CN114686524B - 一种利用基因编辑生产1龄雌性黄鳍鲷的方法 - Google Patents
一种利用基因编辑生产1龄雌性黄鳍鲷的方法 Download PDFInfo
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Abstract
本发明公开了一种规模化生产全雌性黄鳍鲷的方法,包括以下步骤:(S1)F0代嵌合体雄鱼的获得;(S2)测交和基因突变个体的筛选;(S3)1龄雌性黄鳍鲷的生产和鉴定。该方法以黄鳍鲷雌雄同体生殖特性为基础,结合性别功能基因nanos2利用和CRISPR/Cas9基因编辑技术,为黄鳍鲷养殖提供遗传改良的新品系或新品种。
Description
技术领域
本发明属于水产动物遗传育种与功能基因利用技术领域,具体涉及一种利用基因编辑生产1龄雌性黄鳍鲷的方法,尤其是涉及一种利用CRISPR/Cas9基因编辑生产1龄雌性黄鳍鲷的方法。
背景技术
黄鳍鲷(Acanthopagrus latus),属于鲈形目(Perciformes),鲷科(Sparidae),棘鲷属(Acanthopagrus),又称为黄脚立、赤翅、鲛腊鱼、黄鳍鲷。体长椭圆形,扁而窄,体高,头部尖,尾双叉形。黄鳍鲷的分布贯穿印度西太平洋,从波斯湾沿着印度海岸到菲律宾,从澳大利亚到日本,我国主要分布广东、福建、广西等的沿海城市。黄鳍鲷盐度适应能力强,海水、咸淡水均生长良好,所以在养殖业中占有重要地位。黄鳍鲷属于雌雄同体雄性先熟鱼类,没有性染色体,存在性反转现象,1龄和2龄黄鳍鲷全部为功能性雄鱼,直到第三个繁殖季节,才有部分成鱼性逆转为功能性雌鱼。这种性别分化方式和性逆转生殖特点造成了黄鳍鲷生产实践中雌鱼数量缺乏,给黄鳍鲷的人工繁育和遗传改良带来了很大的困难。且黄鳍鲷群体中雌鱼个体比雄鱼大,导致养殖群体中规格不整齐,饲料利用率低。因此,进行黄鳍鲷性别控制技术的研究,生产1龄雌性黄鳍鲷,将大幅提高黄鳍鲷的养殖效率,为下一步培育全雌性黄鳍鲷品系奠定基础。迄今,国内外还没有见到有关黄鳍鲷性别控制技术的研究。
CRISPR/Cas9基因编辑技术是近年来发展起来基因组定向编辑和改造技术,主要是利用靶向特异的guide RNA,引导Cas9蛋白酶对靶向DNA序列完成特异性切割。该技术操作简单,对基因组切割效率高,目前已在多种鱼类中得到应用,包括斑马鱼、青鳉、黄颡鱼、半滑舌鳎、尼罗罗非鱼、斑点叉尾鮰、白斑狗鱼、虹鳟、鲤鱼、黄鳝等。但是,迄今为止,还没有在黄鳍鲷中实现基因编辑操作。因此,黄鳍鲷的基因功能和遗传改良育种等方面研究进展缓慢。
Nanos2是Nanos基因家族成员之一,可以编码一种保守的RNA结合蛋白,其主要在原始生殖细胞和精原细胞中表达,是一种重要的雄性生殖调控因子。目前,Nanos2已在小鼠、果蝇、斑马鱼、鸡等多个物种中进行了研究,证明nanos2对于雄性的发生和雌性的抑制具有重要的调控作用,但对于雌雄同体鱼类中精卵巢结构发生和发育的影响,还没有报到,仍需进一步研究。
发明内容
本发明的目的在于提供一种利用基因编辑生产1(岁)龄雌性黄鳍鲷的方法,该方法以黄鳍鲷雌雄同体生殖特性、性别功能基因nanos2利用和CRISPR/Cas9基因编辑技术为基础,为黄鳍鲷养殖提供遗传改良的新品系或新品种。
本发明的上述目的可以通过以下技术方案来实现:一种利用基因编辑生产1龄雌性黄鳍鲷的方法,包括以下步骤:
(S1)F0代嵌合体雄鱼的获得:利用CRISPR/Cas9基因编辑方法,采用显微注射的方式,将nanos2靶基因的gRNA和Cas9蛋白的mRNA混合物导入黄鳍鲷1细胞期的受精卵的动物极中,孵化后即可获得靶基因突变的F0代嵌合体黄鳍鲷雄鱼;
(S2)测交和基因突变个体的筛选:将步骤(S1)中获得的F0代嵌合体黄鳍鲷雄鱼与野生型雌鱼交配,从后代中筛选具有nanos2有义突变的F1代杂合子个体(nanos2+/-);
(S3)1龄雌性黄鳍鲷的生产和鉴定:将F1代杂合子个体(nanos2+/-)养殖到性成熟,其中一部分个体正常发育为杂合子雄鱼(nanos2+/-),一部分个体自然性逆转为F1代杂合子雌鱼(nanos2+/-),选取F1代杂合子雌鱼(nanos2+/-)和F1代杂合子雄鱼(nanos2+/-)交配,筛选得到F2代纯合子突变体个体(nanos2-/-),所述F2代纯合子突变体个体(nanos2-/-)1龄性成熟时,性反转为功能性雌鱼。
本发明通过研究黄鳍鲷的生殖特性,证明了nanos2基因对黄鳍鲷性腺发育的功能影响,从而建立一种生产1(岁)龄雌性黄鳍鲷的步骤和方法,为黄鳍鲷的性别控制和全雌黄鳍鲷育种研究提供技术支撑。
在上述利用基因编辑生产1龄雌性黄鳍鲷的方法中:
优选的,步骤(S1)中合成所述nanos2靶基因的gRNA的引物包括正向引物nanos2-gRNA-F和反向引物gRNA-R,所述正向引物nanos2-gRNA-F的序列如SEQ ID NO:1所示,所述反向引物gRNA-R的序列如SEQ ID NO:2所示。
具体的:
gRNA的合成所需的正向引物nanos2-gRNA-F为:
nanos2-gRNA-F:TAATACGACTCACTATAGGGCTCCCTCCTGCCCGACGGTTTTAGAGCTAGAAATAGC。
反向引物gRNA-R为:
gRNA-R: AGCACCGACTCGGTGCCACT。
优选的,步骤(S1)中所述nanos2靶基因的gRNA和所述Cas9蛋白的终浓度分别约为30~40 ng/µL和150~200 ng/µL,更佳为40 ng/µL和200 ng/µL,每颗受精卵中所述nanos2靶基因的gRNA和Cas9蛋白的mRNA混合物的注射量约为1~2nL,更佳为1nL。
优选的,步骤(S1)中采用显微注射的方式,将nanos2靶基因的gRNA和Cas9蛋白的mRNA混合物导入黄鳍鲷1细胞期的受精卵的动物极中,需在受精后40分钟内完成注射。
室温条件下,受精卵1细胞期只能维持大约40分钟,因此需在受精后40分钟内完成注射。
优选的,步骤(S2)中将F0代嵌合体黄鳍鲷雄鱼与野生型雌鱼按照1:1~2的数量配比交配,更佳为1:1。
优选的,步骤(S2)中所述具有nanos2有义突变的F1代杂合子个体(nanos2+/-)是突变比例最高的突变类型为5个碱基缺失的F1代杂合子个体(nanos2+/-)。
本发明共检测到5种突变类型,即在靶序列位置附近出现不同程度的碱基缺失,总体突变率约为25%,其中比例最高的突变类型为5碱基的缺失,本申请选择突变比例最高的突变类型5个碱基缺失的F1代杂合子个体(nanos2+/-)进行下一步。
优选的,步骤(S2)中用于检测具有nanos2有义突变的F1代杂合子个体(nanos2+/-)的PCR引物,包括正向引物nanos2-F1和反向引物nanos2-R1,其中所述正向引物nanos2-F1的序列如SEQ ID NO:3所示,所述反向引物nanos2-R1的序列如SEQ ID NO:4所示。
具体的:
正向引物nanos2-F1为:GCGCAATTTCTCCCCCAAAA;
反向引物nanos2-R1为:GTCTCTCGCTGTCCATCGTC。
与现有技术相比,本发明具有以下优点:
(1)目前尚无进行黄鳍鲷胚胎的显微操作技术,也没有进行黄鳍鲷内源基因编辑的相关技术报道,本发明首次在黄鳍鲷中建立内源基因精准编辑技术,获得高突变率的黄鳍鲷胚胎;
(2)本发明利用了功能基因nanos2,有效的调控黄鳍鲷性腺发育,生产雌性黄鳍鲷。
附图说明
图1为实施例1中CRISPR/Cas9敲除nanos2基因靶向序列的选择及突变检测;
图2为实施例1中1龄野生型和1龄F2代纯合突变体(nanos2-/-)性腺发育切片图。
具体实施方式
本发明所述技术方案,如未特别说明,均为本领域的常规方案,所述试剂或材料,如未特别说明,均来源于商业渠道。
实施例1
本实施例提供的一种利用基因编辑生产1龄(1岁龄)雌性黄鳍鲷的方法,包括以下步骤:
(1)黄鳍鲷人工繁殖和1细胞期受精卵的获取
挑选性成熟的黄鳍鲷亲本,暂养于30立方米的水泥池中,雌雄鱼按4μg/kg的剂量注射促黄体素释放激素A3(Luteinizing hormone releasing hormone A3, LHRHA3,宁波第二激素厂,中国)和1000单位/kg剂量的注射人绒毛膜促性腺激素(HumanChorionicGonadotropin,hCG,宁波第二激素厂)。注射后,将亲鱼放回水温22~25℃和盐度22‰~25‰的水泥池中,等待32~36小时,即可将雌鱼捞出进行挤卵,并同时挤出雄鱼的精子进行人工受精。先将精卵子混合均匀,然后用盐度25‰的海水激活精子,完成授精,并用于显微注射。
(2)构建黄鳍鲷nanos2基因的guide RNA和Cas9 mRNA
根据已经验证的黄鳍鲷的nanos2转录本(Gene ID: 119008502)及黄鳍鲷的基因组序列(PRJEB40700),使用CRISPR/Cas9靶序列设计网站(http://zifit.partners.org/ZiFiT/)进行nanos2基因敲除靶标的设计,主要设计和筛选原则如下:(1)靶序列长度为19~24个碱基,5’端以GG或G幵头,靶序列后紧跟以NGG基序;(2)靶序列在外显子区,最好在靠近蛋白编码区5’端,以便使蛋白彻底变性;(3)用本地blast程序对靶序列进行全基因组检索,E-value设置上限为1E-10,blast到的非靶序列碱基一致性小于14 nt,以减少脱靶。
gRNA的合成所需的正向引物为:
nanos2-gRNA-F:TAATACGACTCACTATAGGGCTCCCTCCTGCCCGACGGTTTTAGAGCTAGAAATAGC(如SEQ ID NO:1所示),其中前17个碱基为T7启动子序列,后面20个碱基为gRNA骨架5’端部分序列,中间20个碱基为nanos2基因的靶标序列(图1中A图);
反向引物为gRNA-R: AGCACCGACTCGGTGCCACT(如SEQ ID NO:2所示),为gRNA骨架3’端部分序列。
gRNA的体外转录使用T7 High Yield RNA Transcription Kit(诺唯赞,中国)试剂盒。然后用超微量分光光度计Nanodrop 2000进行浓度检测,并用1.0%琼脂糖凝胶电泳分析其完整性,最后放到-80℃冰箱备用。
PCR反应体系包括:2xTaq Plus Master Mix II (Vazyme, China) 5 μL,nanos2-gRNA-F和gRNA-R引物各0.5 μL(10μM),含gRNA骨架序列的质粒(DR274)模板0.5μL(100ng),ddH2O 3.5 μL。
PCR扩增反应条件:先95℃5 min; 95℃30 s, 50℃退火20 s,72℃延伸10 s,共37个循环; 最后72℃延伸1 min。
扩增产物经1.0%琼脂糖凝胶分离,并将符合预期大小的条带切胶,使用E.Z.N.A胶回收试剂盒(Omega,美国)进行回收,并用DEPC水进行溶解,部分样品送北京擎科生物科技有限公司进行测序。待测序核实准确后,即可开始体外转录。
gRNA的体外转录使用T7 High Yield RNA Transcription Kit(Vazyme,China)进行,主要步骤包括:向无酶PCR管加入10 x reaction buffer 2 μL,ATP,GTP,UTP和CTPsolution各2 μL,RNA polymerase mix 2 μL,PCR产物回收模板200~500ng,补DEPC水到20μL,混匀后,37℃孵育3~4 h,加入1 μL DNase I,37℃孵育15 min,消化DNA模板。
最后,使用HiPure RNA Pure Micro Kit(Magen,China)进行纯化回收,主要步骤包括:首先用DEPC水调整样品体积到100 μL,然后加入300 μl Buffer RP,涡旋混匀10 s,室温静置5 min,加入600 μL无水乙醇,涡旋混匀15 s,将HiPureRNA 柱子套在收集管上,把前面得到的混合液转移至柱子中,10,000 g 离心30~60 秒,弃滤液,加入500 μL BufferRW2(已用无水乙醇稀释)至柱子中,静置2 min,10,000 g 离心30~60 秒,重复此步骤一次,然后将柱子套到1.5mL离心管中,加入15~30 μLRNase Free Water 至柱子膜中央,放置1min,然后13,000 g 离心1 min即可。
然后用超微量分光光度计Nanodrop 2000进行浓度检测,并用1.0%琼脂糖凝胶电泳分析其完整性,最后放到-80℃冰箱备用。
将Cas9质粒用XbaI限制性内切酶进行线性化处理,即37℃水浴3 h,然后取少量反应液进行琼脂糖凝胶电泳确认质粒是否完全切开,对于完全切开后的质粒,使用GenElute™ PCR 纯化试剂盒(Sigma,Germany)进行直接回收。然后使用mMESSAGEmMACHINE T7 Kit(ambion)进行Cas9 mRNA的体外转录,主要操作步骤包括:向无酶PCR管中加入10 xreaction buffer 2 μL,2 x NTP/CAP 10 μL,T7 Enzyme Mix 2 μL,RNase inhibitor 0.5μL,线性化Cas9质粒模板800ng~1 μg,然后补DEPC水到20 μL,37℃孵育2-3 h,加入1 μLTURBO DNase,37℃孵育15 min。向得到反应体系中30 μL LiCl和30 μLDEPC水,-20℃放置1h以上,4℃离心30 min,然后吸去上清液,然后加入用DEPC水配置的70%乙醇,上下颠倒数次,然后4℃离心5 min,吸去上清液,室温晾干15 min,加入20 μL DEPC水进行溶解,轻轻吹打溶解后,测定RNA的浓度并电泳检测其完整性,最后放到-80℃冰箱备用。
(3)黄鳍鲷受精卵显微注射和胚胎孵育
将刚授精的黄鳍鲷受精卵均匀摆放到1%琼脂糖凝胶制备的凹槽中,在体视显微镜下观察细胞的动物极,并调整到合适的倍数。然后将Cas9 mRNA和nanos2-gRNA混合,并加入少量无RNA酶的酚红,终浓度Cas9 mRNA和nanos2-gRNA分别调整为200 ng/µL和40ng/µL,加入玻璃注射针中。玻璃注射针采用WPI品牌水平拉针仪(PUL-1000)拉制,拉针参数为heat:710,Force: 300,Distance: 9.00,Delay: 0。最后使用WPI品牌的显微注射系统(PV820)对1细胞期受精卵动物极进行注射,使用氮气加压,每颗受精卵注射量为1nL左右,室温条件下,受精卵1细胞期只能维持大约40分钟,需在受精后40分钟内完成注射。
将显微注射后的黄鳍鲷胚胎转入盐度23‰的海水中孵育,显微注射后的胚胎比较脆弱,剧烈震动容易导致其死亡。静置孵育12小时后即可转入20 L左右的桶用充氧泵充氧孵化,直至破膜。孵化期间及时挑死卵,并每天换2/3的水一次,出膜后,将破膜后的黄鳍鲷小苗转入半立方米体积的桶中养殖,破膜第3-4天后,观察到黄鳍鲷苗能够快速平游,即可逐渐放入适口饵料喂养,持续喂养至3月龄以上。
(4)显微注射黄鳍鲷胚胎突变检测
为检测靶向突变效率,在显微注射24小时后,随机选取20颗左右的显微注射胚胎进行突变检测。首先用PCR标本处理液(东盛,中国)对胚胎进行DNA粗提,主要步骤是取20颗显微注射胚胎(Mutant)和野生型胚胎(WT)放入1.5 mL离心管中,加入180 µL ExtractionSolution,涡旋振荡,95℃温浴15min,加入20µL Neutralization Solution,充分振荡,5000rpm离心2min,上清即为DNA粗提液。
根据黄鳍鲷nanos2基因靶向编辑位点上下游序列设计PCR引物,正向引物nanos2-F1为:GCGCAATTTCTCCCCCAAAA(如SEQ ID NO:3所示),反向引物nanos2-R1为:GTCTCTCGCTGTCCATCGTC(如SEQ ID NO:4所示)。
PCR反应体系包括:2xTaq Plus Master Mix II (诺唯赞)10µL,Nanos2-F1和Nanos2-R1引物各1µL(10μM),上述粗提DNA模板2 µL,ddH2O 6µL。
PCR扩增反应条件:先95℃5 min;95℃30 s,55℃退火30 s,72℃延伸30 s,共35个循环;最后72℃延伸5 min。
扩增产物经1.0%琼脂糖凝胶分离,并将符合预期大小的条带切胶,使用E.Z.N.A胶回收试剂盒(Omega,美国)进行回收,并用DEPC水进行溶解。
样品送北京擎科生物科技有限公司进行直接测序,测序结果显示在靶序列位置出现套峰,说明靶序列发生改变,PCR在靶序列位置扩增出多种不同的序列,即此位置发生了突变。并通过TA克隆的方法连接到pGEM-T Easy 载体上,并转化到Trelief™ 5αChemically Competent Cell(擎科,中国)感受态细胞中,主要转化步骤包括:向100 μL冰上融化的感受态细胞中加入连接产物,轻轻混匀,冰上静置5 min,然后42℃水浴热激45~60s,迅速转移至冰上,静置2 min,向离心管中加入500 μL不含抗生素的无菌液体LB培养基,37℃摇床培养45分钟,5000 g离心2 min,弃掉450μL上清液,加入IPTG(200mg/ml)4 μL,X-gal(20mg/ml)40 μL,均匀涂布到含Amp抗生素的固体培养基上,37℃培养箱倒置培养过夜,并随机挑取30个白斑菌落进行测序。
结果共检测到5种突变类型,即在靶序列位置附近出现不同程度的碱基缺失,总体突变率约为25%,其中比例最高的突变类型为5碱基的缺失(图1中B图)。
(5)测交
检测后的F0代嵌合体亲本养在水泥池中,水深约1.5米,循环水专池养殖。每天三次投喂海水鱼商品饲料,养至1龄性成熟。选取健康无病,个体较大的性成熟F0代雄鱼,与性成熟的野生型雌鱼进行1:1配对。雌雄亲鱼按4μg/kg的剂量注射LHRHA3(宁波第二激素厂)和1000单位/kg剂量的注射hCG(宁波第二激素厂)。注射后,将亲鱼放回水温22~25℃和盐度22‰~25‰的水泥池中,等待32~36小时,即可将雌鱼捞出进行挤卵,同时挤出F0代雄鱼的精子完成授精过程,获得F1代黄鳍鲷子代。
(6)突变个体筛选和繁殖传代
F1代子代正常养殖到3月龄,剪取F1代黄鳍鲷尾鳍2g,利用醋酸氨法进行DNA提取,对F1代个体进行突变位点检测,检测方法如同步骤(4),筛选获得5个碱基缺失的的杂合子黄鳍鲷个体(nanos2+/-),进一步养殖到2龄性成熟。在繁殖季节,其中一部分个体正常发育为功能性雄鱼,少数个体自然性逆转为功能性雌鱼,选取健康无病,个体较大性成熟的20对个体作为繁殖父母本,交配繁殖。雌雄亲鱼按4μg/kg的剂量注射LHRHA3(宁波第二激素厂)和1000单位/kg剂量注射HCG(宁波第二激素厂)。注射后,将雌雄亲鱼放回水温22~25℃和盐度22‰~25‰的水泥池中,进行自然产卵授精,等待32~36小时,收集水面上层的黄鳍鲷受精卵,进行孵化培育,方法如同步骤(3),获得F2代黄鳍鲷子代。
(7)1龄雌性黄鳍鲷的生产和F2代纯合子性腺发育分析
F2代子代养至3月龄,剪取尾鳍鳍条,提取DNA,进行突变位点基因型鉴定,检测方法如同步骤(4),得到5碱基缺失的纯合F2代子代(nanos2-/-),将纯合子F2代子代(nanos2-/-)养至1龄(1岁龄)性成熟,经切片检验,其性腺全部发育为卵巢(图2)。
本发明不局限于上述特定的实施方案范围内,上述实施方案仅仅是为了能够对本发明的使用过程进行详细地说明,而且有相等功能的生产方法和技术细节也属于本发明内容的一部分。事实上,本领域技术人员根据前文的描述,就能够根据各自需要找到不同的调整方案,这些调整都应在本文所附的权利要求书的范围内。
序列表
<110> 中山大学
<120> 一种利用基因编辑生产1龄雌性黄鳍鲷的方法
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Claims (5)
1.一种利用基因编辑生产1龄雌性黄鳍鲷的方法,其特征是包括以下步骤:
(S1)F0代嵌合体雄鱼的获得:利用CRISPR/Cas9基因编辑方法,采用显微注射的方式,将nanos2靶基因的gRNA和Cas9蛋白的mRNA混合物导入黄鳍鲷1细胞期的受精卵的动物极中,孵化后即可获得靶基因突变的F0代嵌合体黄鳍鲷雄鱼;
(S2)测交和基因突变个体的筛选:将步骤(S1)中获得的F0代嵌合体黄鳍鲷雄鱼与野生型雌鱼交配,从后代中筛选具有nanos2有义突变的F1代杂合子个体(nanos2+/-);
(S3)1龄雌性黄鳍鲷的生产和鉴定:将F1代杂合子个体(nanos2+/-)养殖到性成熟,其中一部分个体正常发育为杂合子雄鱼(nanos2+/-),一部分个体自然性逆转为F1代杂合子雌鱼(nanos2+/-),选取F1代杂合子雌鱼(nanos2+/-)和F1代杂合子雄鱼(nanos2+/-)交配,筛选得到F2代纯合子突变体个体(nanos2-/-),所述F2代纯合子突变体个体(nanos2-/-)1龄性成熟时,性反转为功能性雌鱼;
步骤(S1)中合成所述nanos2靶基因的gRNA的引物包括正向引物nanos2-gRNA-F和反向引物gRNA-R,所述正向引物nanos2-gRNA-F的序列如SEQ ID NO:1所示,所述反向引物gRNA-R的序列如SEQ ID NO:2所示。
2.根据权利要求1所述利用基因编辑生产1龄雌性黄鳍鲷的方法,其特征是:步骤(S1)中所述nanos2靶基因的gRNA和所述Cas9蛋白的终浓度分别为30~40 ng/µL和150~200 ng/µL,每颗受精卵中所述nanos2靶基因的gRNA和Cas9蛋白的mRNA混合物的注射量为1~2nL。
3.根据权利要求1所述利用基因编辑生产1龄雌性黄鳍鲷的方法,其特征是:步骤(S1)中采用显微注射的方式,将nanos2靶基因的gRNA和Cas9蛋白的mRNA混合物导入黄鳍鲷1细胞期的受精卵的动物极中,需在受精后40分钟内完成注射。
4.根据权利要求1所述利用基因编辑生产1龄雌性黄鳍鲷的方法,其特征是:步骤(S2)中将F0代嵌合体黄鳍鲷雄鱼与野生型雌鱼按照1:1~2的数量配比交配。
5.根据权利要求1所述利用基因编辑生产1龄雌性黄鳍鲷的方法,其特征是:步骤(S2)中用于检测具有nanos2有义突变的F1代杂合子个体(nanos2+/-)的PCR引物,包括正向引物nanos2-F1和反向引物nanos2-R1,其中所述正向引物nanos2-F1的序列如SEQ ID NO:3所示,所述反向引物nanos2-R1的序列如SEQ ID NO:4所示。
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