CN117247971A - 家蚕非受体型酪氨酸磷酸酶13在高丝量品种选育中的应用 - Google Patents
家蚕非受体型酪氨酸磷酸酶13在高丝量品种选育中的应用 Download PDFInfo
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Abstract
本发明属于生物技术领域,具体涉及家蚕非受体型酪氨酸磷酸酶13在高丝量品种选育中的应用。本发明公开了BmPTPN13基因是一个提高雄蚕茧丝产量的基因靶标;本发明在家蚕中敲除BmPTPN13基因,会提高雄蚕的茧层率,增加茧丝产量。因此BmPTPN13敲除可以用于创制家蚕高丝产量突变体,应用于家蚕高丝产量新品种的选育。
Description
技术领域
本发明属于生物技术领域,具体涉及家蚕非受体型酪氨酸磷酸酶13在高丝量品种选育中的应用。
背景技术
家蚕的茧层率是蚕业生产中的重要经济性状,茧层率越高,丝量越多,因此茧层率决定了该行业的产量和经济效益。因此选育高茧层率的家蚕实用品种一直是蚕业研究的重要内容。传统家蚕杂交育种技术茧层率的杂种优势率在-3.82%~5.63%之间,收效甚微。
与传统杂交育种方式相比基因编辑技术的出现极大改善了这一现状,该技术可以更高效、更便捷地从分子水平通过靶向某个基因从而调控家蚕丝腺的生长发育过程,进而改善家蚕的蚕丝经济性状。
非受体型酪氨酸磷酸酶13(PTPN13),可以通过调控不同的下游因子的磷酸化水平来调控细胞的增殖分化和周期进展。
蚕丝由中部丝腺分泌的丝胶和后部丝腺分泌的丝素组成,其中丝胶占蚕丝量的20%~30%,因此后部丝腺的大小直接决定着蚕丝的产量。增加丝腺大小或者促进相关蛋白质合成可有效的提高家蚕茧层率。
发明内容
有鉴于此,本发明对家蚕非受体型酪氨酸磷酸酶13(BmPTPN13)全长序列进行了克隆和鉴定,并提供了一种敲除该基因获得雄蚕丝量提高的方法。
本发明的目的一:提供家蚕BmPTPN13基因或BmPTPN13蛋白在高丝量家蚕品种选育中的应用,所述家蚕为雄蚕。
具体的,在家蚕育种时抑制SEQ ID NO:1所示家蚕BmPTPN13基因和/或SEQ ID NO:2所示家蚕BmPTPN13蛋白的表达以培育高茧层率家蚕,进而得到家蚕高丝量品种。
本发明的目的二:提供利用基因编辑技术敲除BmPTPN13基因提高雄蚕丝量的方法。
通过靶向BmPTPN13基因,抑制其表达,或者破坏BmPTPN13基因结构,可以提高中部丝腺及后部丝腺的大小,显著提高茧丝的丝素相对含量,提高全茧量和茧层率,可用于在蚕业生产中培育蚕丝生产所需要的丝素含量高、丝胶少、出丝率高的家蚕材料和品种。具体可利用ZFNs、TALENs、CRISPR/Cas9或其变体介导靶标BmPTPN13基因的敲除。
本发明的目的三:提供一种创制家蚕高丝量突变体的方法,具体是利用基因编辑技术敲除雄蚕中SEQ ID NO.1所示BmPTPN13基因和/或抑制SEQ ID NO.2所示BmPTPN13蛋白的表达。
本发明目的四:一种利用CRISPR-Cas9基因编辑技术敲除BmPTPN13基因创制家蚕高丝量突变体的方法在选育高丝量家蚕新品种中的应用。
为达到上述目的,本发明提供如下技术方案:
通过在家蚕中敲除BmPTPN13基因,筛选,获得敲除BmPTPN13基因的家蚕突变个体,突变的雄蚕的茧层率提高,即获得了高丝量家蚕。
(1)所述敲除BmPTPN13基因表达的方法是通过CRISPR-Cas9介导敲除家蚕BmPTPN13基因。CRISPR-Cas9介导敲除家蚕BmPTPN13基因由Cas9表达载体与靶标BmPTPN13基因的sgRNA的双元转基因系统介导。
所述sgRNA的位点序列如SEQ ID NO:3所示。所述sgRNA表达载体由以下方法制备:将SEQ ID NO:4和SEQ ID NO:5所示核苷酸序列退火形成双链连入sgRNA表达载体。
(2)构建Cas9表达载体,将Cas9表达载体转染进家蚕,制备Cas9转基因家蚕;然后将构建的靶标BmPTPN13基因的sgRNA表达载体,进行家蚕蚕卵的显微注射,制备sgRNA转基因家蚕;
将sgRNA转基因家蚕和Cas9转基因家蚕进行杂交、筛选,PCR检测,筛选到基因编辑技术敲除BmPTPN13基因的家蚕突变体。
本发明的有益效果在于:
本发明公开了BmPTPN13基因是一个提高雄蚕茧层率的基因靶标;本发明在家蚕中敲除BmPTPN13基因,会提高雄蚕的茧层率和茧丝产量,因此BmPTPN13基因敲除可以用于创制高丝量突变体,应用于家蚕高丝量新品种的选育。
本发明的其他优点、目标和特征在某种程度上将在随后的说明书中进行阐述,并且在某种程度上,基于对下文的考察研究对本领域技术人员而言将是显而易见的,或者可以从本发明的实践中得到教导。本发明的目标和其他优点可以通过下面的说明书来实现和获得。
附图说明
为了使本发明的目的、技术方案和优点更加清楚,下面将结合附图对本发明作优选的详细描述,其中:
图1为构建成功的sgRNA表达载体骨架图。
图2为双元转基因阳性个体图片(A1、A4为Cas9的荧光;A2、A3为gRNA的荧光)。
图3为CRISPR-Cas9基因编辑敲除BmPTPN13基因的家蚕突变位点检测图以及家蚕敲除突变体丝腺中BmPTPN13蛋白的表达。
图4为基因编辑技术敲除BmPTPN13基因的家蚕表型观察图;(L5D4:五龄第六天;WT:野生型家蚕;BmPTPN13KO:敲除BmPTPN13家蚕)。
具体实施方式
以下通过特定的具体实例说明本发明的实施方式,本领域技术人员可由本说明书所揭露的内容轻易地了解本发明的其他优点与功效。本发明还可以通过另外不同的具体实施方式加以实施或应用,本说明书中的各项细节也可以基于不同观点与应用,在没有背离本发明的精神下进行各种修饰或改变。
实施例1sgRNA稳定转基因系的获得
从NCBI获得家蚕基因BmPTPN13的CDS全长序列(SEQ ID NO:1),并在CCTop网站设计其sgRNA位点,获得的sgRNA位点序列:5’-GGTCGCCGTGCCTGTCTCCAAGG-3’(AGG为PAM基序)(SEQ ID NO:3)。
合成序列F:5’-AAGTGGTCGCCGTGCCTGTCTCCA -3’(SEQ ID NO:4)和R:5’-AAACTGGAGACAGGCACGGCGACC-3’(SEQ ID NO:5),将SEQ ID NO:4和SEQ ID NO:5所示核苷酸序列退火后形成双链gRNA,用限制性内切酶AarI酶切piggyBac[3×
P3-EGFP-SV40-TTTTTT-U6]基础载体,将退火后的双链gRNA连接到酶切后的piggyBac[3×P3-EGFP-SV40-TTTTTT-U6]基础载体上(基础载体由西南大学前沿交叉科学研究院生物学研究中心提供,Yuanyuan Liu.Tissue-specific genome editing oflaminA/C in the posterior silk glandsof Bombyx mori.2017),构建piggyBac[3×P3-EGFP-SV40-TTTTTT-gRNA-U6]敲除载体,图1为敲除载体结构图。
实施例2CRISPR-Cas9编辑技术敲除BmPTPN13家蚕突变体的获得
将piggyBac-[3×P3-EGFP-SV40-TTTTTT-gRNA-U6]敲除载体质粒和辅助helper质粒按照1:1的摩尔比进行混合,注射进刚产下2h内的非滞育蚕卵中;待蚁蚕孵化后,常规桑叶饲养G0代家蚕。化蛾后进行同圈交配,得到G1代个体;常规催青G1代蚕卵。用桑叶饲养至三龄后,将幼虫置于荧光显微镜下观筛选眼部发射绿色荧光的幼虫,即为BmPTPN13-sgRNA阳性G1代转基因家蚕个体(图2A2);常规饲养至化蛾后与眼部发红光的后部丝腺Cas9成虫(图2A1)交配产卵制种F1代。后部丝腺Cas9转基因成虫个体制备过程如下:克隆FibH启动子并连接到pUC57-Cas9中,然后进行AscI单酶切后插入到piggyBac[3×P3-DsRed]载体主干中,构建piggyBac[3×P3-RFP,FibH-Cas9](FibH-Cas9)质粒,注射蚕卵后得到Cas9转基因个体(后部丝腺Cas9成虫由西南大学前沿交叉科学研究院生物学研究中心提供,pUC57-Cas9载体参见Ma S,CRISPR/Cas9 mediated multiplex genome editing and heritablemutagenesis of BmKu70 in Bombyx mori.Sci Rep.2014.;piggyBac[3×P3-DsRed]载体参见Tomita M,Transgenic silkworms produce recombinant human type IIIprocollagen in cocoons.Nat Biotechnol.2003.)。蚕卵出蚁后,桑叶饲养转基因家蚕个体至三龄后将幼虫置于荧光显微镜下筛选到眼睛同时发射绿色荧光和红色荧光的幼虫即为F1代BmPTPN13基因敲除阳性个体(图2A3、A4)。化蛾后进一步通过荧光筛选确认。
分别通过分子水平对CRISPR-Cas9编辑技术敲除BmPTPN13基因家蚕进行突变位点检测以及蛋白水平对BmPTPN13的表达量进行检测。具体的,选取了五龄第六天的敲除BmPTPN13基因家蚕雄蚕丝腺材料提取基因组和蛋白。
将提取的蛋白与5×上样缓冲液混合均匀后在95℃变性10分钟,上样于12.5%SDS-聚丙稀酰胺胶并进行Western blot分析。所用的抗体制备过程如下:首先依据BmPTPN13氨基酸序列设计合成一条多肽CRKLTKDNNHYEQPI(SEQ ID NO:8),然后分三次免疫新西兰大白兔之后收集抗体血清后进行抗体纯化和鉴定,然后得到BmPTPN13多克隆抗体。检测结果表明,与对照相比,敲除BmPTPN13基因家蚕中BmPTPN13蛋白表达量明显低于野生型(图3)。
将提取的基因组作为PCR扩增反应的模板,以BmPTPN13基因的CDS序列为参照设计了一对检测引物F:5’-GCGCTTCGGAAACATTACATTTTGTGGG-3’(SEQ ID NO:6)和R:5’-ACGCGTGTTGAAACTTATGTTGCCTTGT-3’(SEQ ID NO:7),采用该对引物对敲除BmPTPN13基因的家蚕雄蚕丝腺的基因组进行PCR扩增,将扩增产物回收后进行T克隆然后挑斑测序,测序结果显示BmPTPN13基因敲除阳性个体产生敲入、缺失、突变三种敲除形式(图3)。值得注意的是,本实施例中的检测引物必须特异,无非特异性扩增现象出现,其PCR产物可直接用于测序分析。
实施例3CRISPR-Cas9敲除BmPTPN13家蚕突变体的的表型观察
将筛选获得的敲除BmPTPN13基因的家蚕突变体与野生型家蚕在相同条件饲养,发现敲除BmPTPN13基因的家蚕的大蚕期体型(四龄和五龄)和雄蚕茧型和茧壳重均大于野生型,解剖了五龄六天的雄蚕发现敲除BmPTPN13基因的家蚕幼虫的丝腺明显大于野生型(图4)。这说明敲除BmPTPN13家蚕丝腺中BmPTPN13表达的缺失会导致雄蚕的茧层率上升。
本发明实施例中以Cas9介导靶标BmPTPN13基因敲除,本领域技术人员公知,只要能够敲除BmPTPN13基因的任何手段均可实现本发明的发明目的,如:ZFNs、TALENs和CRISPR/Cas9或其变体介导靶标BmPTPN13基因的敲除。
最后说明的是,以上实施例仅用以说明本发明的技术方案而非限制,尽管参照较佳实施例对本发明进行了详细说明,本领域的普通技术人员应当理解,可以对本发明的技术方案进行修改或者等同替换,而不脱离本技术方案的宗旨和范围,其均应涵盖在本发明的权利要求范围当中。
Claims (10)
1.家蚕BmPTPN13基因或BmPTPN13蛋白在高丝量家蚕品种选育中的应用,其特征在于,通过抑制SEQ ID NO:1所示家蚕BmPTPN13基因和/或SEQ ID NO:2所示家蚕BmPTPN13蛋白的表达以培育高丝量家蚕品种,所述家蚕为雄蚕。
2.根据权利要求1所述的应用,其特征在于,利用基因编辑技术敲除BmPTPN13基因。
3.根据权利要求2所述的应用,其特征在于,利用ZFNs、TALENs、CRISPR/Cas9或其变体介导敲除BmPTPN13基因。
4.一种创制家蚕高丝量突变体的方法,其特征在于,敲除家蚕中SEQ ID NO:1所示BmPTPN13基因和/或抑制SEQ ID NO:2所示BmPTPN13蛋白的表达,所述家蚕为雄蚕。
5.根据权利要求4所述的方法,其特征在于,利用基因编辑技术敲除BmPTPN13基因。
6.根据权利要求5所述的方法,其特征在于,利用ZFNs、TALENs、CRISPR/Cas9或其变体介导敲除BmPTPN13基因。
7.根据权利要求6所述的方法,其特征在于,采用CRISPR/Cas9敲除BmPTPN13基因包括以下步骤:
步骤1:构建sgRNA表达载体,所述sgRNA表达载体中含有靶向家蚕BmPTPN13基因的gRNA,所述gRNA由SEQ ID NO:4和SEQ ID NO:5所示的引物制备得到;将sgRNA表达载体转染进家蚕,筛选得到sgRNA转基因家蚕;
步骤2:构建Cas9表达载体,将Cas9表达载体转染进家蚕,筛选得到Cas9转基因家蚕;
步骤3:将sgRNA转基因家蚕和Cas9转基因家蚕进行杂交、筛选,得到敲除BmPTPN13基因的家蚕,即为家蚕高丝量突变体。
8.权利要求4-7任一项所述的方法在选育高丝量家蚕新品种中的应用。
9.家蚕BmPTPN13基因或BmPTPN13蛋白在增大家蚕茧型、茧壳重和丝腺中的应用,其特征在于,通过抑制SEQ ID NO:1所示家蚕BmPTPN13基因和/或SEQ ID NO:2所示家蚕BmPTPN13蛋白的表达以增大家蚕茧型、茧壳重和丝腺,所述家蚕为雄蚕。
10.家蚕BmPTPN13基因或BmPTPN13蛋白在提高家蚕茧层率中的应用,其特征在于,通过抑制SEQ ID NO:1所示家蚕BmPTPN13基因和/或SEQ ID NO:2所示家蚕BmPTPN13蛋白的表达以提高家蚕茧层率,所述家蚕为雄蚕。
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