CN101366944A - Mrpink在抑制罗氏沼虾精子活性中的应用 - Google Patents
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Abstract
本发明提供了罗氏沼虾雄性生殖系统特异性Kazal型蛋白酶抑制MRPINK在抑制罗氏沼虾精子活性中的应用。在研究中发现，在罗氏沼虾的精子中添加MRPINK能有效的抑制精子活性，使精卵不能受精。在精子的冻存中添加类似的蛋白酶抑制剂，抑制精子内酶活性，使精子不活化，对精子的低温储存具有重要的借鉴意义。
Description
(一)技术领域
本发明涉及罗氏沼虾雄性生殖系统特异性Kazal型蛋白酶抑制剂MRPINK在抑制罗氏沼虾精子活性中的应用。
(二)背景技术
生物的性别、生殖现象是生物学研究的主要内容之一，它涉及到许多重大的生物学问题，比如性别决定、遗传、发育和进化等，因此历来倍受关注。目前对于性别、生殖的分子机制研究主要集中在人类和模式生物中，而对于一般水生生物这方面的研究很少，尤其在甲壳类中，已知的相关分子信息更加缺乏。
虾类作为具有重要经济价值的水生生物，在分子水平的研究开展得相对较少，因此，大量获取水生动物性别和生殖相关基因资源来研究它们的生殖生物学、遗传育种等显得尤为必要。虾类同龄雌雄性别个体的大小、生长速度相差悬殊，性别与生殖制约着它们的产值。若能培育出单性虾，并进行单性化养殖可以大幅度提高上市商品规格和养殖产量，因此虾类性别控制一直是遗传育种研究的热点之一。目前有关这方面的认识仅仅局限在形态结构和一般的组织化学、生化成份和基本的生理指标，在基因水平的认识和可操作性则几乎为零。
(三)发明内容
本发明则是通过对罗氏沼虾雄性生殖相关基因进行克隆、表达特征分析以及定位功能等研究，提供了罗氏沼虾雄性生殖系统特异性Kazal型蛋白酶抑制剂MRPINK在抑制罗氏沼虾精子活性中的应用。
本发明采用的技术方案是：
罗氏沼虾雄性生殖系统特异性Kazal型蛋白酶抑制剂MRPINK在抑制罗氏沼虾精子活性中的应用。(有关MRPINK的相关介绍参见文献：Cao JX，Dai JQ，Dai ZM，Yin GL and Yang WJ.A male-reproduction-relatedkazal-type peptidase inhibitor gene in the prawn，Macrobrachium rosenbergii：molecular characterization and expression patterns.Marine Biotechnology.2007，9：45～55.)
所述MRPINK的cDNA序列如下：AGTTGACAGACTGCCAGCTTCCTGTGCAGAATTCTGAACGATAGGCTTAGGTAAGACCTCACAACTAGCAAAATGAAACTGTACATCTTCACCTTCTGCCTAGCCGCGTCAATTCTCGTCGGGTTTTCATCTGGGGAATACCGCTTCCTTGTCTCTCCACTGTGCCCCGTACGTTGTACTCTGAGGTACATCCCTGTCTGTGGCAGCGACGGCCGAACGTACGGGAACAAATGTCACCTGGACAATGCACGTCTCTGTGACAACAGCAATGTTCAGGTAGTTCACGAAGGCGAATGTAAGCAAGACTACTGCGCCCCGGTAGTCCGCTGCCCACCGGTCATAAAAGCCGTATGTGCCAACGACGGCCAAACATACTTGAACGAGTGTTTCGCCAAAGTCGCAGCCTGCGGATTCCCTAACCTGAAGATAGTCCACAGCGGATTATGTGGTCCTAGACCAAAGACTGAAGTATGGTAGCGAGGTGGTGACTGTTGGAAATAGACCATTACGTGAGTGAAAACGGCGATATATGAATATGTTCCATCAATCAAATCAAGTTCTGATCGGCTGTGTACTAATAACTTCGTACATATGGCCTTCTTTCAAGTTCTCCTGATTTCCACCAGATTTATTATATTTTTATGGATATTGGAGGTTTCTGAGATACGCCCAGTTGTCTATTACATCTGAAGAAAAACTTGATCAATAAAGCCTTTGTAACAGGAAAAAAAAAAAA。
具体的，所述应用为：以MRPINK制备精子活性抑制剂，用于罗氏沼虾精子冻存。
优选的，所述精子活性抑制剂为MRPINK。所述MRPINK的氨基酸序列如下：MKLYIFTFCLAASILVGFSSGEYRFLVSPLCPVRCTLRYIPVCGSDGRTYGNKCHLDNARLCDNSNVQVVHEGECKQDYCAPVVRCPPVIKAVCANDGQTYLNECFAKVAACGFPNLKIVHSGLCGPRPKTEVW。
选取罗氏沼虾几乎全组织，包括肌肉、肝胰腺、心脏、鳃、胃、血细胞、雄性生殖系统、卵巢、脑、中枢神经、脂肪体、眼柄、肠和腹部神经进行Northernblotting分析，结果表明MRPINK基因仅表达在雄性生殖系统，而且在输精管中的表达占主导地位，其次是末端壶腹，在精巢中的表达并不是很高。
通过分析表明MRPINK基因mRNA的表达在后幼体期四周内检测不到，此后其表达出现，但表达量随着罗氏沼虾逐渐生长发育并无明显变化。MRPINK基因的表达在罗氏沼虾的不同生长发育时期呈现恒定趋势，表明这一基因更多的与维持内环境稳定和整个组织的完整性相关，而不是调控精子形成或形态分化的特异阶段。
运用原位杂交的实验手段在细胞水平上检测了MRPINK在罗氏沼虾雄性生殖系统中的定位，发现这MRPINK基因的mRNA仅表达在输精管和末端壶腹的内壁上皮分泌细胞中。这一特异性的定位暗示着它可能在精子自身保护、生殖系统中蛋白酶降解及内环境稳定等过程中发挥功能。
MRPINK基因的表达在罗氏沼虾生长发育时期呈现恒定趋势且仅在输精管和末端壶腹的内壁上皮分泌细胞中有表达，表明这一基因在精子自身保护、生殖系统中蛋白酶降解及维持内环境稳定等过程中发挥了重要功能。MRPINK是蛋白酶抑制剂，参与维持雄性生殖系统内环境的稳定，对精子的生理成熟和活力维持可能有十分重要的意义。在我们的研究中发现，在罗氏沼虾的精子中添加MRPINK能有效的抑制精子活性。
人类精子库已应用于临床治疗，但是作为生育能力的储备形式仍然有很大的局限性。目前人类精子储存的办法是将精子冻存在装有液态氮的大罐子中，在超低温情况下保持精子自身能量不被消耗。但在精子冷冻和复苏过程中，温度的变化会对精子产生生物物理损伤和化学损害，使精子细胞膜水、电介质浓缩、紊乱、使不稳固的类脂蛋白变性、以及机械性破坏从而影响精子的生命活力。此外，精子冷冻的价格非常昂贵，需要一套严格的控制系统，以避免出现差错。
在精子的冻存中添加类似的蛋白酶抑制剂，抑制精子内酶活性，使精子不活化，对精子的低温储存具有重要的借鉴意义。
(四)具体实施方式
下面结合具体实施例对本发明进行进一步描述，但本发明的保护范围并不仅限于此：
实施例1：MRPINK基因及其编码的蛋白质的获得
具体可参见文献：Cao JX，Dai JQ，Dai ZM，Yin GL and Yang WJ.Amale-reproduction-related kazal-type peptidase inhibitor gene in the prawn，Macrobrachium rosenbergii：molecular characterization and expressionpatterns.Marine Biotechnology.2007，9：45～55.
①取罗氏沼虾雄性和雌性生殖系统组织，按Trizol试剂盒说明抽提总RNA；
②建立罗氏沼虾雄性生殖系统SSH cDNA文库，筛选差异性表达的基因片段：
A.第一条链的合成：按Oligotex mRNA Kits法，提取PolyA+RNA，以PolyA+RNA为模板反转录合成单链cDNA。
B.双链(ds cDNA)的合成，Rsa I酶消化产生平末端。
C.接头连接：把Rsa I酶切后雄性组织ds cDNA分成两份，分别加上接头1(Adaptor1)和接头2(Adaptor2)，16℃连接过夜，形成两组检测子(Tester)。
D.消减杂交：
第一，分别将Tester1和Tester2的连接产物与RsaI酶消化的过量驱动子(Driver)ds cDNA混合，在98℃下热变性，然后在65～68℃下放置10小时使Tester和Driver的cDNA进行充分地杂交。检测子单链cDNA片段被均化，即平均高、低丰度的cDNA浓度，使其基本相等。
第二，将第一次杂交的产物混匀到一起，然后在68℃继续放置过夜(约16小时)，使Tester1和Tester2之间的目的差异cDNA形成异源杂交子。
E.巢氏PCR扩增差异表达的cDNA。
F.筛选差异性文库：PCR产物经连接转化到TOPO10载体上，构建差异表达基因富集的cDNA文库，筛选差异性表达的基因。
③全长cDNA的获得：
利用FirstChoiceTM RLM-RACE kit合成5’和3’RACE，分别得到487bp的5’端和270bp的3’端，拼接完成后获得全长cDNA，命名为MRPINK基因。
MRPINK cDNA序列如下：
AGTTGACAGACTGCCAGCTTCCTGTGCAGAATTCTGAACGATAGGC
TTAGGTAAGACCTCACAACTAGCAAAATGAAACTGTACATCTTCAC
CTTCTGCCTAGCCGCGTCAATTCTCGTCGGGTTTTCATCTGGGGAAT
ACCGCTTCCTTGTCTCTCCACTGTGCCCCGTACGTTGTACTCTGAGG
TACATCCCTGTCTGTGGCAGCGACGGCCGAACGTACGGGAACAAA
TGTCACCTGGACAATGCACGTCTCTGTGACAACAGCAATGTTCAGG
TAGTTCACGAAGGCGAATGTAAGCAAGACTACTGCGCCCCGGTAG
TCCGCTGCCCACCGGTCATAAAAGCCGTATGTGCCAACGACGGCCA
AACATACTTGAACGAGTGTTTCGCCAAAGTCGCAGCCTGCGGATTC
CCTAACCTGAAGATAGTCCACAGCGGATTATGTGGTCCTAGACCAA
AGACTGAAGTATGGTAGCGAGGTGGTGACTGTTGGAAATAGACCA
TTACGTGAGTGAAAACGGCGATATATGAATATGTTCCATCAATCAA
ATCAAGTTCTGATCGGCTGTGTACTAATAACTTCGTACATATGGCC
TTCTTTCAAGTTCTCCTGATTTCCACCAGATTTATTATATTTTTATGG
ATATTGGAGGTTTCTGAGATACGCCCAGTTGTCTATTACATCTGAA
GAAAAACTTGATCAATAAAGCCTTTGTAACAGGAAAAAAAAAAAA
RPINK cDNA全长736bp，编码区405bp，5’和3’非翻译区长度分别是72bp和247bp，其中3’非翻译区具有多聚腺苷酸信号。该基因已递交到GenBank，其序列号为AY885242。
MRPINK氨基酸序列如下：
MKLYIFTFCLAASILVGFSSGEYRFLVSPLCPVRCTLRYIPVCGSDGRTY
GNKCHLDNARLCDNSNVQVVHEGECKQDYCAPVVRCPPVIKAVCAN
DGQTYLNECFAKVAACGFPNLKIVHSGLCGPRPKTEVW。
它编码135个氨基酸，分子量约14.7kDa，推演的氨基酸序列中具有两个Kazal型domain，且含有21个氨基酸的信号肽。
MRPINK和已知的Kazal型蛋白酶抑制剂在氨基酸序列上的同源性为16～32％，其Kazal型domain序列上有19～52％的同源性，表明这个蛋白属于Kazal家族中的成员。MRPINK作为蛋白酶抑制剂，与已知哺乳动物蛋白酶抑制剂可能具有相似的功能。
实施例2：MRPINK对罗氏沼虾的精子活性的抑制作用
明胶底物实验：
罗氏沼虾精子(挑选成熟雄性罗氏沼虾，摘取壶腹磨碎，离心，计数，使精子浓度为1×106cells/ml)用MRPINK(2mg/ml)28℃预处理1h；未经MRPINK处理的罗氏沼虾精子作为对照组；将处理后样品涂于已含明胶(终浓度为5％)的玻片上，37℃孵育过夜，相差显微镜(400×)下观察。
实验结果表明：95％罗氏沼虾精子会降解明胶形成晕轮(图1-A)，经MRPINK处理的精子不能降解明胶形成晕轮(图1-B)，MRPINK能有效抑制罗氏沼虾精子降解明胶的活性。由此说明，MRPINK对罗氏沼虾的精子活性有抑制作用。
序列表_ST25
SEQUENCE LISTING
<110>浙江大学
<120>MRPINK在抑制罗氏沼虾精子活性中的应用
<130>
<160>2
<170>PatentIn version 3.4
<210>1
<211>736
<212>DNA
<213>Macrobrachium rosenbergii
<400>1
<210>2
<211>134
<212>PRT
<213>Macrobrachium rosenbergii
<400>2
Claims (5)
1.罗氏沼虾雄性生殖系统特异性Kazal型蛋白酶抑制剂MRPINK在抑制罗氏沼虾精子活性中的应用。
2.如权利要求1所述的应用，其特征在于所述MRPINK的cDNA序列如下
AGTTGACAGACTGCCAGCTTCCTGTGCAGAATTCTGAACGATAG
GCTTAGGTAAGACCTCACAACTAGCAAAATGAAACTGTACATCT
TCACCTTCTGCCTAGCCGCGTCAATTCTCGTCGGGTTTTCATCTG
GGGAATACCGCTTCCTTGTCTCTCCACTGTGCCCCGTACGTTGTA
CTCTGAGGTACATCCCTGTCTGTGGCAGCGACGGCCGAACGTAC
GGGAACAAATGTCACCTGGACAATGCACGTCTCTGTGACAACAG
CAATGTTCAGGTAGTTCACGAAGGCGAATGTAAGCAAGACTACT
GCGCCCCGGTAGTCCGCTGCCCACCGGTCATAAAAGCCGTATGT
GCCAACGACGGCCAAACATACTTGAACGAGTGTTTCGCCAAAGT
CGCAGCCTGCGGATTCCCTAACCTGAAGATAGTCCACAGCGGAT
TATGTGGTCCTAGACCAAAGACTGAAGTATGGTAGCGAGGTGGT
GACTGTTGGAAATAGACCATTACGTGAGTGAAAACGGCGATATA
TGAATATGTTCCATCAATCAAATCAAGTTCTGATCGGCTGTGTAC
TAATAACTTCGTACATATGGCCTTCTTTCAAGTTCTCCTGATTTCC
ACCAGATTTATTATATTTTTATGGATATTGGAGGTTTCTGAGATA
CGCCCAGTTGTCTATTACATCTGAAGAAAAACTTGATCAATAAA
GCCTTTGTAACAGGAAAAAAAAAAAA。
3.如权利要求2所述的应用，其特征在于所述应用为：以MRPINK制备精子活性抑制剂，用于罗氏沼虾精子冻存。
4.如权利要求3所述的应用，其特征在于所述精子活性抑制剂为MRPINK。
5.如权利要求1～4之一所述的应用，其特征在于所述MRPINK的氨基酸序列如下：
MKLYIFTFCLAASILVGFSSGEYRFLVSPLCPVRCTLRYIPVCGSDGRT
YGNKCHLDNARLCDNSNVQVVHEGECKQDYCAPVVRCPPVIKAVC
ANDGQTYLNECFAKVAACGFPNLKIVHSGLCGPRPKTEVW。
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104745584A (zh) * | 2015-04-12 | 2015-07-01 | 浙江万里学院 | 一种用于干扰罗氏沼虾雄性生殖的核酸分子及其制备方法 |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104745584A (zh) * | 2015-04-12 | 2015-07-01 | 浙江万里学院 | 一种用于干扰罗氏沼虾雄性生殖的核酸分子及其制备方法 |
| CN104745584B (zh) * | 2015-04-12 | 2017-07-04 | 浙江万里学院 | 一种用于干扰罗氏沼虾雄性生殖的核酸分子及其制备方法 |
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