摘要
性类固醇激素与生物生殖、配子排放以及性腺发育密切相关,Srd5a1基因在性类固醇激素合成过程中扮演重要角色。为了探究性类固醇激素合成相关酶基因Srd5a1对雌性三角帆蚌卵巢发育的影响,应用RACE克隆得到了该基因全长cDNA序列,通过实时荧光定量(qRT-PCR)、原位杂交技术以及不同浓度17β-雌二醇(E2)和17α-甲基睾酮(MT)激素处理来探究基因的表达特性。结果显示,Srd5a1 cDNA全长2 224 bp,包括446 bp 5′-UTR、953 bp 3′-UTR和825 bp ORF,编码274个氨基酸。Srd5a1基因在性腺中的表达呈二态性,在卵巢中表达量更高;且在卵巢排放期的表达量最高,极显著高于其他各时期。原位杂交结果显示,Srd5a1基因在卵巢卵母细胞、卵泡以及精巢精母细胞中有表达。不同浓度E2和MT处理24 d后,Srd5a1基因的表达发生了变化。综上,Srd5a1基因可能在雌性三角帆蚌卵巢卵母细胞发育成熟和配子排放中发挥一定作用,对探索三角帆蚌性腺生长发育具有重要意义,同时为实现三角帆蚌单性化养殖提供理论参考价值。
三角帆蚌(Hyriopsis cumingii)是我国最特殊的淡水育珠蚌之一,雄性三角帆蚌所产珍珠经济效益更
类固醇5α还原酶家族有3个成员:SRD5A1、SRD5A2和SRD5A3,分别由Srd5a1、Srd5a2和Srd5a3基因编
目前,有研究者已在头足纲、双壳纲和腹足纲等软体动物中发现了性类固醇激素物质的存在。此外,有些性类固醇激素能够参与贝类生殖调控和性腺发育过
实验所用三角帆蚌由浙江省金华市武义养殖基地提供,实验室暂养2~5 d,水温为(26±2) ℃。用微型注射器抽取12~36月龄三角帆蚌的部分性腺组织液于载玻片上,镜检辨别雌雄。分别选取12~36月龄健康的三角帆蚌雌雄各3只,4~8月龄幼蚌雌雄各6只,对其进行性腺组织取样;分别采集生长健康的24月龄雌雄三角帆蚌的7个组织:闭壳肌、斧足、外套膜、鳃、肝脏、性腺和肾脏;根据形态学观
取体积相同(5 mm×5 mm×5 mm)的新鲜雌雄性腺组织于4%多聚甲醛中固定2 h,实验样品设3个平行,后转入70%乙醇中4 ℃保存备用。梯度脱水,包埋浸蜡,制作厚度为6~8 μm的石蜡切片。37 ℃烘箱烘干12 h,-20 ℃保存备用。根据H.E染色试剂盒(北京索莱宝科技有限公司)说明书进行染色,树胶封片。Leica DM 2500显微镜(Leica,德国)拍照。
冷冻的性腺组织RNA按照Trizol方法提取,分别使用NanoDrop 2000C(Thermo Fisher Scientific,美国)和1%琼脂糖凝胶电泳检测其质量与完整性。cDNA体外反转录根据试剂盒(RR047A-1,TaKaRa)进行。最终将其稀释5倍,于-20 ℃保存备用。
用Primer Premier 5.0软件进行引物设计(
引物名称 Primer name | 序列5′-3′ Sequence (5′-3′) | 目的 Application |
---|---|---|
Srd5a1-F1 | TACATCCAGGGTGGCTTCCT | 序列验证 |
Srd5a1-R1 | AACCATCAGCTCTACCGCAG | |
Srd5a1-F2 | ATGTCGTGCCGAGTTCTAATG | |
Srd5a1-R2 | CGGATGTCTCCCCTGTTTTT | |
Srd5a1-3' | ATGTGTTGGGCGAGTTGAGACT | 3'克隆 |
qSrd5a1-F | ACAGGGGAGACATCCGTGTA | 荧光定量 |
qSrd5a1-R | TGGACTGACCATGTGGCTACT | |
EFl-αF | GGAACTTCCCAGGCAGACTGTGC | |
EFl-αR | TCAAAACGGGCCGCAGAGAAT | |
I-Srd5a1-F | ACAGGGGAGACATCCGTGTA | 原位杂交 |
I-Srd5a1-R |
TAATACGACTCACTATAGGG TGGACTGACCATGTGGCTACT |
在Srd5a1基因的ORF区设计引物,以EF1-α作为内参基因,引物序列见
引物通过Primer 5.0设计,T7启动子序列(TAATACGACTCACTATAGGG)加在荧光定量下游引物5′端(
实验采用乙醇溶解激素后直接浸泡的方法进行,激素为17β-雌二醇(17β-Estradiol,E2)和17α-甲基睾酮(17α-Methyltestosterone,MT),得到的浓度40 ng/L和200 ng/L参考PUINEA
三角帆蚌Srd5a1基因 cDNA全长为2 224 bp(登录号:OP750480),包括446 bp 5′-UTR、953 bp 3′-UTR和825 bp ORF,共编码氨基酸274个(
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图1 Srd5a1 cDNA序列及其编码的氨基酸序列
Fig.1 Srd5a1 cDNA sequence and encoded amino acid sequence
起始密码子和终止密码子用黑色方框框出;绿色方框内为NADPH绑定域;红色方框内为非规范聚腺苷酸化信号。
The black box showed the start and stop codons; The green box showed the NADPH binding domain; The red box showed the non-canonical polyadenylation signal.
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图2 Srd5a1氨基酸的多序列比对
Fig.2 Multiple comparisons of Srd5a1 amino acid
组织定量结果表明,Srd5a1基因在7个组织中均有表达。其中,Srd5a1基因在性腺中存在差异性表达,卵巢中的表达量极显著高于精巢(P<0.01);在鳃和斧足组织中表达较高,在外套膜中表达最低(
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图3 Srd5a1基因在24月龄雌雄三角帆蚌各组织中的表达
Fig.3 Expression of Srd5a1 in different tissues of 24-month-old male and female H.cumingii
**表示雌雄间差异极显著(P<0.01)。
** indicates highly significant differences between males and females (P<0.01).
与12月龄性腺组织表达相比,Srd5a1在性腺早期发育阶段(4~8月龄)的整体表达水平较低,呈现先下降后上升再下降的波动性变化,其在5月龄和8月龄的表达量相对较低,与其他各月龄存在显著差异(
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图4 Srd5a1 mRNA在4~8月龄性腺组织中的表达
Fig.4 Expression of Srd5a1 mRNA in the gonads of 4-8 months old
图中字母不同代表各月龄之间存在显著性差异。
Different letters in the graph represent significant differences between months of age.
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图5 Srd5a1 mRNA在12~36月龄性腺组织中的表达
Fig.5 Expression of Srd5a1 mRNA in the gonads of 12-36 months old
*表示卵巢和精巢间差异显著(P<0.05);**表示卵巢和精巢间差异极显著(P<0.01)。
* indicated significant difference between ovary and testis (P<0.05); ** indicated significant difference between ovary and testis (P<0.01).
Srd5a1基因在卵巢整个发育过程中均有表达,其表达量呈现先上升后下降的趋势。随着卵母细胞体积增大,数目增多,Srd5a1基因的表达显著上调,并在配子排放时达到峰值,配子排放后滤泡开始萎缩,Srd5a1基因的表达迅速下降至最低值(
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图6 Srd5a1 mRNA在卵巢不同发育时期中的表达
Fig.6 Expression of Srd5a1 mRNA in different developmental periods of the ovary
图中不同的字母代表不同时期之间差异具有显著性。
Different letters in the graph represent significant differences between periods.
Srd5a1 mRNA在卵巢卵母细胞细胞质以及卵泡上有强烈的阳性信号,且随着卵母细胞卵黄的积累,其阳性信号更为强烈;在精巢精母细胞中检测到其微弱的阳性信号(图版)。
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1.精巢空白组;2.精巢实验组;3.卵巢空白组;4.卵巢实验组;紫色为阳性信号。Sp.精子; Sc.精母细胞; Fc.卵泡; Oc.卵母细胞; Og.卵原细胞; Nu.细胞核。
1. Testis blank control group; 2. Testis experimental group; 3. Ovarian blank control group; 4. Ovarian experimental group; Purple color is the positive signal. Sp. Sperm; Sc. Spermatocyte; Fc. Follicles; Oc. Oocyte; Og. Oogonium; Nu. Nucleus.
图版 Srd5a1 mRNA在雌雄性腺中的表达定位
Plate Localization of Srd5a1 mRNA expression in male and female gonads
用不同浓度的激素处理24 d,结果显示:在低浓度40 ng/L E2处理条件下,第1天,Srd5a1基因表达迅速上升,在处理6 d后其表达量出现极显著下降,第12天时,回升至正常水平;第18天时,出现显著性下降;第24天时,又回升至正常水平,Srd5a1基因的表达变化规律以6 d为1个周期。在高浓度200 ng/L E2处理条件下,第1~3天,Srd5a1基因表达显著性下降,第6~18天,其表达量开始回升,显著高于空白对照组;第24天时,Srd5a1基因的表达回落至较低水平(
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图7 不同浓度17β-雌二醇处理下Srd5a1基因在卵巢中的表达
Fig.7 Expression of Srd5a1 gene in ovary treated with E2 at different concentrations
*表示显著差异(P<0.05);**极显著差异(P<0.01)。
* indicates significant differences (P<0.05); ** indicates highly significant differences (P<0.01).
在低浓度40 ng/L MT处理条件下,Srd5a1基因的表达只在第1天有显著性升高,其他各时间点处,该基因表达量没有显著变化。高浓度200 ng/L MT处理24 d,Srd5a1基因的整体表达水平出现显著性下降(
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图8 不同浓度17α-甲基睾酮处理下Srd5a1基因在卵巢中的表达
Fig.8 Expression of Srd5a1 gene in ovary treated with MT at different concentrations
*表示显著差异(P<0.05);**表示极显著差异(P<0.01)。
* indicates significant differences (P<0.05); ** indicates highly significant differences (P<0.01).
SRD5A有3种分别由Srd5a1基因、Srd5a2基因和Srd5a3基因所编码的同工
芮彩
近年来,外源性类固醇激素能够影响水生生物性腺发育的相关报道越来越多。有研
综上所述,本研究首次从三角帆蚌中克隆了性类固醇激素合成相关酶基因Srd5a1。通过研究其表达特性,发现Srd5a1基因可能与雌性三角帆蚌卵巢发育、成熟以及配子排放密切相关;利用不同浓度外源雌雄激素处理雌性三角帆蚌后,发现Srd5a1基因参与三角帆蚌内源性类固醇激素的合成过程,外源激素可能通过正负调控Srd5a1基因的表达影响雌性三角帆蚌的性腺发育过程。研究结果为三角帆蚌性腺生长发育及其生殖内分泌机理提供参考,同时为实现三角帆蚌单性化养殖提供理论参考价值。
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