CN116622717B - 脊尾白虾雄性性别分化控制基因EcIAG及引导RNA与应用 - Google Patents
脊尾白虾雄性性别分化控制基因EcIAG及引导RNA与应用 Download PDFInfo
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Abstract
本发明属于虾蟹类基因工程技术领域,涉及脊尾白虾雄性性别分化控制基因EcIAG及引导RNA与应用。本发明所涉及的脊尾白虾IAG(EcIAG)基因序列为列表中SEQ ID No.1所示的核苷酸序列;基于该序列设计合成的引导RNA分别为序列列表中SEQ ID No.2和SEQ ID No.3所示的核糖核酸序列,将EcIAG‑gRNA1、EcIAG‑gRNA2与商品化Cas9蛋白共同混合后注射脊尾白虾的受精卵,可以特异性靶向EcIAG基因第二外显子的两个PAM识别位点,对两个PAM位点中间的DNA序列进行切割,实现EcIAG基因的敲除,在EcIAG基因敲除个体观察到脊尾白虾的性逆转。
Description
技术领域
本发明涉及虾蟹类基因工程技术领域,具体涉及一种用于虾蟹类性逆转的技术及其应用,具体为脊尾白虾雄性性别分化控制基因EcIAG及引导RNA与应用。
背景技术
甲壳动物存在性别二态性,即雌雄个体的生长速率之间存在差异,通过性别调控获得单性化群体,进行单性化养殖是提高产量安全环保的途径。
甲壳动物获得单性化群体的方法大致分为两类,一类是直接获得单性化群体;另一类是间接获得单性化群体。直接获得单性化群体不需要考虑物种的染色体性别决定类型,可以直接应用于所有的甲壳动物。现有的直接获得单性化群体一般有四种途径:(1)摘除雄性个体的促雄性腺(AG),或者将AG移植到雌性个体中。在雄性罗氏沼虾发育的早期,摘除双边促雄性腺后,可以观察到卵子发生和输卵管发育。(2)直接干扰胰岛素样促雄性腺素(IAG)基因,或者抑制IAG的分泌。自从IAG在雄性甲壳动物的性别分化中的关键作用被证明以后,利用IAG基因进行性别的调控就变成了重点关注问题。在罗氏沼虾中,证明干扰IAG基因可以获得可育的雌性;在小龙虾中,干扰IAG基因可以引起精巢退化。(3)使用激素进行刺激。在短沟对虾中,使用17β-雌二醇刺激无节幼体,会有71.88%的概率导致雌性化。(4)多倍体诱导。在中国明对虾的三倍体诱导中,会出现精子形态异常的状况,导致雌雄比例可以达到4:1。
间接获得单性化群体中的目的是获得没有受激素和化学物质影响的单性群体。一般可以分为两步,第一步将纯合子的性别进行反转,变成可育的相反性别的个体,然后与纯合子的个体进行交配,第二代产生均为纯合子性别的群体。在罗氏沼虾中,雄性的生长速率要高于雌性,在养殖过程中雄性单性化养殖可以带来更多的收益。罗氏沼虾的性别决定系统为ZW/ZZ,其中雄性为纯合子ZZ,在雄性个体发育的早期,使用微手术摘除促雄性腺,将ZZ基因型的雄性个体变成可育的假雌性,然后再与ZZ基因型的雄性个体进行交配,最终获得单一的雄性群体。通过双链RNA干扰IAG基因也可以得到可育的假雌性个体,其与野生雄性进行杂交,可获得全雄群体。
基因编辑是实现物种遗传改变的重要手段之一,目前主要使用的是CRISPR/Cas9基因编辑技术,利用该技术可以实现调控经济性状关键基因的敲入、敲除和基因型的改变,实现目标性状的改良。基因编辑育种不受遗传背景影响,靶向性高,性状改良速度快,能够实现新种质的分子设计,被认为是未来的核心育种技术。鱼类的基因编辑技术研究进展较快,基因编辑获得的美国的大马哈鱼和日本的虹鳟均已上市。在淡水鱼类如鲤鱼、鲫鱼、黄颡鱼以及海水鱼类的鲆鲽类中也相继建立了基因编辑技术,并获得了生长快、抗病、无肌间刺、性别逆转等表型的编辑个体。然而,在甲壳动物中,基因编辑技术仍存在基因导入效率低、胚胎存活率低、基因编辑效率低等瓶颈问题,应用进展缓慢,仅在脊尾白虾等极少数物种中有成功的报道,至今未见利用基因编辑技术实现甲壳动物性别逆转的报道。
脊尾白虾(Exopalaemon carinicauda),属于甲壳动物亚门十足目(Decapoda),是一种经济价值较高的海产虾类,同时也是十足目动物研究模型之一,具有环境适应性强、繁殖能力强、易于人工饲养、雌虾可连续产卵、胚胎大且透明等优势。脊尾白虾基因编辑研究可用于揭示十足目甲壳动物的发育、生长、代谢与繁殖等生命活动规律。
本发明通过基因编辑实验证实了该基因的突变可以引起脊尾白虾性别的逆转,是一种实现其性别控制的有效手段。另外,基于该基因在甲壳动物中的功能保守性,也可以采用此方法实现其它虾蟹类经济甲壳动物的性逆转。
发明内容
本发明描述了一种基于脊尾白虾雄性性别分化控制基因EcIAG的性逆转技术及其应用,本发明针对上述缺陷,现通过基因编辑实验证实了该基因的突变可以引起脊尾白虾性别的逆转,是一种实现其性别控制的有效手段。
本发明技术方案如下:
提取脊尾白虾总RNA,反转录获得cDNA,基于EcIAG基因核苷酸序列设计引物EcIAG-FL-F:TCTCTGTCTCTCTCAGCTTG和EcIAG-FL-R:ACTTGATCTGAAGGTCAGTCTT。以脊尾白虾cDNA做模版进行PCR扩增获得EcIAG的全长序列,其序列特征为:
SEQ ID No.1:
ATGTCAACCTGTAATATCGGAGTCAAGAAGGTGTTCTTTTGTGTTTTGTTGGCGTTATGCTTGATGCAGCCTTCTTCCGGCTACACCATTGAATGTCTTTCTGTTGACTTTGATTGTGGCGATATTCCGCAAACCCTTGCATCAGTTTGCAGGGTATACAAACCATTCGTCCCCTTCAATCAACCCTTCATTACCAAAAGGCGGCGTTCTGTGAGCAACTCCACCCAACTTCCAATTGAGTGTTCCTCGCCCTTCTTCCACCCACGAGCCACCCACCTGACGAAGGCAAAGGCTGACGAAGGCAGTGTCATGGCGTTGGAGATGCCAAGTGAGATTCGAGACATGTTTATAAGTCAGGAGAAAGCAAACATGATGCTTCAGTCGAATCGCAGACTCCGACGGCATGGGCAAAGAAATACGCCGAGGGATGAATGTTGTGCCGTCAAAGATTGCTGCACCTTTGAGGAAGTCGCCGAATATTGTGTTGAAGTGCGTCCCGGAGCCCTAACCTGCGAAAGACCACAGGACGGTTCCTCGCCTATGGTCCCCAATTGCACACCTGCCGTTCCGGAGAGCTAG。
使用在线工具CRISPRdirect(http://crispr.dbcls.jp/)进行EcIAG基因编辑靶向位点的选择。以引导RNA(gRNA)框架质粒为模板,分别以EcIAG-sgRNA1-F:TAATACGACTCACTATAGTCAACAGAAAGACATTCAAGTTTTAGAGCTAGAAATAGC和EcIAG-sgRNA2-F:TAATACGACTCACTATAGGTTGATTGAAGGGGACGAAGTTTTAGAGCTAGAAATAGC 为正向引物,以gRNA-R:AAAAAAGCACCGACTCGGTGCCA为反向引物,进行PCR扩增出gRNA模板。对gRNA的PCR产物进行体外转录,合成gRNA (EcIAG-gRNA1和EcIAG-gRNA2),其序列特征分别为:
SEQ ID No.2 (EcIAG-gRNA1):
GTCAACAGAAAGACATTCAAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTT
SEQ ID No.3 (EcIAG-gRNA2):
GGTTGATTGAAGGGGACGAAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTT。
所述的脊尾白虾雄性性别分化控制基因EcIAG基因的应用。基于脊尾白虾EcIAG基因序列获得脊尾白虾体外转录的gRNA,混合商品化的Cas 9蛋白后,将基于脊尾白虾雄性性别分化控制基因EcIAG序列设计并获得体外转录的所述引导RNA (EcIAG-gRNA1和EcIAG-gRNA2),混合Cas9蛋白,制备gRNA和Cas9蛋白混合后的终浓度均为1000 ng/μL的混合液,每个脊尾白虾受精卵注射0.5 nL,可以识别并切割EcIAG基因第二外显子中两个引导RNA识别位点之间的DNA序列,在编辑个体发育至成体时期观察到遗传性别为雄性的个体发育为雌性,实现脊尾白虾的性逆转,在发育至成体时观察到遗传雄性的个体性逆转为雌性,具有纳精囊、卵巢等雌性性征。
本发明的优点为确定了一种脊尾白虾雄性性别分化控制基因EcIAG及其在脊尾白虾性别控制过程中的作用,其次通过本发明可获得有效的调控脊尾白虾雄性性别分化基因EcIAG的引导RNA,可用于控制脊尾白虾的性别分化。
附图说明
图1为原始的脊尾白虾的EcIAG的全长cDNA序列和氨基酸序列。
图2为IAG多序列比对图。
图3为EcIAG突变体(Hetm/HomM)测序与野生型(WT)测序色谱图的比较图,其中图3中的(a)为EcIAG杂合突变体(Hetm)测序与野生型(WT)测序色谱图的比较图;图3中的(b)为EcIAG纯合突变体(HomM)测序与野生型(WT)测序色谱图的比较图。
图4为EcIAG纯和突变体与野生型氨基酸序列比对图。
图5为EcIAG 纯和突变体的性别表型图。
具体实施方式
下面结合实施例对本发明作进一步详细说明。
实施例
一种脊尾白虾雄性性别分化控制基因EcIAG及其编码基因,序列如下:
(1) SEQ ID No.1 的信息 (参见序列表)
(a)序列特征
*长度:579碱基对
*类型:核苷酸
*链型:双链
*拓扑结构:线性
(b)分子类型:核酸
序列描述:SEQ ID No.1
ATGTCAACCTGTAATATCGGAGTCAAGAAGGTGTTCTTTTGTGTTTTGTTGGCGTTATGCTTGATGCAGCCTTCTTCCGGCTACACCATTGAATGTCTTTCTGTTGACTTTGATTGTGGCGATATTCCGCAAACCCTTGCATCAGTTTGCAGGGTATACAAACCATTCGTCCCCTTCAATCAACCCTTCATTACCAAAAGGCGGCGTTCTGTGAGCAACTCCACCCAACTTCCAATTGAGTGTTCCTCGCCCTTCTTCCACCCACGAGCCACCCACCTGACGAAGGCAAAGGCTGACGAAGGCAGTGTCATGGCGTTGGAGATGCCAAGTGAGATTCGAGACATGTTTATAAGTCAGGAGAAAGCAAACATGATGCTTCAGTCGAATCGCAGACTCCGACGGCATGGGCAAAGAAATACGCCGAGGGATGAATGTTGTGCCGTCAAAGATTGCTGCACCTTTGAGGAAGTCGCCGAATATTGTGTTGAAGTGCGTCCCGGAGCCCTAACCTGCGAAAGACCACAGGACGGTTCCTCGCCTATGGTCCCCAATTGCACACCTGCCGTTCCGGAGAGCTAG。
(2) SEQ ID No.2的信息
(a)序列特征
*长度:57碱基
*类型:核苷酸
*链型:单链
*拓扑结构:线性
(b)分子类型:核糖核酸
序列描述:SEQ ID No.2
TAATACGACTCACTATAGTCAACAGAAAGACATTCAAGTTTTAGAGCTAGAAATAGC。
(3) SEQ ID No.3的信息
(a)序列特征
*长度:57碱基
*类型:核苷酸
*链型:单链
*拓扑结构:线性
(b)分子类型:核糖核酸
序列描述:SEQ ID No.3
TAATACGACTCACTATAGGTTGATTGAAGGGGACGAAGTTTTAGAGCTAGAAATAGC。
下面实施例中所使用的技术,包括RNA的提取、cDNA合成,PCR扩增与检测,以及gRNA合成等分子生物学技术,除非特别说明,均为本领域内技术人员已知的常规技术。所使用的仪器设备、试剂等,除非本说明书特别注明,均为本领域技术人员可通过公关途径或商业途径获得。
实施例1脊尾白虾雄性性别分化控制基因EcIAG的克隆与分析
(1)脊尾白虾总RNA提取
总RNA提取方法参照RNA提取试剂RNAiso Plus(Takara,货号9108)提供说明书。提取雄性脊尾白虾头胸部的总RNA,使用NanoDrop 2000分光光度计(Thermo)测定RNA浓度为4.2 μg/μl,然后进行琼脂糖凝胶电泳检验,显示其未降解,-80℃保存备用。
(2)cDNA反转录
对RNA样品的DNase I消化提取与反转录合成第一链cDNA参见RevertAidFirstStrand cDNA Synthesis Kit(Thermo,K1622,https://www.thermofisher.cn/order/catalog/product/K1621)。
(3)EcIAG基因全长cDNA克隆
根据EcIAG基因核酸序列设计引物EcIAG-FL-F/R验证预测的EcIAG基因全长cDNA序列,利用EcIAG-FL-F/R进行PCR扩增,PCR体系参照Ex Taq酶(Takara,DRR001A)使用说明书。PCR反应程序:94 ℃预变性5 min;94 ℃变性10 s,56 ℃退火30 s,72 ℃复性延伸45s,38个循环;72℃延伸10 min。扩增完毕后,琼脂糖凝胶电泳鉴定后,利用DNA凝胶回收试剂盒(AxyGen,AP-GX-50)纯化回收目的片段,即为PCR产物。
引物序列如下:
EcIAG-FL-F:TCTCTGTCTCTCTCAGCTTG
EcIAG-FL-R:ACTTGATCTGAAGGTCAGTCTT
(4)PCR产物的克隆:将PCR产物连接到pMD19-T载体上(TaKaRa,6013),转化大肠杆菌DH5α感受态细胞(TransGen Biotech,CD201-01)中,37 ℃摇床培养过夜、涂板、挑取单克隆菌落、菌落PCR扩增、筛选阳性克隆送上海生工生物工程公司进行测序,详细步骤如下:
a)连接
连接体系如下:
16℃连接4 h。
b)准备平板
LB液体培养基配方:
培养基灭菌后,在超净台中向培养基中添加0.1%(体积比)氨苄抗生素。
c)转化感受态细胞
将连接产物加入100 μL感受态细胞中,混匀,冰浴30 min。42 ℃水浴热激30秒,置于冰上2分钟。加250μL LB液体培养基,200 rpm震荡培养,37℃培养1小时。
d)涂平板,每个直径9cm的平板加入40 μL 浓度为20mg/mL的5-溴-4-氯-3-吲哚基-β-D-半乳糖苷(x-gal)和7 μL浓度为200mg/mL的异丙基硫代半乳糖苷(IPTG),涂匀,置于37℃培养箱中,直至使用。取200 μL菌液涂板,于37℃培养箱培养过夜。
e)阳性克隆检测
放入4℃冰箱显色1h,超净台中挑选白色单克隆至10 μL无菌水中,涡旋混合。取1μL混合液加入9 μL的 PCR体系:5 μL Premix Taq™ (Takara, RR901A), M13引物M13F/R各0.5 μL,4 μL DEPC-H2O。PCR反应程序:95℃预变性5 min;95℃变性30 s,55℃退火30 s,72℃复性延伸1 min,35个循环;72℃延伸10 min。通过琼脂糖凝胶电泳检测,选取条带700bp左右的单克隆,加入到含质量浓度0.1%氨苄抗生素的1.5 ml LB培养基中,37℃培养6 h后,菌液送上海生工生物工程公司进行测序。
M13引物序列如下:
M13F:AGGGTTTTCCCAGTCACG
M13R:GAGCGGATAACAATTTCACAC。
(5)序列分析:利用NCBI(https://www.ncbi.nlm.nih.gov/)将测序所得的EcIAG基因核苷酸序列与转录组测序所得的核苷酸序列比对验证其正确性,结果显示,测序结果一致。利用ExPASy(http://web.expasy.org/translate/)预测并分析该基因的蛋白序列,结果显示,EcIAG基因开放阅读框架全长579 bp,编码192个氨基酸残基(图1),预测分子质量为21.5 KD,理论等电点为7.43。本发明进一步将脊尾白虾EcIAG基因编码序列与贪食沼虾、日本沼虾、罗氏沼虾、Macrobrachium vollenhovenii、太平洋长臂、条纹长臂虾、宽脚长额虾等节肢动物同源基因的氨基酸序列进行比对,确认EcIAG具有IAG同源蛋白中保守的6个半胱氨酸残基(图2)。
实施例2脊尾白虾雄性性别分化控制基因EcIAG的gRNA合成及基因编辑实验
(1)脊尾白虾雄性性别分化控制基因EcIAG的引导RNA(gRNA)合成
a)gRNA框架质粒构建
首先根据gRNA的结构,设计合成gRNA框架序列:
GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTT。
然后将框架序列连入pMD19-T载体(TaKaRa,D102A)的TA克隆位点,获得gRNA框架质粒pMD19-gRNA。
b)gRNA模板的合成
使用在线工具CRISPRdirect(http://crispr.dbcls.jp/)选择EcIAG基因的gRNA位点,设计对应的EcIAG-gRNA1-F和EcIAG-gRNA2-F,同时设计反向引物的gRNA-R。
EcIAG-gRNA1-F:
TAATACGACTCACTATAGTCAACAGAAAGACATTCAAGTTTTAGAGCTAGAAATAGC
EcIAG-gRNA2-F:
TAATACGACTCACTATAGGTTGATTGAAGGGGACGAAGTTTTAGAGCTAGAAATAGC
gRNA-R:AAAAAAGCACCGACTCGGTGCCA。
以gRNA框架质粒pMD19-gRNA为模板,分别以EcIAG-gRNA1-F和EcIAG-gRNA2-F为正向引物,以gRNA-R反向引物,采用ExTaq聚合酶(Takara,RR001A)进行PCR扩增。
PCR体系如下:
PCR循环程序:98℃预变性5 min;98℃10 sec,55℃5 sec,72℃30 sec,35个循环;72℃延伸10 min;4℃保温。PCR产物用琼脂糖凝胶电泳检测扩增gRNA模板大小后,PCR产物测序。剩余PCR产物用作gRNA体外转录模板。
c)gRNA的体外转录与纯化
使用T7体外转录试剂盒TranscriptAid T7High Yield Transcription Kit(Thermo,K0441)对gRNA的PCR产物进行体外转录,合成gRNA。
体外转录体系如下:
转录体系在42℃反应2 h;加入2 μL DNA酶 (QIAGEN,79254)于37℃消化15 min,除去DNA模板;加入2 µL 0.5M EDTA(pH8.0),于65℃反应10 min,终止反应;取1μL用于电泳检测。使用酚/氯仿抽提法(Lee S. Toni, et al., Optimization of phenol-chloroformRNA extraction, MethodsX, 2018, 5: 599-608)从消化后的体系获取gRNA EcIAG-gRNA1和EcIAG-gRNA2。
(2)脊尾白虾EcIAG的基因编辑实验
a)将性腺发育良好的雌雄个体分别15尾混合饲养。当雌虾产卵后,收集刚产的受精卵。
b)将EcIAG-gRNA1和EcIAG-gRNA2与商品化Cas9蛋白(金斯瑞,Z03469, 南京金斯瑞公司生产(金斯瑞生物科技股份有限公司))混合,使EcIAG-gRNA1、EcIAG-gRNA2和Cas9蛋白的终浓度均为1000 ng/μL,每个受精卵注射量约为0.5 nL。
c)注射后的胚胎置于盛无菌海水的培养皿中,在摇床上室温孵育,每天更换无菌海水三次。
d)在受精卵孵育5天后,随机收集185个发育至无节幼体的胚胎,检测基因编辑效率。利用裂解液微萃取的方法(Gao et al., CRISPR/Cas9-mediated mutation on aninsulin-like peptide encoding gene affects the growth of the ridgetail whiteprawnExopalaemon carinicauda, Front Endocrinol, 2022, 13:986491.)对随机挑选的胚胎的基因组DNA进行提取,对EcIAG的两个sgRNA的CRISPR靶点侧翼的基因组区域进行PCR扩增。
所用的检测引物如下:
EcIAG-exon2-F:TAGCTGTAAGTGCAGGTAGAA
EcIAG-exon2-R:CTGCATCACTAACTTTTGGTAAT。
PCR扩增体系如下:
PCR扩增条件如下:
94℃预变性5 min;98℃变性10 sec,57℃退火30 sec,72℃延伸25 s,35个循环;72℃延伸10 min;4℃保温。
利用Sanger测序方法对扩增靶序列得到的产物进行测序,随后用MEGA 6.0软件进行序列比对,用Chromas软件查看测序图谱,根据谱图结果计算编辑效率,约20%的胚胎的EcIAG基因序列中引导RNA识别位置发生了遗传突变。
e)按d)中方法对存活至仔虾的个体(大约30个)进行检测,发现8个个体的EcIAG基因发生了突变,其中3个个体发生了纯和突变,5个个体发生了杂合突变。杂合突变体(HetM) 仅有一条染色体发生突变,其色谱图显示在原间隔相邻基序 (PAM) 位点后出现杂峰,而野生型 (WT) 则没有如图3中的(a)所示。纯合突变体 (HomM) 的两条染色单体均发生了突变,其测序色谱图中原间隔相邻基序(PAM)位点后没有出现多个峰,但序列长度较野生型 (WT) 明显缩短如图3中的(b)所示,在其氨基酸序列中引入大片段缺失突变或翻译提前终止(图4),三个突变体中的HomM1和HomM3突变体氨基酸序列大片段缺失,HomM2突变体氨基酸翻译提前终止。
根据以上获得的突变体的测序结果和据其推导的氨基酸序列比对结果得知,专利中针对EcIAG基因设计的引导RNA (EcIAG-gRNA1和EcIAG-gRNA2)准确的识别了它们的DNA靶序列,造成脊尾白虾的一条染色体或两条染色体上的EcIAG基因核苷酸序列的大片段缺失,进而引起氨基酸序列的大片段缺失或翻译的提前终止,获得了敲除EcIAG的杂合突变体和纯和突变体。
f)在8尾突变体发育至成体后,分别对其表型性别和遗传性别鉴定。其表型性别通过观察是否具有雄性附肢及第五对步足基部是否有突起进行鉴定,具备该特征为雄性,不具备该特征雌性。其遗传性别利用遗传性别探针(于洋,李富花,方小晨,李诗豪,张晓军.一种脊尾白虾遗传性别鉴定的 DNA 标记及其应用.申请号:202310392890.4.申请日:2023.04.13)进行鉴定,所用引物为:
Sex-F:TCACACAGAATGGATGCAACT
Sex-R:TCCATGGGTTTGATCAATCCCT。
遗传雌性的性别遗传标记基因型为CT,遗传雄性为TT。将它们的性别表型和遗传性别进行比较后发现:3尾纯和突变的脊尾白虾的遗传性别全部为雄性,但是其性别表型为雌性,即发生了性别反转(图5);5尾杂合突变体的性别表型与其遗传性别一致,未发生性逆转。
以上结果证明了在脊尾白虾中基于雄性性别分化控制基因EcIAG建立了基于CRISPR/Cas9基因编辑的性逆转技术,实现了遗传性别为雄性的脊尾白虾的性别表型表现为雌性,同时该技术可用于实现其他虾蟹类甲壳动物的性逆转。
Claims (6)
1.一种脊尾白虾雄性性别分化控制基因EcIAG,其特征在于:脊尾白虾雄性性别分化控制基因EcIAG为序列列表中SEQ ID No.1所示的核苷酸序列。
2. 一种权利要求1所述的脊尾白虾雄性性别分化控制基因EcIAG的引导RNA,其特征在于:脊尾白虾雄性性别分化控制基因EcIAG的引导RNA为EcIAG-gRNA1和EcIAG-gRNA2,EcIAG-gRNA1和EcIAG-gRNA2分别为序列列表中SEQ ID No.2和SEQ ID No.3所示的核糖核苷酸序列。
3.一种权利要求2所述脊尾白虾雄性性别分化控制基因EcIAG的引导RNA的获取方法,具体过程如下:
以引导RNA框架质粒pMD19-gRNA为模板,分别以EcIAG-sgRNA1-F:TAATACGACTCACTATAGTCAACAGAAAGACATTCAAGTTTTAGAGCTAGAAATAGC和EcIAG-sgRNA2-F:TAATACGACTCACTATAGGTTGATTGAAGGGGACGAAGTTTTAGAGCTAGAAATAGC 为正向引物,以gRNA-R:AAAAAAGCACCGACTCGGTGCCA为反向引物,进行PCR扩增gRNA模板,对引导RNA的PCR模板进行体外转录,分别合成得到引导RNA EcIAG-sgRNA1和EcIAG-sgRNA2;
gRNA框架质粒构建过程为:
gRNA框架序列为:
GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTT,
将框架序列连入pMD19-T载体(TaKaRa,D102A)的TA克隆位点,即获得gRNA框架质粒pMD19-gRNA。
4.一种权利要求1所述的脊尾白虾雄性性别分化控制基因EcIAG的应用,其特征在于:脊尾白虾雄性性别分化控制基因EcIAG可应用于脊尾白虾性别控制遗传操作的靶基因。
5.按照权利要求4所述的应用,其特征在于:
将基于权利要求1所述脊尾白虾雄性性别分化控制基因EcIAG序列设计并获得体外转录的权利要求2所述引导RNA (EcIAG-gRNA1和EcIAG-gRNA2),混合Cas9蛋白,注射脊尾白虾受精卵后可以识别并切割EcIAG基因第二外显子中两个引导RNA识别位点之间的DNA序列,在编辑个体发育至成体时期观察到遗传性别为雄性的个体发育为雌性,实现脊尾白虾的性逆转。
6.按照权利要求4或5所述的应用,其特征在于:
脊尾白虾雄性性别分化控制基因EcIAG还可以用于实现对虾、螯虾、沼虾或蟹类甲壳动物雄性个体的性逆转。
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