【摘要】：改善奶牛的泌乳性能,提高奶牛的泌乳產(chǎn)量,一直以來都是奶牛遺傳育種工作者的目標(biāo)。有研究表明,MEN1基因可以通過影響垂體的功能、抑制催乳素啟動子活性、催化ERα激活等方面,而對乳腺發(fā)育和性能的改善發(fā)揮重要的調(diào)節(jié)功能。MEN1(multiple endocrine neoplasia type 1)基因,是多發(fā)性內(nèi)分泌腫瘤1型綜合征的關(guān)鍵致病基因,其表達(dá)的蛋白menin不僅可以與大量關(guān)鍵轉(zhuǎn)錄因子相互作用,通過表觀遺傳方式調(diào)控其靶基因的轉(zhuǎn)錄和細(xì)胞表型,也可與大量的細(xì)胞內(nèi)信號通路因子相互作用,廣泛參與機(jī)體正常發(fā)育和代謝的平衡穩(wěn)態(tài)調(diào)節(jié)。本研究擬以MEN1基因作為奶牛泌乳重要候選基因,研究其在奶牛乳腺泌乳過程,尤其是在乳蛋白合成過程中的調(diào)控作用。PI3K/Akt/mTOR和JAK2-STAT5信號通路作為乳腺中調(diào)節(jié)乳蛋白合成的重要信號途徑,研究MEN1基因?qū)ζ湫盘柾穬?nèi)因子的調(diào)控作用對于研究該基因?qū)θ榈鞍缀铣傻恼{(diào)控具有重要意義。因此,本研究以奶牛乳腺上皮細(xì)胞系MAC-T為模型,構(gòu)建及瞬時轉(zhuǎn)染重組質(zhì)粒pEGFP-C2-bMEN1,進(jìn)行MEN1基因過表達(dá)實驗;應(yīng)用RNAi的方法瞬時轉(zhuǎn)染細(xì)胞后,進(jìn)行MEN1基因表達(dá)抑制實驗。后采用qRT-PCR、Western blot技術(shù),在mRNA水平及蛋白水平檢測與乳蛋白合成相關(guān)信號通路基因的表達(dá)變化,在mRNA水平檢測主要酪蛋白之一κ-酪蛋白基因CSNK的表達(dá)變化;同時,選取干奶期及泌乳峰期的奶牛乳腺組織,qRT-PCR技術(shù)檢測不同泌乳時期乳腺組織內(nèi)MEN1基因及乳蛋白合成相關(guān)信號通路基因在mRNA水平的表達(dá)關(guān)系。通過體外試驗和體內(nèi)驗證的聯(lián)合,尋求MEN1基因的表達(dá)對奶牛乳腺內(nèi)乳蛋白合成的調(diào)控關(guān)系,從而為奶牛乳腺泌乳調(diào)節(jié)機(jī)制的研究提供一定的理論基礎(chǔ),為遺傳改良奶牛乳品質(zhì)和乳產(chǎn)量奠定理論基礎(chǔ)。研究結(jié)果表明:(1)構(gòu)建的重組質(zhì)粒pEGFP-C2-bMEN1在MAC-T細(xì)胞內(nèi)成功表達(dá),并在轉(zhuǎn)染24h后檢測發(fā)現(xiàn)MEN1基因在mRNA及蛋白水平的表達(dá)量均極顯著提高(P0.01);(2)qRT-PCR檢測發(fā)現(xiàn),MEN1基因的過表達(dá)可在mRNA水平顯著降低包括Akt、mTOR、S6K1、4E-BP1及STAT5在內(nèi)的與乳蛋白合成相關(guān)基因的表達(dá)(P0.01);Western Blot檢測發(fā)現(xiàn)MEN1基因過表達(dá)也同時有下調(diào)包括Akt、mTOR、S6K1在內(nèi)的蛋白表達(dá)(P0.05)的趨勢;而MEN1基因低表達(dá)則表現(xiàn)出相反的調(diào)節(jié)作用。(3)在MEN1基因過表達(dá)條件下檢測發(fā)現(xiàn),作為主要酪蛋白亞型之一的κ-酪蛋白基因CSNK在mRNA水平表達(dá)顯著降低(P0.05),而MEN1基因的低表達(dá)則能顯著上調(diào)CSNK基因在mRNA水平的表達(dá)(P0.01)。(4)在體內(nèi)奶牛乳腺組織的研究結(jié)果顯示,泌乳高峰期乳腺組織內(nèi)MEN1基因的表達(dá)量低于干奶期乳腺組織內(nèi)的表達(dá);而與干奶期相比,泌乳高峰期的乳腺組織在mRNA水平不僅存在較高水平的酪蛋白基因表達(dá),包括CSNB(P0.05)、CSNAS1、CSNAS2、CSNK(P0.05),同時也有更高水平的信號通路內(nèi)基因包括Akt(P0.05)、mTOR、S6K1、4E-BP1及STAT5(P0.05)的表達(dá),這與我們在奶牛乳腺上皮細(xì)胞中所研究的結(jié)果相一致。綜上所述,MEN1基因可通過負(fù)向調(diào)控包括PI3K/Akt/mTOR信號通路及JAK2-STAT5信號通路在內(nèi)的與乳蛋白合成相關(guān)因子的表達(dá),進(jìn)而參與負(fù)向調(diào)控奶牛乳腺上皮細(xì)胞內(nèi)乳蛋白的合成。
[Abstract]:In order to improve the lactating performance of dairy cows and improve the milk production of dairy cows, it has always been the goal of dairy cattle genetics and breeding workers. Some studies have shown that MEN1 gene can play an important role in regulating the development and performance of mammary gland by affecting the function of the pituitary, inhibiting the activity of prolactin promoter and catalyzing the activation of ER alpha (MU),.MEN1 (mu Ltiple endocrine neoplasia type 1) gene is a key pathogenic gene of multiple endocrine tumor type 1 syndrome. Its expressed protein menin can not only interact with a large number of key transcription factors, but also regulate the transcriptional and cell phenotype of the target gene by epigenetic way, and can also interact with a large number of intracellular signaling pathways. This study intends to use MEN1 gene as an important candidate gene for milk lactating in dairy cows. This study is intended to study the lactating process in dairy cows, especially the regulatory role of.PI3K/Akt/mTOR and JAK2-STAT5 signaling pathway in milk protein synthesis as an important regulation of milk protein synthesis in mammary glands. In the signal pathway, the study of the regulation of the MEN1 gene on its signaling pathway is of great significance to the study of the regulation of the gene for milk protein synthesis. Therefore, this study uses the mammary gland epithelial cell line MAC-T as a model to construct and transiently transfect the recombinant plasmid pEGFP-C2-bMEN1 to carry out the MEN1 gene overexpression experiment; the application of RNAi method. After transient transfection, MEN1 gene expression inhibition experiment was carried out. Then qRT-PCR, Western blot technique was used to detect the expression of signal pathway gene related to milk protein synthesis at mRNA level and protein level, and the expression of the main casein protein gene CSNK in the main casein gene was detected at mRNA level; at the same time, the dry milk period and the lactating peak were selected. The expression of MEN1 gene and milk protein synthesis related signal pathway genes in the mammary gland of different lactation period was detected by qRT-PCR technique in the mammary gland tissue of the period of milk. The regulation relationship between the expression of MEN1 gene expression on milk egg white synthesis in dairy cow's mammary gland was sought through the combination of in vitro and in vivo validation, so as to secrete the cow mammary gland. The study of milk regulation mechanism provided a theoretical basis for genetic improvement of milk quality and milk yield. The results showed that: (1) the recombinant plasmid pEGFP-C2-bMEN1 was successfully expressed in MAC-T cells, and the expression of MEN1 gene at mRNA and protein levels was significantly increased after transfection of 24h (P0.01) (2) qRT-PCR detection found that the overexpression of MEN1 gene could significantly reduce the expression of protein synthesis related genes including Akt, mTOR, S6K1,4E-BP1 and STAT5 (P0.01) at mRNA level, and Western Blot detection found that the overexpression of MEN1 gene also decreased the trend of protein expression including Akt, The expression showed the opposite regulation. (3) it was found that the expression of kappa casein gene CSNK, one of the main casein subtypes, decreased significantly at mRNA level (P0.05), while the low expression of MEN1 gene could significantly increase the expression of CSNK gene in mRNA level (P0.01). (4) the mammary gland tissue of dairy cows in the body (P0.01). (3) The results of the study showed that the expression of MEN1 gene in mammary tissue at peak lactation period was lower than that in the breast tissue during the dry milk period. Compared with the dry milking period, the mammary tissue at the peak period of lactation not only had a higher level of casein gene expression at the mRNA level, including CSNB (P0.05), CSNAS1, CSNAS2, CSNK (P0.05), but also higher levels. The genes in the signal pathway include the expression of Akt (P0.05), mTOR, S6K1,4E-BP1 and STAT5 (P0.05). This is in accordance with the results we have studied in the mammary epithelial cells of dairy cows. To sum up, the MEN1 gene can regulate the expression of protein synthesis related factors, including the PI3K/Akt/mTOR signaling pathway and the JAK2-STAT5 signaling pathway, by negative regulation. It participates in the regulation of milk protein synthesis in dairy cow mammary epithelial cells.
【學(xué)位授予單位】：山東農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：S823

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