CN114015709B - 绵羊ctsd基因在调控初情期启动中的应用 - Google Patents
绵羊ctsd基因在调控初情期启动中的应用 Download PDFInfo
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Abstract
本发明涉及动物生物技术领域,具体涉及绵羊CTSD基因在调控初情期启动中的应用。本发明发现绵羊CTSD基因能够调控动物生殖激素的分泌以及生殖相关基因的表达,增强动物体内CTSD基因的表达能够促进生殖激素的分泌,提高生殖相关基因的表达量,进而促进动物初情期的启动。绵羊CTSD基因的新功能的发现为培育初情期早的绵羊品种提供了基因资源和新方法。
Description
技术领域
本发明涉及动物生物技术领域,具体涉及绵羊CTSD基因在调控初情期启动中的应用。
背景技术
初情期是指母畜初次发情并发生排卵、公畜睾丸具有分泌和生精功能的时期,是动物获得繁殖能力的重要时期。初情期出现的早晚直接关系到动物的繁殖性能,培育初情期早的母畜可以节约饲养成本,提高母畜的利用率。
多浪羊是新疆的优良肉脂兼用型绵羊品种,具有较高的繁殖能力。多浪羊具有初情期早、肉质鲜嫩、产肉多、毛质好、繁殖率高等优良性状。在饲养过程中,初情期早的绵羊可以增加母畜终生产羔数从而降低饲养成本,提高母畜利用率。开发与多浪羊初情期调控相关的基因对于培育初情期早的绵羊具有重要意义。
发明内容
本发明的目的是提供绵羊CTSD基因在调控初情期启动中的应用。
本发明以多浪羊为研究对象,分析其初情期启动机制。以初情期前、中、后的多浪羊下丘脑为试材,利用RNA-seq测序分析技术寻找影响母羊发情的基因,经筛选和验证获得参与初情期调控的相关基因CTSD基因。本发明发现,CTSD基因具有调控生殖激素分泌、生殖相关基因表达的功能,增强CTSD基因基因的表达,生殖激素分泌增加,生殖相关基因表达增强,进而发挥调控初情期启动的功能。
具体地,本发明提供以下技术方案:
第一方面,本发明提供绵羊CTSD基因、其编码蛋白或含有所述CTSD基因的生物材料在调控动物生殖激素分泌中的应用。
以上所述的生殖激素为选自促性腺激素释放激素、促卵泡素、雌激素、孕激素、促黄体素中的一种或多种。
第二方面,本发明提供绵羊CTSD基因、其编码蛋白或含有所述CTSD基因的生物材料在调控动物生殖相关基因表达中的应用。
以上所述的生殖相关基因优选为GnRH基因。
第三方面,本发明提供绵羊CTSD基因、其编码蛋白或含有所述CTSD基因的生物材料在动物初情期调控中的应用。
以上所述的初情期调控为初情期启动的调控。
第四方面,本发明提供绵羊CTSD基因、其编码蛋白或含有所述CTSD基因的生物材料在初情期调控的动物遗传育种或转基因动物构建中的应用。
优选地,所述动物遗传育种、转基因动物构建为初情期早的动物遗传育种或转基因动物构建。
本发明中,绵羊CTSD基因的编码蛋白具有如下任一种氨基酸序列:
(1)如SEQ ID NO.1所示的氨基酸序列;
(2)如SEQ ID NO.1所示的氨基酸序列经一个或多个氨基酸的替换、插入或缺失得到的具有相同功能蛋白的氨基酸序列;
(3)与如SEQ ID NO.1所示的氨基酸序列具有至少90%同源性的氨基酸序列;优选地,所述同源性为至少95%;更优选为99%。
根据上述绵羊CTSD基因的编码蛋白的氨基酸序列,本领域技术人员能够确定绵羊CTSD基因的核苷酸序列。
作为本发明的一种实施方式,绵羊CTSD基因的核苷酸序列如SEQ ID NO.2所示。
本发明中,所述生物材料包括表达盒、载体或宿主细胞,其中,载体可以是质粒、病毒、转座子、噬菌体等,宿主细胞可以是微生物细胞或动物细胞。
以上所述的应用中,通过增强CTSD基因的表达,促进动物生殖激素分泌、增强动物生殖相关基因的表达或促进动物初情期的启动。
本发明中,所述动物优选为羊或鼠,更优选为绵羊或小鼠。
第五方面,本发明提供一种促进动物生殖激素分泌或促进动物初情期启动的方法,该方法包括增强CTSD基因表达的步骤;
所述CTSD基因的编码蛋白具有如下任一种氨基酸序列:
(1)如SEQ ID NO.1所示的氨基酸序列;
(2)如SEQ ID NO.1所示的氨基酸序列经一个或多个氨基酸的替换、插入或缺失得到的具有相同功能蛋白的氨基酸序列;
(3)与如SEQ ID NO.1所示的氨基酸序列具有至少90%同源性的氨基酸序列;优选地,所述同源性为至少95%;更优选为99%。
以上所述的方法中,生殖激素为选自促性腺激素释放激素、促卵泡素、雌激素、孕激素、促黄体素中的一种或多种。
以上所述的方法中,可通过基因工程技术增强动物体内CTSD基因的表达,例如:在动物细胞中导入CTSD基因的过表达载体等。
本发明的有益效果在于:本发明发现了绵羊CTSD基因能够调控动物生殖激素的分泌以及生殖相关基因的表达,增强动物体内CTSD基因的表达能够促进生殖激素的分泌,提高生殖相关基因的表达量,进而促进动物初情期的启动。绵羊CTSD基因的新功能的发现为培育初情期早的绵羊品种提供了基因资源和新方法。
附图说明
图1为本发明实施例2中CTSD基因过表达载体构建的菌落PCR鉴定结果图,其中,M:DL2000,从上到下分别是2000,1000,750,500,250,100bp;1-6分别为不同克隆的菌落PCR产物。
图2为本发明实施例2中CTSD基因过表达载体转染小鼠下丘脑神经元细胞48小时的图片,其中,左侧图片为转染48h明场图,右侧图片为48h荧光图。
图3为本发明实施例2中CTSD基因过表达载体转染小鼠下丘脑神经元细胞后促卵泡素(FSH)的检测结果,其中,CON代表对照组,FSH代表CTSD基因过表达载体转染细胞。
图4为本发明实施例2中CTSD基因过表达载体转染小鼠下丘脑神经元细胞后促黄体素(LH)的检测结果,其中,CON代表对照组,LH代表CTSD基因过表达载体转染细胞。
图5为本发明实施例2中CTSD基因过表达载体转染小鼠下丘脑神经元细胞后孕激素(P)的检测结果,其中,CON代表对照组,P代表CTSD基因过表达载体转染细胞。
图6为本发明实施例2中CTSD基因过表达载体转染小鼠下丘脑神经元细胞后促性腺激素释放激素(GnRH)的检测结果,其中,CON代表对照组,GnRH代表CTSD基因过表达载体转染细胞。
图7为本发明实施例2中CTSD基因过表达载体转染小鼠下丘脑神经元细胞后雌激素(E)的检测结果,其中,CON代表对照组,E代表CTSD基因过表达载体转染细胞。
图8为本发明实施例2中CTSD基因过表达对GnRH基因表达的影响,其中,CON为转染空载体的对照组,CTSD24h、CTSD48h、CTSD72h分别代表CTSD过表达载体转染24h、48h和72h。
具体实施方式
以下实施例用于说明本发明,但不用来限制本发明的范围。
实施例1调控多浪羊初情期的关键基因的筛选和确定
以初情期前、中、后的多浪羊下丘脑为试材,利用RNA-seq测序分析技术寻找响应母羊发情的基因,经筛选和验证获得参与初情期调控的相关基因,具体方法如下:
每日分三个时间段以母羊接受爬跨且外阴有黏液为标准观察鉴定母羊首次发情,对多浪羊初情期前、初情期、初情期后三个阶段的各三个生物学重复进行下丘脑组织采集,并提取RNA,对提取的RNA进行质量检测,构建文库进行转录组测序,获得测序结果并进行分析。
其中,转录组文库质量评估方法为:
(1)通过检验插入片段在基因上的分布,评估mRNA片段化的随机性、mRNA的降解情况。
(2)通过插入片段的长度分布,评估插入片段长度的离散程度。
(3)通过绘制饱和度图,评估文库容量和Mapped Data是否充足。
转录组测序数据的分析方法如下:
(1)对raw reads进行数据质控,从raw reads中删除包含adapter的reads、包含ploy-N的reads和低质量的reads得到clean reads。
(2)使用HISAT2软件将clean reads比对到绵羊参考基因组(Oar_v4.0)上。使用StringTie进行组装并且使用FPKM表示基因的表达量。
(3)使用R包corrplot对三个发情时期内的三个生物学重复样品计算相关性系数。使用R包DEseq2鉴定差异基因,阈值为Fold change≥1.5,P-value≤0.05。
转录组测序分析差异基因分三组,第一组为初情期前和初情期进行差异基因分析,第二组为初情期前和初情期后进行差异基因分析,第三组为初情期和初情期后进行差异基因分析。通过以上三种比较方法最终确定出与多浪羊初情期启动的关键基因。
结果显示,在初情期前与初情期的比较中,共得到575个差异基因,其中上调基因490个,下调基因85个。在初情期前与初情期后的比较中,共得到166个差异基因,其中上调基因96个,下调基因70个。在初情期与初情期后的比较中,共得到648个差异基因,其中上调基因97个,下调基因551个。在初情期启动过程中,上调基因占更大比例,在初情期结束过程中,下调基因占更大比例。
对获得的差异基因进行GO功能注释和KEGG分析,发现了与初情期启动相关的GO条目,包括response to estrogen(GO:0043627),cellular response to gonadotropinstimulus(GO:0071371),copulation(GO:0007620),developmental process involved inreproduction(GO:0003006),female pregnancy(GO:0007565),estrogen receptorbinding(GO:0030331),ovarian follicle development(GO:0001541),mating behavior(GO:0007617),fertilization(GO:0009566),oocyte maturation(GO:0001556),celldifferentiation involved in embryonic placenta development(GO:0060706),gonaddevelopment(GO:0008406),positive regulation of germinal center formation(GO:0002636);与初情期启动相关的KEGG通路有:Estrogen signaling pathway(ko04915),Oxytocin signaling pathway(ko04921),GnRH signaling pathway(ko04912),Progesterone-mediated oocyte maturation(ko04914),Prolactin signaling pathway(ko04917),Ovarian steroidogenesis(ko04913)。
结合GO注释、KEGG分析和筛选,对初步鉴定得到的CTSD等基因进行qPCR验证,结合qPCR检测结果,最终选择CTSD基因作为与初情期启动相关的候选基因进行后续试验。
CTSD基因编码蛋白的氨基酸序列如SEQ ID NO.1所示,CTSD基因的核苷酸序列如SEQ ID NO.2所示。
实施例2CTSD基因过表达促进生殖激素分泌及生殖相关基因表达
为分析CTSD基因与生殖相关的功能,构建CTSD基因的过表达载体并转染小鼠下丘脑神经元细胞,具体方法如下:
1、CTSD基因的过表达载体的构建
(1)CTSD基因的PCR扩增
PCR反应体系如表1所示。
表1 PCR反应体系
组分 | 体积(μl) |
2×KOD-FX Buffer | 25 |
2mM dNTPs | 10 |
DNA | 2 |
正向引物(10μM) | 1.5 |
反向引物(10μM) | 1.5 |
KOD-FX DNA聚合酶 | 1 |
<![CDATA[ddH<sub>2</sub>O]]> | 9 |
总体积 | 50 |
PCR反应程序如表2所示。
表2 PCR反应程序
将PCR扩增产物进行1%琼脂糖凝胶电泳,确定有特异扩增后,再进行3个50μl体系的PCR扩增,回收PCR产物,具体方法如下:
a、在1.5ml离心管(含有PCR反应体积200μl)中加入4倍体积(800μl)的Buffer CP;
b、剧烈震荡,短暂离心;
c、将吸附柱放在收集管里;
d、将步骤c的混合物转入吸附柱(若体积大于750μl,可分多次吸附,每次750μl,离心后,倒掉废液,再把剩余混合物转入吸附柱离心);
e、13000g离心1min,弃滤液;
f、加入700μl洗脱液,13000g离心1min,弃滤液;
g、加入500μl洗脱液,13000g离心1min,弃滤液;
h、13000g离心2min,离心去除吸附柱上的乙醇;
i、将吸附柱转到一个新的1.5ml离心管,在吸附柱中心加入30μl ddH2O,室温放置1min;13000g离心2min,滤液即是回收的DNA。
(2)将PCR产物与表达载体pGL4.18经双酶切后连接,具体方法如下:
双酶切按表3配制反应体系。
表3双酶切反应体系
组分 | 体积(μl) |
<![CDATA[ddH<sub>2</sub>O]]> | 6 |
10×FastDigest Green Buffer | 2 |
pGL4.18 | 10 |
FastDigest HindIII | 1 |
FastDigest XhoI | 1 |
总体积 | 20 |
连接反应按表4配制反应体系。
表4连接反应体系
组分 | 体积(μl) |
5×Reaction Buffer | 4 |
目标片段 | 4 |
载体 | 12 |
总体积 | 20 |
将连接反应体系轻轻摇匀,短暂离心后,于冰上放置30min,得到的连接产物直接转化。
(3)将连接后的过表达载体转化至宿主细胞,方法如下:
a、取出化学感受态细胞大肠杆菌Top10放于冰水混合物中解冻。
b、加入连接产物,得到混合物,冰上放置30min。
c、将混合物置于离心管中,于42℃水浴90s,不可摇动。
d、水浴结束后,快速将离心管移到冰浴上2min,室温放置5min。
e、每管加800μl未添加抗生素的LB液体培养基,37℃振荡45min复苏。
f、8000rpm离心1min,将上清液去掉800μl,重悬后均匀涂布到抗性平板上。
g、平板放置于室温吹干,倒置于37℃培养箱,培养12-16h长出菌落。
h、进行菌落PCR鉴定和测序鉴定。
菌落PCR鉴定结果如图1所示,结合测序鉴定结果,表明CTSD基因的过表达载体构建成功。
2、无内毒素质粒提取
对上述步骤1构建的CTSD基因的过表达载体进行一次中量的无内毒素质粒提取,以免在转染过程中,内毒素对细胞造成伤害,将提取得到的CTSD基因的过表达载体用于后续细胞转染。
3、转染前细胞准备
复苏一管小鼠下丘脑神经元细胞,经过2-3次传代后,待细胞状态较好时用于转导、传代,具体方法如下:
(1)将DMEM+10%FBS培养基、1×PBS和Trypsin-EDTA预先平衡至到37℃。
(2)吸去培养液,加入适量Trypsin-EDTA,轻轻旋转,使Trypsin-EDTA均匀覆盖培养器皿表面,消化1-2分钟,显微镜下可见细胞间隙增大,细胞变圆,用手轻拍培养器皿的壁,立即加入2mL的DMEM+10%FBS完全培养基终止消化。
(3)用吸管吸取液体,轻轻吹打培养器皿表面,反复3-5次,使细胞彻底脱离瓶皿底壁,将细胞移入离心管中。离心(900g,2分钟),吸去上清液。
(4)加入5mL的DMEM+10%FBS培养基重悬细胞沉淀,用吸管轻轻吹打收集的细胞悬液,取样计数。参照计数结果,取5×105cells/well的密度接种到6-well plate备转染。
4、质粒共转染细胞
通常6孔小鼠下丘脑神经元细胞转染3000ng的DNA。转染共分为4组,具体如下:空载体组,CSTD过表达载体转染24h、48h、72h。具体转染方法如下:
(1)接种小鼠下丘脑神经元细胞隔夜后,即可进行转导;转导前,将实验孔的细胞换成1mL新鲜的DMEM培养基后放入37℃、5%CO2、95%相对湿度的培养箱中培养。
(2)按如下步骤制备DNA-Lipo2000复合物(参照lipo2000manual):将质粒3000ng与150μl转染用Opti培养基混合,得到质粒-Opti混合物,将7μl lipo2000与150μl转染用Opti培养基混合,得到lip2000-opti混合物,将质粒-Opti混合物加入到lip2000-opti混合物中,形成DNA-lipo2000的复合体。室温放置5-10min用于复合体形成。(3)弃培养基,每孔小鼠下丘脑神经元细胞加入300μl DNA-lipo2000-opti混合物。
(4)转染6-8h后,换正常培养基培养过夜。
结果显示,CTSD过表达载体成功转染进小鼠下丘脑神经元细胞(图2)。
5、细胞收集、生殖激素水平和生殖相关基因表达检测
(1)分别在转染24、48、72h收集培养上清及细胞,利用酶联免疫标记方法(ELISA)检测CTSD基因过表达载体转染细胞的上清中的GnRH(促性腺激素释放激素)、FSH(促卵泡素)、LH(促黄体素)、E(雌激素)、P(孕激素)的浓度,以转染空载体的小鼠下丘脑神经元细胞作为对照组。
检测结果如图3、图4、图5、图6和图7所示,结果显示,转染CTSD基因过表达载体的细胞在24、48、72h这三个时期的GnRH浓度平均值为23.5066±1.9428mIU/mL,对照组为21.6633±0.5043mIU/mL,相对于对照组,转染CTSD基因过表达载体的细胞的GnRH浓度显著提高(P<0.05);FSH浓度平均值为20.99±2.8465mIU/mL,对照组为18.3433±0.4354mIU/mL,相对于对照组,转染CTSD基因过表达载体的细胞的FSH浓度显著提高(P<0.05);转染CTSD基因过表达载体的细胞的LH浓度平均值为12.27±1.2057mU/ml,对照组为9.9133±0.3604mU/ml,两者差异不显著(P>0.05);E的浓度平均值为42.566±1.7676pg/mL,对照组为36.64±0.6829pg/mL,两者差异不显著(P>0.05);P的浓度平均值为6.0667±1.4438ng/ml,对照组为4.17±0.1014ng/ml,两者差异不显著(P>0.05)。该试验结果说明CTSD基因过表达后,GnRH、FSH、LH、E、P这5种生殖激素整体呈上升趋势,说明CTSD基因有利于生殖激素的分泌。
(2)提取RNA,并利用qPCR分别检测转染后24、48、72h的过表达基因CTSD及GnRH基因的相对表达量。
结果显示,当CTSD转染到48h时,GnRH表达量出现上调,且相较于其他组差异显著(p<0.05)(图8)。
以上结果表明,CTSD过表达促进下丘脑神经元细胞中生殖激素的分泌以及GnRH基因的表达。
虽然,上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述,但在本发明基础上,可以对之作一些修改或改进,这对本领域技术人员而言是显而易见的。因此,在不偏离本发明精神的基础上所做的这些修改或改进,均属于本发明要求保护的范围。
序列表
<110> 塔里木大学
<120> 绵羊CTSD基因在调控初情期启动中的应用
<130> KHP211121169.4
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Claims (9)
1.绵羊CTSD基因、其编码蛋白或含有所述CTSD基因的生物材料在调控动物生殖激素分泌中的应用。
2.根据权利要求1所述的应用,其特征在于,所述生殖激素为选自促性腺激素释放激素、促卵泡素、雌激素、孕激素、促黄体素中的一种或多种。
3.绵羊CTSD基因、其编码蛋白或含有所述CTSD基因的生物材料在调控动物GnRH基因表达中的应用。
4.绵羊CTSD基因、其编码蛋白或含有所述CTSD基因的生物材料在动物初情期调控中的应用。
5.根据权利要求4所述的应用,其特征在于,所述初情期调控为初情期启动的调控。
6.绵羊CTSD基因、其编码蛋白或含有所述CTSD基因的生物材料在初情期调控的动物遗传育种或转基因动物构建中的应用。
7.根据权利要求1~6任一项所述的应用,其特征在于,所述绵羊CTSD基因的编码蛋白的氨基酸序列如SEQ ID NO.1所示。
8.根据权利要求1~6任一项所述的应用,其特征在于,通过增强CTSD基因的表达,促进动物生殖激素分泌、增强动物GnRH基因的表达或促进动物初情期的启动。
9.一种促进动物生殖激素分泌或促进动物初情期启动的方法,其特征在于,包括增强CTSD基因表达的步骤,所述CTSD基因的编码蛋白的氨基酸序列如SEQ ID NO.1所示。
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