CN113122539A - 一种驴Zfy基因的RNA干扰片段、表达载体及其应用 - Google Patents
一种驴Zfy基因的RNA干扰片段、表达载体及其应用 Download PDFInfo
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Abstract
本发明属于分子遗传学的技术领域，本发明提供了一种驴Zfy基因的RNA干扰片段、表达载体及其应用。本发明提供的RNA干扰片段包括如SEQ ID NO：1或SEQ ID NO：2所示的序列。本发明提供的RNA干扰片段，对驴Zfy基因的表达进行人为干扰，使Y精子的生成和发育受阻，为X精子创造更多的受精机会，从而使子一代的母驴比例增加，达到控制后代驴性别的目的。
Description
技术领域
本发明涉及分子遗传学的技术领域，尤其涉及一种驴Zfy基因的RNA干扰片段、表达载体及其应用。
背景技术
实现对家畜的性别控制，意义重大。动物性别控制的意义在于：(1)提高畜牧业生产效益；(2)生产具有利用价值的性别的动物(一般雌性动物的利用价值要高于雄性动物)；(3)避免胚胎移植过程中异性胚胎导致的孪生不育；(4)提高选种强度；(5)预防伴性遗传病的发生等。对动物性别控制的相关研究早在多年前就已经开始。动物的性别控制(sexcontrol)是指通过对动物的正常生殖过程进行人为干预，使成年雌性动物产出人们期望性别后代的一门生物技术。目前国内外家畜进行性别控制的方法主要有三种:(1)分离X精子与Y精子。通过X、Y精子微弱的生物学差异，分离出含两种不同染色体的精子，目前主要采用物理分离法，如采用流式细胞分离仪分离精子的准确率可达到90％，但其分离速度太慢(18mil-lion/h)，且精液价格昂贵，分离后精液不耐冷冻，后代畸形率较高，因而在生产应用上受到限制；(2)胚胎的性别鉴定。胚胎性别鉴定主要是通过鉴定胚胎的性别，从而人工干预出生的性别比例。早期胚胎性别鉴定方法主要有细胞学方法、免疫学方法、分子生物学方法、PCR扩增法、雄性特异性DNA探针法等，但从胚胎上切取部分细胞而后经冷冻、解冻会对胚胎造成一定的损害使胚胎移植的成功率下降，同时劣质胚胎的性别鉴定成功率更低，这就制约了提供预知性别家畜胚胎的商业化进程；(3)控制家畜受精的外部环境。家畜外部环境中的某些因素也是性别决定机制的重要条件，主要包括营养、体液酸碱度、温度、输精时间、年龄胎次、激素水平等。但决定动物性别的因素主要是动物体的内部因素，采用控制外部环境的方法来控制出生动物性别，随机性大，不稳定，且不易控制。如今，随着科学技术水平的发展，从基因水平研究性别控制已经成为一种新的方向，同时随着基因技术的日趋成熟，基因技术的成本也大幅下降，使其在生产应用中大规模推广成为了一种可能。
RNA干扰(RNAInterference)是一种转录后水平的基因沉默现象，是通过体外人工合成双链RNA或体内的双链RNA在细胞内特异性降解其同源mRNA，使相应的基因沉默，达到阻止基因表达的目的，且具有特异、高效、易于操作等优点。
Zfy基因位于哺乳动物Y染色体上，负责编码一个大的酸性链锌指蛋白上的12～13个转录因子的特异性基因。Zfy基因具有特定的核定位信号区域和DNA结合位点，能够特异性的与靶基因结合，定向引导靶基因穿过核膜，定位于精子细胞核内，其编码蛋白作为转录调控因子，与Y精子的发育相关。通过RNA干扰技术，沉默Zfy基因是实现性别控制的可行途径。目前通过沉默Zfy基因能够实现性别控制的动物包括小鼠(CN201110072013.6)、绵羊(CN201510639871.2)、犬(CN201610584301.2)、牛(CN201910069205.8)。
发明内容
本发明的目的在于利用RNA干扰技术对驴后代进行性别控制。
为了实现上述发明目的，本发明提供以下技术方案：
本发明提供了一种RNA干扰片段，所述RNA干扰片段包括如SEQ ID NO：1或SEQ IDNO：2所示的序列。
本发明还提供了一种包括所述RNA干扰片段的表达载体。
本发明还提供了一种所述RNA干扰片段或所述表达载体在制备控制后代驴性别的制剂、试剂盒中的应用。
优选的，所述RNA干扰片段或所述表达载体用于沉默驴精子细胞中的Zfy基因。
本发明提供的RNA干扰片段，对驴Zfy基因的表达进行人为干扰，使Y精子的生成和发育受阻，为X精子创造更多的受精机会，从而使子一代的母驴比例增加，且对精子细胞不会造成损伤也不会降低其活力。本发明在受精前有效控制了后代性别，达到了控制后代驴性别的目的，而且成本低廉。
附图说明
图1为shRNA片段连接到慢病毒骨架载体pLentiLox3.7的图谱示意图；
图2为生精细胞中Zfy基因mRNA表达水平；
图3为生精细胞中Zfx基因mRNA表达水平。
具体实施方式
下面结合实施例对本发明提供的技术方案进行详细的说明，但是不能把它们理解为对本发明保护范围的限定。
1.构建驴Zfy基因的RNA干扰片段和表达载体
本申请经过前期工作筛选出了5对siRNA片段，然后按照载体及所带启动子的要求特点，对每个siRNA片段的5’和3’添加酶切位点，loop环及终止序列，最终形式为：T+正义链19nt靶序列+茎环结构(TTCAAGAGA)+靶序列反向互补序列+RNAPolyIII聚合酶转录中止位点(TTTTTT)+XhoI酶切位点(GAGCT)的shRNA寡核苷酸序列(委托上海生物工程有限公司合成shRNA寡核苷酸序列)，如表1所示。
表1 5对shRNA寡核苷酸序列
合成的shRNA片段经退火合为双链，连接到慢病毒骨架载体pLentiLox3.7，得到表达载体，载体图谱的示意图，如图1所示；然后转化到E.coli DH5α感受态细胞中进行扩增，提取质粒，-20℃保存备用。
2.对驴Zfy基因的体外干扰试验
采集成熟驴睾丸组织，采用两步酶消化法分离出睾丸支持细胞和生精细胞。将分离出的支持细胞和生精细胞放入完全培养液(含有160mL DMEM/F12、30mLFBS、2mL双抗、2mL胰岛素(10μg/mL)、2mL转铁蛋白(10μg/mL)、2mL维生素E(10μg/mL)、200uL维生素A(1000×)、200μL视黄酸(1000×)、200μL维生素C(1000×)、200μL丙酮酸(10-3mol/L)、200μL睾酮(1000×))中在32℃、5％CO2、湿度为95％的条件下培养24h，用钙离子作为转染试剂将步骤1中保存的质粒转染到生精细胞中，每组重复三次。转染64h后提取生精细胞的总RNA，用qRT-PCR检测Zfy和Zfx基因的mRNA表达水平，以空载体为对照组，判定5对干扰片段的干扰效果。试验所用引物如表2所示，qRT-PCR检测生精细胞Zfy和Zfx基因的mRNA表达情况如图2和图3所示。
表2 qRT-PCR检测Zfy和Zfx基因以及内参基因的引物序列
如图2显示，实验组Ea-E干扰载体中Zfy基因mRNA的表达水平低于对照组(空载体组)，且实验组和对照组相比差异显著(P＜0.05)；初步判定Ea-E干扰片段对驴Zfy基因就有沉默效果。如图3显示，实验组Ea-E干扰载体中Zfx基因mRNA的表达水平高于对照组，且实验组和对照组相比差异显著(P＜0.05)。
3.Zfy基因的驴体内干扰试验
在10mL离心管中加入总含量为3.0mg的质粒(为步骤1保存的质粒)及500μL的双抗，用PBS补充体积至6mL。在阜康驴场选取4头成熟健康公驴进行试验，先用1.0*18的10号穿刺针对睾丸皮肤穿刺，再用0.6*122的6号针抽取质粒通过穿刺针进入睾丸内部进行注射，该注射方法避免了因为针头过粗导致睾丸内部的损伤，每头公驴每侧睾丸注射3.0mg，每隔10天注射一次，共注射3次，第3次注射结束后4天可进行配种，每头公驴与15头母驴合圈，进行自然交配，并统计注射公驴的耳号，母驴的耳号、圈号，以及后代驴的公母数量。
表3后代驴性别统计
| 质粒名称 | 雄性 | 雌性 | 雌性比例 |
| Ea-E | 14 | 39 | 73.58％<sup>***</sup> |
由表3得知，经Ea-E质粒干扰后，驴后代中雌性比例为73.58％，同1:1的性别比例相比差异极显著(P＜0.01)。
由以上实施例可知，本发明提供的RNA干扰片段对驴Zfy基因有较好的沉默效应，干扰Zfy基因后，后代仔驴中母驴率为73.58％。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
序列表
<110> 石河子大学
<120> 一种驴Zfy基因的RNA干扰片段、表达载体及其应用
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 55
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
tggatacaga gttggaaatt ttcaagagaa atttccaact ctgtatcctt ttttc 55
<210> 2
<211> 59
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
tcgagaaaaa aggatacaga gttggaaatt tctcttgaaa atttccaact ctgtatcca 59
Claims (4)
1.一种RNA干扰片段，其特征在于，所述RNA干扰片段包括如SEQ ID NO：1或SEQ ID NO：2所示的序列。
2.一种包括权利要求1所述RNA干扰片段的表达载体。
3.一种权利要求1所述RNA干扰片段或权利要求2所述表达载体在制备控制后代驴性别的制剂、试剂盒中的应用。
4.如权利要求3所述的应用，其特征在于，所述RNA干扰片段或所述表达载体用于沉默驴精子细胞中的Zfy基因。
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