CN109988763A - 中华绒螯蟹性逆转siRNA及其应用 - Google Patents
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Abstract
本发明涉及中华绒螯蟹雄蟹的性别发育调控技术，具体的说是一种中华绒螯蟹性逆转siRNA及其应用。中华绒螯蟹雄蟹性逆转siRNA的正义链的核苷酸序列如SEQ ID NO.1所示，反义链的核苷酸序列如SEQ ID NO.2所示。本发明从中华绒螯蟹促雄腺转录组中筛选到了中华绒螯蟹IAG基因片段，并克隆了其全长cDNA序列，根据中华绒螯蟹IAG基因序列设计并合成siRNA，在性别分化期导入到中华绒螯蟹体内，干扰促雄腺细胞中IAG基因的表达，从而实现雄蟹的性逆转，与手术摘除促雄腺相比，本发明采用显微注射的方法，对中华绒螯蟹的身体创伤小，避免了手术后伤口出现感染而死亡；且注射剂量极低，不易引发受试动物的免疫应激反应，从而提高了性逆转的成功率。
Description
技术领域
本发明涉及中华绒螯蟹雄蟹的性别发育调控技术，具体的说是一种中华绒螯蟹性逆转siRNA及其应用。
背景技术
促雄腺(insulin-like androgenic gland,IAG)是雄性甲壳动物特有的器官，其分泌的促雄腺激素对甲壳动物的性别分化具有重要调控作用[1-4]。在雌性日本绒螯蟹体内植入促雄腺能够促使雌蟹产生类似雄性的附肢[5,6]，但移植的手术难度大，促雄腺存活率低；向雌性中华绒螯蟹体内注射促雄腺提取物也能够使雄蟹产生雄性附肢[7]。
RNAi技术又被形象地称为基因敲除(Knock-down)或基因沉默(Gene silencing)，是由dsRNA诱发的同源mRNA高效特异性降解的现象。1995年,Guo和Kemphues[8]利用反义RNA阻断线虫(Caenorhabditis elegans)的part-1基因表达时,意外发现作为对照组的正义RNA也可以抑制part-1基因的表达；1998年,Fire等[9]分别将part-1基因的正义、反义和dsRNA导入线虫,发现dsRNA的沉默效果明显高于单链RNA，将这种由dsRNA抑制特定基因表达的现象称为RNAi。随后,RNAi以其独有的高效性、特异性、稳定性和可传播性迅速成为研究基因功能的重要工具。
发明内容
本发明目的在于提供一种中华绒螯蟹性逆转siRNA及其应用。
为实现上述目的，本发明采用技术方案为：
一种中华绒螯蟹性逆转siRNA，中华绒螯蟹雄蟹性逆转siRNA的正义链的核苷酸序列如SEQ ID NO.1所示，反义链的核苷酸序列如SEQ ID NO.2所示。
SEQ ID NO.1为GGAAUGAUUGGGAACACCU；
SEQ ID NO.2为AGGUGUUCCCAAUCAUUCC。
为了增强上述siRNA双链复合体的稳定性，用dTdT取代了3'端的两个碱基突出；即为GGAAUGAUUGGGAACACCUTT；
AGGUGUUCCCAAUCAUUCCTT。
一种中华绒螯蟹性逆转siRNA的应用，所述siRNA在中华绒螯蟹性逆转中的应用。
所述siRNA在中华绒螯蟹雄蟹性逆转中的应用。
具体为：将所述siRNA经转染试剂将其溶剂，而后按核酸用量为2.5mg/kg体重的标准，将siRNA与转染试剂复合物从Ⅲ期幼蟹第五步足基部关节膜处通过现有的转染手段导入，即实现中华绒螯蟹雄蟹性逆转。siRNA与转染试剂按照每100ug核酸溶于50ul转染试剂的比例混合，转染试剂也可使用其它的阳离子脂质体试剂。如RNAi-Mate等。
一种含有编码所述siRNA的RNA性逆转试剂盒。
本发明所具有的优点：本发明从中华绒螯蟹促雄腺转录组中筛选到了中华绒螯蟹IAG基因片段，并克隆了其全长cDNA序列，根据中华绒螯蟹IAG基因序列设计并合成siRNA，在性别分化期导入到中华绒螯蟹体内，干扰雄蟹体内IAG基因的表达，从而实现雄蟹的性逆转，与手术摘除促雄腺相比，有效降低感染死亡率，避免了受试动物的免疫应激反应，提高了性逆转的成功率。
附图说明
图1为本发明实施例提供的3种siRMNA干扰后中华绒螯蟹IAG基因的相对表达量图。
图2为本发明实施例提供的采用本发明方法实现雄性幼蟹发生性逆转的照片图。
图3为本发明实施例提供的RNAi后雄蟹的雄性附肢发育受到抑制图；其中，A.正常方发育附肢，B.附肢发育受到抑制。
具体实施方式
以下结合实例对本发明的具体实施方式做进一步说明，应当指出的是，此处所描述的具体实施方式只是为了说明和解释本发明，并不局限于本发明。
实施例1
利用转录组技术获得中华绒螯蟹IAG基因片段，并克隆了其全长cDNA序列，根据中华绒螯蟹IAG基因序列(登录号：MF098773)设计并合成siRNA；在线siRNA设计软件(http:// sidirect2.rnai.jp/)设计备选siRNA序列，从候选的siRNA中排除含有SNP位点的和位于非开放阅读框架的片段，去除含有简并碱基、毒性结构域、重复序列、高GC/低GC序列，从热力学、高级结构等打分，列出从高到低的序列，将siRNA片段进行Blast分析，去除非特异结合的序列，该设计委托上海吉玛基因公司合成，共合成三个siRNA和一个阴性对照dsRNA(参见表1)；为增强序列稳定性，用dTdT取代了3'端的两个碱基突出；同时采用胆固醇锁核酸二硝基苯酚等化学基团对双链siRNA的正义或反义链进行化学修饰，增强siRNA在受试动物活体内的稳定性。
表1不同siRNA及对照组的序列
实施例2
将上述实施例获得的siRNA用EntransterTM-in vivo(Engreen Biosystem Co,Lid.)转染试剂将其溶剂，核酸用量按照2.5mg/kg体重的标准，将siRNA与转染试剂复合物从中华绒螯蟹雄蟹的Ⅲ期幼蟹第五步足基部关节膜处，利用毛细管注入到射精管附近，注射前和注射12小时后，分别取雄蟹阴茎基部周围组织做荧光定量PCR验证，所用qRT-PCR引物见表2。
表2 qRT-PCR所用引物序列
反应体系如下：
25μL qRT-PCR反应体系包括：
PCR反应程序为：
设置阴性对照，每组3个生物学重复，β-actin基因做为内参基因，qRT-PCR扩增结束后，分别对所得产物进行溶解曲线分析，经检测，siRNA-229、siRNA-305、siRNA-391干扰前后，中华绒螯蟹IAG基因的荧光定量检测结果见表3。
表3.IAG基因的荧光定量检测结果
利用2-⊿⊿CT比较CT法计算IAG基因的相对表达量，检测siRNA的干扰效率，结果见表4
表4. 3种siRNA的干扰效果
经检测，分别注射siRNA-229、siRNA-305、siRNA-391十二小时后，中华绒螯蟹IAG基因的表达量分别降低了26％、5.4％、67％(图1)。可以看出，siRNA-391的干扰效果显著。
实施例3
取上述siRNA-391用EntransterTM-in vivo(Engreen Biosystem Co,Lid.)转染试剂按比例将其溶剂，待用；
实验：取中华绒螯蟹Ⅲ期幼蟹雄性90只，核酸用量按照2.5mg/kg体重的标准，将siRNA与转染试剂复合物从第五步足基部关节膜处注射入射精管周围，待Ⅲ期幼蟹蜕壳为Ⅳ期和Ⅴ期幼蟹后第二天分别通过现有的转染手段各重复导入一次，待Ⅴ期幼蟹蜕壳后观察腹部形态和雄性附肢的变化，由观察结果可见：Ⅴ期幼蟹蜕壳后，90只实验中华绒螯蟹有9只雄蟹腹部的形状由狭长形变成了三角形，说明9只雄蟹发生了性逆转，变为雌蟹(图2)，性逆转成功率为10％。
实施例4
取上述siRNA-391用EntransterTM-in vivo(Engreen Biosystem Co,Lid.)转染试剂按比例将其溶剂，取30只大眼幼体，分别浸泡到10μM siRNA与转染试剂复合物中30秒，转入烧杯分别培养，变态为Ⅰ期幼蟹后，观察雄性附肢的发育情况(参见图3)。实验结果发现：30大眼幼体浸泡后，死亡12只，死亡率高；最后观察到有3只雄性Ⅰ期幼蟹的雄性附肢发育受到抑制。
参考文献：
[1]Nagamine C,Knight AW,Maggenti A,and Paxman G.Effects of androgenicgland ablation on male primary and secondary sexual characteristics in themalaysian prawn,Macrobrachium rosenbergii(decapoda,palaemonidae),with firstevidence of induced feminization in a nonhermaphroditic decapod,General andComparative Endocrinology,1980,41(4):423-441
[2]Sagi A,Cohen D,and Milner Y.Effect of androgenic gland ablation onmorphotypic differentiation and sexual characteristics of male freshwaterprawns,Macrobrachium rosenbergii,General and Comparative Endocrinology,1990,77(1):15-22
[3]Ventura T,Manor R,Aflalo E D,Weil S,Raviv S,Glazer L,and SagiA.Temporal silencing of an androgenic gland-specific insulin-like geneaffecting phenotypical gender differences and spermatogenesis,Endocrinology,2009,150(3):1278-1286
[4]Ventura T,Manor R,Aflalo E D,Weil S,Rosen O,and Sagi A.Timingsexual differentiation:Full functional sex reversal achieved throughsilencing of a single insulin-like gene in the prawn,Macrobrachiumrosenbergii,Biology of Reproduction,2012,86(3):90
[5]LEE T,SHIGESAWA R,YAMAZAKI F.Partial masculinization of femaleEriocheir japonicus(Brachyura,Grapsidae)by androgenic gland implantation[J].Aquaculture Science,1993,41(3):311-319.
[6]Hong L,Li C S,Feng Z C,et al.Masculinization of female Eriocheirsinensis by injecting the extract of androgenic gland of E.sinensis andScylla paramamosain[J].Journal of Fisheries of China,2006,30(5):577-585.
[7]肖美南,徐佳念,蔡生力,et al.中华绒螯蟹促雄性腺素分离、纯化的初步研究[J].上海海洋大学学报,2010,19(1):28-33.
[8]Guo S,Kemphues K J.par-1,a gene required for establishing polarityin C.elegans embryos,encodes a putative Ser/Thr kinase that is asymmetricallydistributed[J].Cell,1995,81(4):611-620
[9]Fire A,Xu S,Montgomery M K,etal.Potent and specific geneticinterference by double-strand ed RNA in Caenorhabditis elegans[J].Nature,1998,391(6669):806-811.
序列表
<110> 潍坊科技学院
<120> 中华绒螯蟹性逆转siRNA及其应用
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 19
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 1
ggaaugauug ggaacaccu 19
<210> 2
<211> 19
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 2
agguguuccc aaucauucc 19
Claims (4)
1.一种中华绒螯蟹性逆转siRNA，其特征在于：中华绒螯蟹雄蟹性逆转siRNA的正义链的核苷酸序列如SEQ ID NO.1所示，反义链的核苷酸序列如SEQ ID NO.2所示。
2.一种权利要求1所述的中华绒螯蟹性逆转siRNA的应用，其特征在于：所述siRNA在中华绒螯蟹性逆转中的应用。
3.按权利要求2所述的中华绒螯蟹性逆转siRNA的应用，其特征在于：所述siRNA在中华绒螯蟹雄蟹性逆转中的应用。
4.一种含有编码权利要求1所述siRNA的RNA性逆转试剂盒。
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109536506A (zh) * | 2018-12-10 | 2019-03-29 | 潍坊科技学院 | 一种调控日本沼虾生长发育的类胰岛素受体基因及其应用 |
| CN110699358A (zh) * | 2019-11-20 | 2020-01-17 | 天津师范大学 | 一种提高中华绒螯蟹抗病性的双链rna及其应用 |
| CN111387105A (zh) * | 2020-03-12 | 2020-07-10 | 浙江省淡水水产研究所 | 一种全雄罗氏沼虾制种的方法 |
| CN112544516A (zh) * | 2020-12-08 | 2021-03-26 | 汕头大学 | 一种拟穴青蟹人工性逆转方法 |
| CN115161346A (zh) * | 2022-02-07 | 2022-10-11 | 天津师范大学 | 一种向中华绒螯蟹胚胎导入外源物质的方法与应用 |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009098683A2 (en) * | 2008-02-04 | 2009-08-13 | Ben-Gurion University Of The Negev Research And Development Authority | Insulin-like gene in prawns and uses thereof |
| US20150096499A1 (en) * | 2013-10-04 | 2015-04-09 | Siu Ming Chan | Moderation of crustacean androgenic gland peptide for mono sex culture |
-
2019
- 2019-03-27 CN CN201910237820.5A patent/CN109988763A/zh active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009098683A2 (en) * | 2008-02-04 | 2009-08-13 | Ben-Gurion University Of The Negev Research And Development Authority | Insulin-like gene in prawns and uses thereof |
| CN101981046A (zh) * | 2008-02-04 | 2011-02-23 | 内盖夫研究与发展部本-古里安大学 | 对虾中的胰岛素样基因和其用途 |
| US20150096499A1 (en) * | 2013-10-04 | 2015-04-09 | Siu Ming Chan | Moderation of crustacean androgenic gland peptide for mono sex culture |
Non-Patent Citations (2)
| Title |
|---|
| FU CHUNPENG等: "Molecular characteristics and abundance of insulin-like androgenic gland hormone and effects of RNA interference in Eriocheir sinensis", 《ANIMAL REPRODUCTION SCIENCE》 * |
| 付春鹏: "基于转录组和miRNAs分析中华绒螯蟹促雄腺的相关基因及其调控机制", 《中国博士学位论文全文数据库 农业科技辑》 * |
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| CN109536506B (zh) * | 2018-12-10 | 2022-03-22 | 潍坊科技学院 | 一种调控日本沼虾生长发育的类胰岛素受体基因及其应用 |
| CN110699358A (zh) * | 2019-11-20 | 2020-01-17 | 天津师范大学 | 一种提高中华绒螯蟹抗病性的双链rna及其应用 |
| CN110699358B (zh) * | 2019-11-20 | 2023-05-02 | 天津师范大学 | 一种提高中华绒螯蟹抗病性的双链rna及其应用 |
| CN111387105A (zh) * | 2020-03-12 | 2020-07-10 | 浙江省淡水水产研究所 | 一种全雄罗氏沼虾制种的方法 |
| CN111387105B (zh) * | 2020-03-12 | 2022-02-15 | 浙江省淡水水产研究所 | 一种全雄罗氏沼虾制种的方法 |
| CN112544516A (zh) * | 2020-12-08 | 2021-03-26 | 汕头大学 | 一种拟穴青蟹人工性逆转方法 |
| CN115161346A (zh) * | 2022-02-07 | 2022-10-11 | 天津师范大学 | 一种向中华绒螯蟹胚胎导入外源物质的方法与应用 |
| CN115161346B (zh) * | 2022-02-07 | 2023-05-26 | 天津师范大学 | 一种向中华绒螯蟹胚胎导入外源物质的方法与应用 |
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