CN116622715B - 一种编码鸡nkb的基因及其应用 - Google Patents
一种编码鸡nkb的基因及其应用 Download PDFInfo
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Abstract
本发明公开了一种编码鸡NKB的基因,为TAC3基因,TAC3基因的核苷酸序列如SEQ ID NO.1所示,TAC3基因编码的氨基酸序列如SEQ ID NO.2所示。本发明还公开了一种编码鸡NKB的基因的应用。本发明采用上述的一种编码鸡NKB的基因及其应用,研究鸡NKB系统是否参与介导雌激素对GnRH的反馈调控,进一步完善鸟类繁殖调控理论,快速有效地提高常规选育技术以及提高畜牧业经济效益。
Description
技术领域
本发明涉及生物技术领域,尤其是涉及一种编码鸡NKB的基因及其应用。
背景技术
神经激肽B(NKB)是速激肽家族中重要成员,在感觉传导,免疫反应,造血,癌症,抗抑郁,以及激素调节中起重要作用。NKB及其受体主要分布于中枢神经系统,尤其是下丘脑中。早在上世纪90年代初期,人们就注意到NKB及其速激肽家族的其他成员对不同的哺乳动物HPG轴元件有调控作用,且NKB在接近GnRH神经元的ARC表达,推断NKB可能直接调控GnRH的分泌。但直到2009年,当发现人TAC3或TACR3突变可导致先天性低促性腺激素性功能减退综合症(iHH),才真正开始了NKB对GnRH分泌的作用及其机理的研究。用NK3R选择性激动剂对大鼠、小鼠、绵羊、山羊、牛以及猴进行灌注,可诱导LH的分泌。由于在下丘脑弓状核的NKB和NK3R神经元与表达ERα的神经元共存,且NKB免疫性神经元投射到GnRH神经元末梢,表明在哺乳动物中NKB可以调控GnRH和LH的分泌,并且具有物种,甚至是物种内同品种的特异性。然而,鸟类远古祖先的全基因组通过两次大规模的重排,使得编码NKB的基因TAC3仅有EST序列,NKB系统在鸟类中是否介导雌激素对GnRH的研究还是空白。此外,下丘脑-垂体-性腺(HPG)轴是脊椎动物繁殖调控的普遍规律。促性腺激素释放激素(GnRH)是这一轴上首个也是最重要的一个调控信号分子,对调控HPG轴下游其他元件的活动和维持动物正常的繁殖周期具有重要的作用。因此,研究鸡NKB系统是否参与介导雌激素对GnRH的反馈调控,有望进一步完善鸟类繁殖调控理论,快速有效提高常规选育技术以及提高畜牧业经济效益。
发明内容
本发明的目的是提供一种编码鸡NKB的基因及其应用,研究鸡NKB系统是否参与介导雌激素对GnRH的反馈调控,进一步完善鸟类繁殖调控理论,快速有效地提高常规选育技术以及提高畜牧业经济效益。
为实现上述目的,本发明提供了一种编码鸡NKB的基因,为TAC3基因,TAC3基因的核苷酸序列如SEQ ID NO.1所示,TAC3基因编码的氨基酸序列如SEQ ID NO.2所示。
一种编码鸡NKB的基因的克隆方法,包括以下步骤:
S1、NKB编码序列的分段扩增即TAC3基因的分段扩增
以蛋鸡下丘脑cDNA为模板,进行TAC3基因的PCR扩增,TAC3基因分4段进行扩增,PCR产物序列间均交叉重叠,确保扩增出TAC3基因编码区全长序列;
S2、PCR产物纯化回收
将TAC3基因的4种PCR产物回收,然后进行测序;
S3、扩增片段的拼接
将TAC3基因的4个扩增片段进行拼接,得到完整的编码区全长序列。
优选的,PCR扩增过程中所需引物包括F1、R1、F2、R2、F3、R3、F4和R4,其核苷酸序列如SEQ ID NO.3-10所示。
优选的,PCR扩增过程中所用的缓冲体系包括
2x PCR buffer for KOD FX Neo 25μL
dNTP 10μL
KOD FX Neo 1μL
上游引物1.5μL
下游引物1.5μL
模板cDNA 2μL
RNase水9μL。
优选的,PCR扩增过程中的反应程序为94℃,5min,1个循环;98℃,10s,68℃,15s,35个循环。
一种编码鸡NKB的基因在调控GnRH表达中的应用。
应用包括以下步骤:
S1、构建蛋鸡活体卵巢缺失模型
A:假手术组,B:卵巢摘除组,C:卵巢摘除+0.5mg/kg雌激素处理组,D:卵巢摘除+5mg/kg雌激素处理组;
S2、蛋鸡血清中E2和LH水平的测定
步骤S1中的蛋鸡每隔2h收集一次血样,连续收集6次,血液在室温中静置20min后,3000rpm离心20min以收集血清,然后使用鸡E2、LH酶联免疫分析试剂盒进行检测。
优选的,步骤S2中蛋鸡血清中E2和LH水平的测定包括以下步骤:
a、标准品的加样:设置标准品孔和样品孔,标准品孔添加不同浓度的激素的标准品50μL;
b、加样:分别设置空白对照孔和待测样品孔,其中空白对照孔不加样品及酶标试剂,其余步骤操作相同;待测样品孔为先在酶标包被板上加入适量的40μL样品稀释液,然后再加待测样品,使待测样本最终稀释10倍;
c、加酶:在待测样品孔和标准品孔的每孔中加入100μL酶标试剂;
d、温育:封板后在37℃培养箱,培育60min;
e、配液:用等倍的蒸馏水稀释浓缩洗涤液后备用;
f、洗涤:小心揭开封板膜并购弃去液体,甩干,每孔加满洗涤液,静置30s后弃去,如此重复5次,最后拍干孔中废液;
g、显色:每孔按照次序加入50μL显色剂A和50μL显色剂B,随后轻微震荡混匀,在37℃避光显色15min;
h、终止:每孔加入50μL终止液,终止反应,此时蓝色立刻转为黄色;
i、测定:以空白孔作为对照进行调零处理,在450nm波长下依序测量各孔的OD值,测定应在加终止液后15min以内完成;
g、计算:以标准物的浓度为横坐标,OD值为纵坐标,绘制出标准曲线,根据测得的样品OD值和稀释倍数计算相关激素的实际浓度。
本发明所述的一种编码鸡NKB的基因及其应用的优点和积极效果是:
1、本发明中首次在鸡体内克隆出NKB的编码序列即TAC3基因,并进一步分析了其组织表达特性、基因结构和利用不同物种氨基酸序列构建系统进化树,同时对其初步应用也进行了研究。
2、本发明中首次在鸡体内克隆出NKB的编码序列即TAC3基因,研究了鸡NKB系统参与调控GnRH的表达机制,完善了鸟类繁殖调控理论,提高了常规选育技术和畜牧业经济效益。
下面通过附图和实施例,对本发明的技术方案做进一步的详细描述。
附图说明
图1为本发明实施例1中TAC3基因的PCR示意图;
图2为本发明实施例1中TAC3基因在15周龄和30周龄蛋鸡不同组织中的相对表达量;
图3为本发明实施例1中TAC3的基因结构和系统发育树;
图4为本发明实施例3中雌激素对TAC3和GnRH表达的影响;
图5为本发明实施例3中雌激素对LH分泌的影响;
图6为本发明实施例2中ESR1与NKB、NK3R与GnRH的共定位分析。
具体实施方式
以下通过附图和实施例对本发明的技术方案作进一步说明。
除非另外定义,本发明使用的技术术语或者科学术语应当为本发明所属领域内具有一般技能的人士所理解的通常意义。
实施例1
编码鸡NKB的基因克隆及序列分析
1.NKB编码序列的分段扩增即TAC3基因的扩增
以海兰褐蛋鸡下丘脑cDNA样品为模板,使用KOD-Plus-Neo DNA polymerase进行TAC3基因的PCR扩增。TAC3基因分4段进行扩增,PCR产物序列间均交叉重叠,确保扩增出TAC3 CDS区全长序列,扩增过程中所需引物的核苷酸序列如SEQ ID NO.3-10所示,扩增过程中的缓冲体系如表1所示,反应程序如表2所示。
表1缓冲体系
表2反应程序
2.PCR产物纯化回收
将TAC3基因的4种PCR产物回收,然后进行测序。
3.扩增片段的拼接
将TAC3基因的4个扩增片段进行拼接,得到完整的编码区全长序列。
4.序列分析
TAC3基因的核苷酸和其编码的氨基酸序列用NCBI在线BLAST分析,NKB蛋白理化参数用ProtParm在线软件分析,基因结构分析用在线软件GSDS,Mega7.0进行NKB基因进化树的构建。
TAC3基因经PCR反应后用1.5%琼脂糖凝胶电泳,结果如图1所示,其中,图1中①、②、③、④分别表示TAC3基因的4段PCR产物,①、②、③、④中M为DL700 DNA Marker实验组,①、②、③、④中1、2、3为生物学重复。将与预期大小一致的片段经过纯化后进行测序,TAC3基因的四个扩增片段经过拼接之后可获得完整的CDS区全长序列,序列比对后发现与NCBI网站注释的XM_040694044.1序列完全一致,其编码区全长序列为399bp,核苷酸序列如SEQID NO.1所示,能够编码132个氨基酸,氨基酸序列如SEQ ID NO.2所示。
TAC3基因的时空表达谱结果如图2所示,其在不同组织中广泛表达。TAC3基因在生殖相关的器官(下丘脑、垂体、卵巢)、脾脏和肺脏中表达量较高,而在心脏、肝脏、肾脏中表达量较低。此外,30周龄时,TAC3基因在下丘脑、垂体、卵巢中的表达水平显著高于15周龄(P<0.05),然而在肝脏、脾脏和肾脏中30周龄时TAC3基因的表达量显著低于15周龄(P<0.05)。众所周知,雌激素的分泌水平在蛋鸡产蛋高峰期最高,TAC3基因在30W下丘脑的表达水平显著高于15W,暗示其可能受到雌激素的影响。
NKB蛋白理化性质分析表明,鸡NKB蛋白的分子量是13268.15,理论等电点PI为11.57。该蛋白的分子式为C577H931N189O164S4,原子总数是1865个,总共由132个氨基酸组成,其中(Asp+Glu)负电荷残基总数为9,(Arg+Lys)正电荷残基总数为17,不稳定指数为70.29,表明该蛋白质不稳定,如表3所示。
表3NKB蛋白理化性质分析
利用在线网站GSDS对鸡TAC3基因外显子-内含子结构进行分析,利用MEGA7.0邻接法(Neighbor-joining)构建编码NKB(TAC3)基因在不同物种中的系统进化树,如图3所示。结果显示,TAC3基因有4个外显子和3个内含子组成。系统发育的结果显示其与鸟类(鸭、斑马雀、鸲鹟)聚为一支,爪蟾、绿毛龟聚为一支,大鼠、小鼠、猪、人聚为一支,与斑马鱼亲缘关系最远。
实施例2
细胞双标记免疫荧光染色分析NKB与ESR1,NK3R与GnRH的定位关系
为了探索NKB和雌激素受体ESR1,NKB的受体NK3R与GnRH之间的关系,我们采用双标记细胞免疫荧光的方法对下丘脑神经元中NKB与ESR1,NK3R与GnRH进行了共定位分析。具体的操作步骤如下:
(1)细胞培养:细胞铺板培养前先在六孔板中放置灭菌盖玻片,使细胞贴于盖玻片上,待细胞浓度达到70%以上时用37℃的PBS清洗三次;
(2)细胞固定:向6孔板中加入2mL细胞固定液固定细胞30min后使用PBS清洗三次,每次清洗5min;
(3)细胞破膜:加入破膜液在室温下破膜10min后用PBS清洗3次;
(4)画圈血清封闭:用组化笔在爬片四周画圈,在圈内滴加3%BSA孵育10min;
(5)加一抗:弃去封闭液,加入按一定比例配好的一抗4℃孵育过夜;
(6)加二抗:用PBS清洗3次,每次1min。然后加入二抗覆盖细胞,在避光室温条件下孵育50min;
(7)加入第二种一抗:操作同步骤(5);
(8)加入二抗,操作同步骤(6);
(9)DAPI复染细胞核:加入DAPI染液,避光室温孵育10min;
(10)使用PBS(PH=7.4)轻轻清洗3次,每次1min,随后加入抗荧光淬灭封片剂封片;
(11)镜检拍照:在荧光显微镜下观察爬片,并采集图像。
结果如图6所示,结果表明红色的ESR1与绿色的NKB,红色的NK3R与绿色的GnRH共定位于下丘脑相同的神经元中(黄色),为NKB介导雌激素反馈调控GnRH的表达奠定解剖学基础。
实施例3
编码鸡NKB的基因在调控GnRH表达中的应用
1.构建蛋鸡活体卵巢缺失模型
将32只14周龄海兰褐蛋鸡随机分为以下4组,A:假手术组(仅在手术部位做相同的切口),B:卵巢摘除组(OVX),C:卵巢摘除和0.5mg/kg雌激素处理组(OVX+E2(0.5mg/kg)),D:卵巢摘除和5mg/kg雌激素处理组(OVX+E2(5mg/kg))。
这些鸡在试验之前有7d适应期,在此期间提供充足的饮水和饲料。所有鸡在手术前1天都接受了抗生素的肌肉注射,同时12h断食,2h断水。在术前30min肌肉注射200μL酚磺乙胺注射液以减少出血,根据鸡的体重使用速眠新II(0.25mL/kg)肌肉注射对鸡进行麻醉。
手术过程为:将鸡右侧位绑定在手术台上,取下手术部位的羽毛,并使用碘酒对手术部位的皮肤进行消毒。在倒数第一根和第二根肋骨间上1/3处做1~2cm的切口,使用扩张器撑开切口处皮肤,显露腹膜腔。使用钎匙仔细小心的移开肠道,暴露出卵巢,并配合使用手术细钳完全取出卵巢,如卵巢未被彻底取出,可配合使用电烙铁灼烧剩余的卵巢。术后连续三天肌肉注射抗生素,并连续7d在饮水中添加多维和维生素K3粉,继续饲喂到20周龄。
2.鸡血清中E2和LH水平的测定
经四组处理后的鸡每隔2h收集一次血样,连续收集6次,血液在室温中静置20min后,3000rpm离心20min以收集血清。然后使用鸡E2、LH酶联免疫分析试剂盒进行检测,具体步骤如下:
a、标准品的加样:设置标准品孔和样品孔,标准品孔添加不同浓度的激的标准品50μL;
b、加样:分别设置空白对照孔和待测样品孔,其中空白对照孔不加样品及酶标试剂,其余各步骤操作相同;待测样品孔为先在酶标包被板上加入适量的40μL样品稀释液,然后再加待测样品,使待测样本最终稀释10倍;
c、加酶:在待测样品孔和标准品孔的每孔中加入100μL酶标试剂;
d、温育:封板后在37℃培养箱,培育60min;
e、配液:用等倍的蒸馏水稀释浓缩洗涤液后备用;
f、洗涤:小心揭开封板膜并购弃去液体,甩干,每孔加满洗涤液,静置30s后弃去,如此重复5次,最后拍干孔中废液;
g、显色:每孔按照次序加入50μL显色剂A和50μL显色剂B,随后轻微震荡混匀,在37℃避光显色15min;
h、终止:每孔加入50μL终止液,终止反应(此时蓝色立刻转为黄色);
i、测定:以空白孔作为对照进行调零处理,在450nm波长下依序测量各孔的吸光度(OD值)。测定应在加终止液后15min以内完成;
g、计算:以标准物的浓度为横坐标,OD值为纵坐标,利用CurveExpert软件绘制出标准曲线,根据测得的样品OD值和稀释倍数计算相关激素的实际浓度。
通过构建蛋鸡活体卵巢缺失模型,结果发现A组卵巢发育正常,存在着明显的等级以及等级前卵泡。然而摘除卵巢的B、C、D三组无发育的卵泡和卵巢基质,表明卵巢摘除试验成功。对摘除卵巢的B、C、D三组蛋鸡卵巢缺失模型注射低、高剂量雌激素后,采用qRT-PCR和ELISA的方法检测了TAC3和GnRH的表达以及LH的分泌,结果发现摘除卵巢并注射低、高剂量雌激素后,能够抑制由摘除卵巢引起的TAC3和GnRH mRNA水平的上升(如图4所示)以及抑制LH的分泌(如图5所示),并且注射高剂量雌激素能够使LH恢复至正常的分泌水平。此外,释放到垂体门静脉系统的GnRH分泌模式与LH分泌模式相关,且LH的分泌模式和分泌水平可以反应GnRH的分泌模式和分泌水平。由于GnRH是经由门静脉系统直接作用于垂体,无法在血清中检测到GnRH,我们期望以LH的分泌水平来间接体现GnRH的分泌水平。综上所述,NKB能够介导雌激素对GnRH的调控。
因此,本发明采用上述的一种编码鸡NKB的基因及其应用,研究鸡NKB系统是否参与介导雌激素对GnRH的反馈调控,进一步完善鸟类繁殖调控理论,快速有效地提高常规选育技术以及提高畜牧业经济效益。
最后应说明的是:以上实施例仅用以说明本发明的技术方案而非对其进行限制,本领域的普通技术人员应当理解:其依然可以对本发明的技术方案进行修改或者等同替换,而这些修改或者等同替换亦不能使修改后的技术方案脱离本发明技术方案的精神和范围。
Claims (2)
1.一种编码鸡NKB的基因在调控GnRH表达中的应用,其特征在于,编码鸡NKB的基因为TAC3基因,TAC3基因的核苷酸序列如SEQ ID NO.1所示,TAC3基因编码的氨基酸序列如SEQID NO.2所示;
所述应用,包括以下步骤:
S1、构建蛋鸡活体卵巢缺失模型
A:假手术组,B:卵巢摘除组,C:卵巢摘除+0.5mg/kg雌激素处理组,D:卵巢摘除+5mg/kg雌激素处理组;
S2、蛋鸡血清中E2和LH水平的测定
步骤S1中的蛋鸡每隔2h收集一次血样,连续收集6次,血液在室温中静置20min后,3000rpm离心20min以收集血清,然后使用鸡E2、LH酶联免疫分析试剂盒进行检测。
2.根据权利要求1所述的应用,其特征在于:所述步骤S2中蛋鸡血清中E2和LH水平的测定包括以下步骤:
a、标准品的加样:设置标准品孔和样品孔,标准品孔添加不同浓度的激素的标准品50μL;
b、加样:分别设置空白对照孔和待测样品孔,其中空白对照孔不加样品及酶标试剂,其余步骤操作相同;待测样品孔为先在酶标包被板上加入适量的40μL样品稀释液,然后再加待测样品,使待测样本最终稀释10倍;
c、加酶:在待测样品孔和标准品孔的每孔中加入100μL酶标试剂;
d、温育:封板后在37℃培养箱,培育60min;
e、配液:用等倍的蒸馏水稀释浓缩洗涤液后备用;
f、洗涤:小心揭开封板膜并购弃去液体,甩干,每孔加满洗涤液,静置30s后弃去,如此重复5次,最后拍干孔中废液;
g、显色:每孔按照次序加入50μL显色剂A和50μL显色剂B,随后轻微震荡混匀,在37℃避光显色15min;
h、终止:每孔加入50μL终止液,终止反应,此时蓝色立刻转为黄色;
i、测定:以空白孔作为对照进行调零处理,在450nm波长下依序测量各孔的OD值,测定应在加终止液后15min以内完成;
g、计算:以标准物的浓度为横坐标,OD值为纵坐标,绘制出标准曲线,根据测得的样品OD值和稀释倍数计算相关激素的实际浓度。
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