綿羊PRLR,PRLH和PRLHR基因的克隆及其在下丘腦—垂體—性腺中表達(dá)的研究
發(fā)布時間：2018-11-19 14:54
【摘要】：催乳素(PRL)是由垂體前葉合成的多肽激素,通過與其受體(PRLR)結(jié)合而發(fā)揮眾多的生理功能,研究也發(fā)現(xiàn)催乳素釋放激素(PRLH)及其受體(PRLHR)與PRL的分泌有關(guān)。本研究以綿羊為實驗材料,以下丘腦-垂體-性腺軸為研究對象,運(yùn)用分子生物學(xué)、免疫組化、熒光定量的方法對PRLR及PRLHR的具體表達(dá)情況作初步研究,其結(jié)果如下:(1)成功克隆了PRLR,PRLH和PRLHR。對PRLR基因序列進(jìn)行同源性比對和進(jìn)化樹建立顯示,其與藏羚羊的基因序列最接近;預(yù)測PRLR基因編碼581個氨基酸的蛋白序列,其分子量為65.23kDa,等電點(diǎn)為p I5.12,且PRLR蛋白不太穩(wěn)定;預(yù)測PRLHR編碼含有370個氨基酸的蛋白序列,其分子量為41.05kDa,等電點(diǎn)為p I9.41,且PRLHR蛋白不穩(wěn)定。對PRLH和PRLHR基因序列進(jìn)行同源性比對和進(jìn)化樹建立顯示,綿羊的PRLH與山羊的同源性最高,而PRLHR則與藏羚羊的同源性最高。(2)三個基因在綿羊的四種組織中均有表達(dá)。PRLR以表達(dá)量最少的子宮為標(biāo)準(zhǔn)計算其他組織的表達(dá)量,結(jié)果卵巢的表達(dá)量顯著高于其他組織(P0.05),其表達(dá)量由高到低依次是卵巢下丘腦垂體子宮。PRLH以表達(dá)最少的下丘腦為標(biāo)準(zhǔn),子宮的表達(dá)量顯著高于其他組織(P0.05),組織的表達(dá)量由高到低依次是子宮卵巢垂體下丘腦;PRLHR以表達(dá)量最少子宮為標(biāo)準(zhǔn),發(fā)現(xiàn)子宮和垂體之間的表達(dá)差異顯著(P0.05);組織的表達(dá)量由高到低依次是垂體卵巢下丘腦子宮。(3)PRLR和PRLHR在綿羊的垂體、下丘腦、子宮和卵巢組織中的不同細(xì)胞中均有一定量的表達(dá)。PRLR和PRLHR在垂體上遠(yuǎn)側(cè)端的陽性反應(yīng)著色深,陽性細(xì)胞多呈圓形或卵圓形;同時它們在下丘腦中均有陽性反應(yīng),但染色強(qiáng)度都較輕;PRLR在子宮腔、子宮內(nèi)膜以及子宮腺等部位均有陽性產(chǎn)物,而PRLHR只在子宮內(nèi)膜處陽性反應(yīng)明顯,其他部位很少反應(yīng);卵巢中PRLR陽性細(xì)胞主要分布于卵母細(xì)胞和顆粒細(xì)胞中,相反PRLHR的陽性產(chǎn)物較少;除此之外PRLR的陽性反應(yīng)區(qū)域還包括上述組織的血管和紅細(xì)胞。
[Abstract]:Prolactin (PRL) is a polypeptide hormone synthesized from the anterior pituitary gland and plays a large number of physiological functions by binding to its receptor (PRLR). It has also been found that the prolactin releasing hormone (PRLH) and its receptor (PRLHR) are related to the secretion of PRL. In this study, sheep were used as experimental materials and hypothalamus-pituitary-gonad axis was used to study the specific expression of PRLR and PRLHR by molecular biology, immunohistochemistry and fluorescence quantitative method. The results are as follows: (1) PRLR,PRLH and PRLHR. were cloned successfully. The homology alignment of PRLR gene sequence and the establishment of evolutionary tree showed that the sequence of PRLR gene was the closest to that of Tibetan antelope. The protein sequence of 581 amino acids encoded by PRLR gene was predicted. Its molecular weight was 65.23 kDa, the isoelectric point was Pi 5.12, and the PRLR protein was not stable. It is predicted that PRLHR encodes 370 amino acids with molecular weight of 41.05 kDa, isoelectric point of Pi 9.41, and instability of PRLHR protein. The homology alignment of PRLH and PRLHR gene sequence and the establishment of evolutionary tree showed that the homology of PRLH between sheep and goat was the highest. However, PRLHR had the highest homology with Tibetan antelope. (2) three genes were expressed in four tissues of sheep. PRLR was used to calculate the expression of other tissues according to the least expressed uterus. Results the expression of ovary was significantly higher than that of other tissues (P0.05), and the expression of ovarian hypothalamus-pituitary uterus was in turn from high to low. The expression of PRLH was based on the hypothalamus, and the expression of uterus was significantly higher than that of other tissues (P0.05). The order of tissue expression from high to low was the hypothalamus of uterus, ovary and pituitary. The expression of PRLHR was the lowest in uterus, and the difference between uterus and pituitary was significant (P0.05). The order of tissue expression from high to low is pituitary ovary hypothalamic uterus. (3) PRLR and PRLHR in the pituitary and hypothalamus of sheep. The positive reaction of PRLR and PRLHR in the distal end of pituitary gland was deep and the positive cells were round or oval. At the same time, they all had positive reaction in hypothalamus, but the intensity of staining was light, PRLR had positive products in uterine cavity, endometrium and uterus gland, but PRLHR only showed positive reaction in endometrium, but few in other parts. PRLR positive cells were mainly distributed in oocytes and granulosa cells, on the contrary, the positive products of PRLHR were less. In addition, the positive regions of PRLR also included the blood vessels and erythrocytes of the above tissues.
【學(xué)位授予單位】：甘肅農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S826
本文編號：2342678
[Abstract]:Prolactin (PRL) is a polypeptide hormone synthesized from the anterior pituitary gland and plays a large number of physiological functions by binding to its receptor (PRLR). It has also been found that the prolactin releasing hormone (PRLH) and its receptor (PRLHR) are related to the secretion of PRL. In this study, sheep were used as experimental materials and hypothalamus-pituitary-gonad axis was used to study the specific expression of PRLR and PRLHR by molecular biology, immunohistochemistry and fluorescence quantitative method. The results are as follows: (1) PRLR,PRLH and PRLHR. were cloned successfully. The homology alignment of PRLR gene sequence and the establishment of evolutionary tree showed that the sequence of PRLR gene was the closest to that of Tibetan antelope. The protein sequence of 581 amino acids encoded by PRLR gene was predicted. Its molecular weight was 65.23 kDa, the isoelectric point was Pi 5.12, and the PRLR protein was not stable. It is predicted that PRLHR encodes 370 amino acids with molecular weight of 41.05 kDa, isoelectric point of Pi 9.41, and instability of PRLHR protein. The homology alignment of PRLH and PRLHR gene sequence and the establishment of evolutionary tree showed that the homology of PRLH between sheep and goat was the highest. However, PRLHR had the highest homology with Tibetan antelope. (2) three genes were expressed in four tissues of sheep. PRLR was used to calculate the expression of other tissues according to the least expressed uterus. Results the expression of ovary was significantly higher than that of other tissues (P0.05), and the expression of ovarian hypothalamus-pituitary uterus was in turn from high to low. The expression of PRLH was based on the hypothalamus, and the expression of uterus was significantly higher than that of other tissues (P0.05). The order of tissue expression from high to low was the hypothalamus of uterus, ovary and pituitary. The expression of PRLHR was the lowest in uterus, and the difference between uterus and pituitary was significant (P0.05). The order of tissue expression from high to low is pituitary ovary hypothalamic uterus. (3) PRLR and PRLHR in the pituitary and hypothalamus of sheep. The positive reaction of PRLR and PRLHR in the distal end of pituitary gland was deep and the positive cells were round or oval. At the same time, they all had positive reaction in hypothalamus, but the intensity of staining was light, PRLR had positive products in uterine cavity, endometrium and uterus gland, but PRLHR only showed positive reaction in endometrium, but few in other parts. PRLR positive cells were mainly distributed in oocytes and granulosa cells, on the contrary, the positive products of PRLHR were less. In addition, the positive regions of PRLR also included the blood vessels and erythrocytes of the above tissues.
【學(xué)位授予單位】：甘肅農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S826
【共引文獻(xiàn)】
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1 生冉;閆素梅;;催乳素與其他激素對乳腺內(nèi)乳成分合成的協(xié)同調(diào)節(jié)作用[J];動物營養(yǎng)學(xué)報;2014年06期
2 陳駿;黎開燕;李艷;李文勝;;養(yǎng)血生乳口服液促進(jìn)產(chǎn)后泌乳和產(chǎn)褥復(fù)舊的臨床觀察[J];現(xiàn)代中西醫(yī)結(jié)合雜志;2014年14期
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2 秦姣;卡麥角林對黃毛鼠的不育效果及其作用機(jī)理[D];中國農(nóng)業(yè)大學(xué);2015年
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1 王慧;PRLR基因在西農(nóng)薩能羊乳腺上皮細(xì)胞中的RNA干擾研究[D];西北農(nóng)林科技大學(xué);2013年
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