CN116376919A - 干扰许氏平由性腺正常发育的基因片段、重组菌及其应用 - Google Patents
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Abstract
本发明涉及一种干扰许氏平由性腺正常发育的基因片段、重组菌及其应用，属于分子生物学领域，所述干扰片段的核苷酸序列如SEQ ID NO.1所示。本发明还提供包含所述基因片段的重组菌，将所述重组菌投喂处于原始生殖细胞分化期许氏平由仔鱼能够敲降原始生殖细胞发育关键基因dnd的表达，诱导产生性腺发育不良的个体，为许氏平由不育受体的获取提供了一种方法。
Description
技术领域
本发明属于分子生物学领域，具体涉及一种干扰许氏平由性腺正常发育的基因片段、重组菌及其应用。
背景技术
许氏平由（Sebastes schlegelii）隶属于鲉形目，鲉科，平鲉属，又称黑鲪，俗称黑鱼、黑寨、黑老婆等，是分布于太平洋西北部的一种近海底层鱼类。因其具有肉质细嫩，味道鲜美，能在北方自然越冬等优点，已成为我国北方重要的海水养殖经济鱼种。许氏平由生长性状呈现明显的性别二态性，同龄雌性个体比雄性个体大约25%。因此，开展许氏平由全雌群体养殖，可以提高养殖产量，具有良好的产业前景，将显著提升许氏平由的水产养殖效益。许氏平由为XY性别决定，通过过表达amhy产生XX伪雄鱼是诱导全雌群体的重要手段。然而，伪雄鱼存在性腺发育不良，精子存活率低等问题，制约着全雌群体的创建。生殖干细胞移植技术通过选择雌性个体，将其卵原细胞分离，并移植到雄性不育受体中，雄性受体可于交尾时产生全部为X型精子，待其与野生雌鱼交配后，可产生全雌群体。然而，生殖干细胞移植技术存在其技术瓶颈：由于可育受体含有自身的生殖细胞，当供体的生殖细胞成功嵌合到受体性腺时，受体的内源生殖细胞会同供体生殖细胞相互竞争发育所需的微环境，从而导致受体产生供体配子的比例很低。而不育受体可以实现仅产生供体来源的配子这一要求。因此，构建不育受体对于生殖干细胞移植技术的成功至关重要。
目前获取不育受体的途径包括人工诱导三倍体、种间杂交或使用激素处理导致性腺发育受阻等三种。然而前两种方法的高时间成本和经济成本，以及激素处理对环境的威胁，很大程度上限制了其在养殖过程中的应用。
发明内容
本发明的目的在于提供一种干扰许氏平由性腺正常发育的基因片段、重组菌及其应用，所述重组菌能够在原始生殖细胞分化期敲降许氏平由关键基因dnd的表达，能够诱导产生性腺发育受阻的个体，为许氏平由不育受体的获取提供了一种新型手段，同时也为许氏平由的原始生殖细胞移植提供了新的方法。
本发明通过以下方案来实现上述目的：一种干扰许氏平由性腺正常发育的基因片段，所述基因片段的核苷酸序列如SEQ ID NO.1所示。
一种干扰许氏平由性腺正常发育的重组菌，所述重组菌包括如SEQ ID NO.1所示基因片段、L4440线虫RNAi干扰载体和HT115菌株。
所述基因片段的核苷酸序列为：
gatgatggagaacaagcagagccaggtgctgaatgttgagcgggtgaaggcactggaaacctggctgaaagccaccaatacaaatctgaaacaagtaaacggccagaggaagtatggaggaccacctgaggtgtgggacgggccgtccccaggagcccgctgtgaggtcttcatcagccagatccctcgggacacctacgaggatctgctgattcccctcttcagctccgtggggcccctgtgggagttccggctcatgatgaatttcagcggccagaaccgcggcttcgcctacggcaaatacggctcgccggccgtagctactgaagccatccgcctgctgcacggtcacatgctggagcccggcttctgcctcagtgtccgccgcagcacggagaagagacacctctgtatcggaaacct。
本发明还提供所述重组菌的制备方法，将所述基因片段的核苷酸序列SEQ IDNO.1构建到L4440线虫RNAi干扰载体上，然后导入大肠杆菌HT115菌株，获得所述重组菌。
本发明还提供所述重组菌在干扰许氏平由性腺发育中的应用，所述应用方法如下：将所述重组菌培养扩繁至OD值 0.4，诱导后，按照1mL重悬培养基/200mL初始菌量的比例重悬菌体，按照1mL/200条鱼/天的比例吸取重悬后的诱导菌，混入饵料，对处于原始生殖细胞分化期许氏平由仔鱼进行投喂，投喂周期为90天。
进一步，投喂时间为从许氏平由幼体生后5天开始，至出生后95天结束。
进一步，所述的饵料依次为轮虫、卤虫和颗粒饲料。
本发明与现有技术相比的有益效果：（1）本发明建立了一种全新的获取不育受体的技术，相比于传统的人工诱导产生三倍体、种间杂交等技术，具有省时省力、经济高效的特点。（2）本发明所述干扰片段能够抑制原始生殖细胞标记基因的表达，获得性腺发育不良的许氏平由苗种，本发明方法相对于激素诱导的性腺不育方式，特异性和目的性更强，避免了激素使用带来的环境污染隐患。
附图说明
图1 为dnd敲降组及对照组许氏平由的转录组数据分析情况图；
图2 为敲降组及对照组许氏平由的性腺外观图：a、b为对照组许氏平由精巢外观图，c、d为dnd基因敲降组许氏平由精巢外观图；e、f为对照组许氏平由卵巢外观图； g、h为dnd基因敲降组许氏平由卵巢外观图；
图3 为dnd敲降组及对照组许氏平由的性腺组织切片HE染色图：a、b为敲降组许氏平由精巢形态图，c、d为对照组许氏平由精巢形态图；e、f为敲降组许氏平由卵巢形态图，g、h为对照组许氏平由卵巢形态图；
图4 为dnd敲降组及对照组许氏平由孵化后270天的生长情况统计。
具体实施方式
下面通过实施例来对本发明的技术方案做进一步解释，但本发明的保护范围不受实施例任何形式上的限制。
实施例1：本实施例的实施对象是原始生殖细胞迁移、分化期的许氏平由仔鱼，在出生后120天解剖获取性腺进行形态学观察，并利用转录组测序进一步验证dnd的表达情况。
（1）许氏平由dnd基因干扰片段的选择：利用siRNA预测网站http://sidirect2.rnai.jp/，输入dnd基因ORF序列，选择目的区域，本实施例所选区域序列如SEQID NO.1所示，该区域所预测siRNA如表1所示；
表1. dnd基因干扰片段预测siRNA
（2）许氏平由dnd基因L4440-dnd干扰载体的构建：根据选取片段的序列特征，选取限制性内切酶Xba I和Hind III形成dnd基因干扰片段与L4440干扰载体的连接切口。随后使用T4连接酶将干扰片段构建到L4440线虫RNAi干扰载体上。克隆所用引物如表2所示，正向引物中6-11为Xba I内切酶切割序列TCTAGA，反向引物中6-11为Hind III内切酶切割序列AAGCTT；
表2 dnd基因干扰片段克隆引物
3）L4440-dnd干扰载体阳性HT115菌株的筛选：将构建好的L4440-dnd干扰载体转化至大肠杆菌HT115菌株中，使用四环素和氨苄青霉素两种抗性的固体平板培养基进行涂板，生长12-14h后，挑取单菌落，并进行sanger测序验证，验证结果证明测序序列与dnd基因干扰片段一致：
Sanger测序验证序列：
gcgggtgaggactggaaacctggctgaaagccaccaatacaaatctgaaacaagtaaacggccagaggaagtatggaggaccacctgaggtgtgggacgggccgtccccaggagcccgctgtgaggtcttcatcagccagatccctcgggacacctacgaggatctgctgattcccctcttcagctccgtggggcccctctgggagttccggctcatgatgaatttcagcggccagaaccgcggcttcgcctacggcaaatacggctcgccggccgtagctactgaagccatccgcctgctgcacggtcacatgctggagcccggcttctgcctcagtgtccgccgcagcacggagaagagacacctctgtatcggaaacctaagcttatcgataccgtcgacctcgagg。
（4）dnd基因敲降片段dsRNA的诱导表达：选取验证L4440-dnd阳性的HT115菌株，使用含有双抗的LB液体培养基扩繁至0.4 OD，加入IPTG诱导剂诱导dsRNA表达（IPTG终浓度为0.5mM），持续4h，并提取RNA进行验证；所述双抗的LB液体培养基成分及终浓度：胰蛋白胨10g/L、酵母提取物5g/L、NaCl 10g/L、氨苄青霉素 100mg/L和四环素 25mg/L。
（5）菌体富集：诱导结束后，4000rpm离心5min收集菌体，按照1mL重悬培养基/200mL初始菌量的比例重悬菌体；所述的重悬培养基也就是双抗的LB液体培养基。
（6）原始生殖细胞迁移、分化期许氏平由仔鱼的获取：选取处于妊娠期的健康的许氏平由雌鱼于车间内养殖，模拟自然条件下较长时间的光照，待其自然生产。
（7）人工诱导许氏平由性腺发育不良，即敲降组，同时设置对照组，敲降组和对照组各个养殖阶段投喂的饵料一样，敲降组用含有L4440-dnd干扰载体的诱导菌处理饵料，对照组用含有空载L4440的诱导菌处理饵料；其主要步骤如下所述：
①待许氏平由仔鱼产出后，取6000尾左右仔鱼放入直径1.5m，高1m，体积约为1.8m3的鱼池中，使其适应5天；
②取10mL重悬后的诱导菌混合轮虫投喂产出后5天的许氏平由仔鱼，用量为每天6000尾许氏平由仔鱼用量为10ml重悬后的诱导菌；
③投喂轮虫20天后，许氏平由饵料由轮虫更换为卤虫，处理方式同②；
④40天后，许氏平由饲料由卤虫更换为商业化颗粒饲料，处理方式如下：每天取出10mL重悬后的诱导菌，喷洒至颗粒饲料上，晒微晾干后，投喂许氏平由，用量为每天10ml/6000尾；
⑤本实施例的性腺不育处理，处理至仔鱼出生后95天；
样品获取：在仔鱼出生后120天，随机挑选对照组与敲降组许氏平由个体，剪少量尾鳍固定于95%乙醇中用于许氏平由遗传性别的鉴定，取性腺一部分置于液氮中速冻，用于RNA提取，基因表达量的分析；一部分固定于波恩氏液中用于许氏平由性腺的组织形态学观察。基因的TPM值如图1所示，敲降组dnd的TPM平均值为48.59，而对照组dnd的TPM平均值为80.22。该结果表明，敲降组dnd的表达相比对照组下调。性腺外观结果如图2所示，其中对照组精巢大小较为一致且发育良好（a-b），而敲降组精巢有明显的缺失和萎缩（c-d）；对照组卵巢大小较为一致且发育良好（e-f），而敲降组卵巢有明显的缺失和萎缩（g-h）。形态学鉴定结果如图3所示，敲降组精巢出现了明显的空洞（a-b）且精原细胞明显减少，不同于雄性对照组的精巢结构（c-d）；敲降组卵巢出现了较多的纤维状结构，且卵原细胞明显减少（e-f），不同于雌性对照组的卵巢结构（g-h）。
（8）由图1可以看出，投喂dnd敲降重组菌后，显著降低了许氏平由个体dnd的表达量。由图2、3可以看出，敲降组个体的性腺在外观和组织形态上出现了明显的发育不良情况。
（9）由图4敲降组个体与对照组相比可以看出，dnd敲降组许氏平由的平均体重为43.42g，对照组许氏平由的平均体重为50.49g；dnd敲降组许氏平由的平均体长为11.16cm，对照组许氏平由的平均体长为11.56cm。统计结果显示，敲降许氏平由的dnd基因，体重和体长与对照组相比并无显著差异。即投喂dnd敲降重组菌后，并不影响个体的生长性状。
Claims (6)
1.一种干扰许氏平由性腺正常发育的基因片段，其特征在于，所述基因片段的核苷酸序列如SEQ ID NO.1所示。
2.一种干扰许氏平由性腺正常发育的重组菌，其特征在于，所述重组菌包括如权利要求1中所述SEQ ID NO.1所示的基因片段、L4440线虫RNAi干扰载体和HT115菌株。
3.一种重组菌在干扰许氏平由性腺发育中的应用，其特征在于，应用方法如下：将权利要求2所述重组菌培养扩繁至OD值 0.4，诱导后，按照1mL重悬培养基/200mL初始菌量的比例重悬菌体，按照1mL/200条鱼/天的比例吸取重悬后的诱导菌，混入饵料，对处于原始生殖细胞分化期许氏平由仔鱼进行投喂，投喂周期为90天。
4.根据权利要求3所述一种重组菌在干扰许氏平由性腺发育中的应用，其特征在于，投喂时间为从许氏平由幼体生后5天开始，至出生后95天结束。
5.根据权利要求3所述一种重组菌在干扰许氏平由性腺发育中的应用，其特征在于，所述的饵料依次为轮虫、卤虫和颗粒饲料。
6.干扰许氏平由性腺正常发育重组菌的制备方法，其特征在于，将权利要求1所述的基因片段构建到L4440线虫RNAi干扰载体上，然后导入大肠杆菌HT115菌株，获得所述重组菌。
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