过雌核生殖产生F1代。
本实施例中，在F1代中筛选出15尾ptpn6-A三个等位基因均提前终止的突变基因型个体，6尾ptpn6-B三个等位基因均提前终止的突变基因型个体，以及35尾ptpn6-A和ptpn6-B一个或两个等位基因提前终止的突变基因型个体。其中ptpn6-A一个或两个等位基因提前终止的F1代突变个体能正常性成熟，而编辑3个ptpn6-A等位基因(n＝15)以及编辑ptpn6-B的1个等位基因(n＝7)、2个等位基因(n＝3)和3个等位基因(n＝6)的突变基因型个体，没能正常产卵。
因此，ptpn6-A一个或两个等位基因提前终止的F1代突变个体的成熟卵，再次通过雌核生殖获得分别编辑ptpn6-A的1个等位基因(ptpn6-A+/+/Δ2)和2个等位基因(ptpn6-AΔ82/+/Δ82)的F2代群体。
上述筛选等位基因突变型的方法为：剪取F1代成鱼的鳍条保存于无水乙醇，使用磁珠法动物组织基因组DNA纯化试剂盒(杭州比格飞序生物科技有限公司)分别单独提取鳍条基因组DNA。将每尾成鱼鳍条的基因组DNA作为模板，同时分别用ptpn6-A靶位点检测引物和ptpn6-B靶位点检测引物进行PCR扩增。扩增的PCR产物经过Sanger测序后，野生型的Sanger测序峰图在靶位点附近上下游区域都是单峰，CRISPR/cas9编辑过实验鱼的Sanger测序峰图在靶位点附近区域出现套峰，初步判断该靶位点处发生敲除，之后将纯化PCR产物克隆并进行Sanger测序获得突变序列，通过突变序列与野生型序列进行比较，最终确定并选择所需的等位基因的突变型。
7验证
选用新鲜制备的CaHV病毒悬液，用PBS缓冲液按100、101、102、103、104稀释倍数于冰上进行梯度稀释。将60尾健康的银鲫随机分成6组(每组10尾)，按梯度稀释的病毒滤液分别注射，每尾腹腔注射500μL病毒滤液稀释液，对照组注射等量的PBS缓冲液。每日观察，统计死亡率，计算半致死剂量。
将各F系野生型银鲫及敲除品系(F2代群体)银鲫暂养至实验室的35cm×23.5cm×36cm水族箱中，养殖水温逐步过渡到22℃(±1℃)，饲养两周以观察实验鱼有无异常。
实验鱼腹腔分别注射500μL CaHV过滤后的病毒悬液(1.942×107病毒粒子)，F系野生型个体作为对照组，每尾注射等量的PBS缓冲液。每个感染组设置3组平行，每个平行组有20尾个体。养殖用水保持在22℃(±1℃)，并不间断过滤，每天更换一次，及时捞出死亡个体以保持水体洁净。感染后每天记录银鲫死亡情况，一直统计到8dpi(days postinfection)，最终用Mantel-Cox分析鱼体存活率。
如图2所示，野生型银鲫F系(WT-F)在感染第3天的时候开始出现死亡，但死亡率较平缓，直到第6天时存活率还有20％，第7天之后有10％的存活率，第8天时全部死亡。突变系ptpn6-AΔ82/+/Δ82存活率类似于WT-F，而突变系ptpn6-A+/+/Δ2在感染的第四天才开始死亡，至第7天时存活率为20％，到第8天时存活率为10％。上述的结果表明，F2代中编辑ptpn6-A的1个等位基因(ptpn6-A+/+/Δ2)群体抗病能力相比野生型银鲫F系提高，提高幅度约10％。
实施例2
在F0代，为检测每个靶位点的突变效率，对两个部分同源基因ptpn6-A和ptpn6-B分别设计特异的靶位点检测引物，其中ptpn6-A靶位点检测引物(片段大小约652bp)为：
ptpn6-A-F：5′-CCAGTTTATGTTTGGGGACATGG-3′，
ptpn6-A-R：5′-TGTGTATTT ATGCATCCGTGGGG-3′；
ptpn6-B靶位点检测引物(片段大小约735bp)：
ptpn6-B-F：5′-GTTGTATCTCGGCCAAATGTTGTC-3′，
ptpn6-B-R：5′-GGGGCCTCATTAATTGGTATATGG-3′。
随机选取8尾CRISPR/cas9编辑过的F0代1dph银鲫鱼苗和3尾野生型1dph银鲫鱼苗，使用磁珠法动物组织基因组DNA纯化试剂盒(杭州比格飞序生物科技有限公司)分别单独提取鱼苗基因组DNA。将每尾鱼苗的基因组DNA作为模板，同时分别用ptpn6-A靶位点检测引物和ptpn6-B靶位点检测引物进行PCR扩增。
50μL PCR扩增体系：5×FastPfu Fly Buffer 10μL，2.5mM dNTPs4μL，F和R引物各1μL，基因组DNA模板2μL，/>FastPfu Fly DNA Polymerase(Transgen)1μL，无菌水31μL。
PCR扩增程序：95℃预变性2min；95℃变性20s，56℃退火20s，72℃延伸20s，38个循环；最后72℃延伸5min。
通过在1.2％的琼脂糖凝胶上用合适的DNA染色剂电泳分析1～2μL PCR产物，验证PCR产物条带大小正确后，将其余未纯化PCR产物送到测序公司(武汉艾康健生物科技有限公司)进行Sanger测序。
如图3所示，扩增的PCR产物经过Sanger测序后，野生型的Sanger测序峰图在靶位点附近上下游区域都是单峰，CRISPR/cas9编辑过实验鱼的Sanger测序峰图在靶位点附近区域出现套峰，初步判断该靶位点处发生敲除。之后将纯化PCR产物克隆并进行Sanger测序获得突变序列，通过突变序列与野生型序列进行比较，最终确定靶位点突变效率。
实施例1中，8尾CRISPR/cas9编辑过的F0代1dph银鲫鱼苗，6尾分别在ptpn6-A靶位点和ptpn6-B靶位点都发生了突变，ptpn6基因敲除成功率为75％。
以上实施方式仅用以说明本发明的技术方案，而并非对本发明的限制；尽管参照前述实施方式对本发明进行了详细的说明，本领域的普通技术人员应当理解：凡在本发明创造的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明创造的保护范围之内。
Claims (7)
1.一种抗鲫疱疹病毒银鲫的创制方法，其特征在于，利用CRISPR/Cas9基因编辑技术，向银鲫受精卵注射含有ptpn6-A和ptpn6-B各自两个特异敲除靶位点的sgRNA与Cas9 mRNA混合物获得F0代嵌合体，F0代银鲫饲养至性成熟后经过雌核生殖获得F1代，筛选F1代中产生ptpn6-A一个或2个等位基因提前终止突变的个体再次进行雌核生殖获得F2代，F2代中出现ptpn6-A的1个等位基因突变型的银鲫，即为所需抗鲫疱疹病毒银鲫；所述ptpn6-A的两个靶位点序列如SEQ ID NO:1和SEQ ID NO:2所示，ptpn6-B的两个靶位点序列如SEQ ID NO:3和SEQ ID NO:4所示。
2.如权利要求1所述的一种抗鲫疱疹病毒银鲫的创制方法，其特征在于，包括以下步骤：
S1.获取浓度为1000～1500ng/μL的ptpn6-A和ptpn6-B各自两个特异敲除靶位点的ptpn6-A-sgRNA1、ptpn6-A-sgRNA2、ptpn6-B-sgRNA1、ptpn6-B-sgRNA2，制备浓度为1500～2000ng/μL的Cas9 mRNA，备用；
S2.分别取S1中的ptpn6-A-sgRNA1、ptpn6-A-sgRNA2、ptpn6-B-sgRNA1、ptpn6-B-sgRNA2和Cas9 mRNA各1μL混合，用于注射受精卵，5μL混合物注射500～2000个银鲫受精卵；
S3.显微注射完成后待受精卵孵化，获得F0代嵌合体，F0代养至性成熟后经过雌核生殖获得F1代；
S4.取F1代中产生ptpn6-A一个或2个等位基因提前终止突变的银鲫，再次通过雌核生殖方式获得F2代，F2代中出现的ptpn6-A 1个等位基因突变型的银鲫，即为所需抗鲫疱疹病毒银鲫。
3.如权利要求1或2所述的一种抗鲫疱疹病毒银鲫的创制方法，其特征在于，所述受精卵由银鲫成熟的卵子与兴国红鲤雄鱼的精子混合受精得到。
4.如权利要求2所述的一种抗鲫疱疹病毒银鲫的创制方法，其特征在于，所述步骤S3、S4中的雌核生殖的具体操作为：将银鲫成熟的卵子与兴国红鲤雄鱼的精子混合受精并孵化，产生银鲫全雌性子代。
5.如权利要求2所述的一种抗鲫疱疹病毒银鲫的创制方法，其特征在于，所述步骤S4中，筛选F1代中产生ptpn6-A一个或2个等位基因提前终止突变的银鲫的方法为：分别单独提取F1代成鱼鳍条基因组DNA，将每尾成鱼鳍条的基因组DNA作为模板，同时分别用ptpn6-A靶位点检测引物和ptpn6-B靶位点检测引物进行PCR扩增，扩增的PCR产物经Sanger测序获得突变序列，将突变序列与野生型序列进行比较，从而确定并选择所需的等位基因的突变型银鲫。
6.如权利要求2所述的一种抗鲫疱疹病毒银鲫的创制方法，其特征在于，所述ptpn6-A和ptpn6-B的sgRNA的制备方法为：分别合成ptpn6-A和ptpn6-B各自两个特异靶位点引物，以所述靶位点引物和pUC19-gRNA质粒为模板，进行PCR扩增得到ptpn6-A-sgRNA1、ptpn6-A-sgRNA2、ptpn6-B-sgRNA1和ptpn6-B-sgRNA2片段，再对所述四个片段进行纯化及回收，得到最终所需的ptpn6-A-sgRNA1、ptpn6-A-sgRNA2、ptpn6-B-sgRNA1和ptpn6-B-sgRNA2；
所述ptpn6-A的两个特异靶位点引物的正向引物序列如SEQ ID NO:5或SEQ ID NO:6所示，所述ptpn6-B的两个特异靶位点引物的正向引物序列如SEQ ID NO:7或SEQ ID NO:8所示，ptpn6-A和ptpn6-B的特异靶位点引物的反向引物序列如SEQ ID NO:9所示。
7.如权利要求6所述的一种抗鲫疱疹病毒银鲫的创制方法，其特征在于，所述纯化及回收通过体外转录和氯化锂沉淀法进行。
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