MEN1基因参与调控奶牛乳腺上皮细胞内乳蛋白的合成
发布时间：2018-08-08 13:04
【摘要】：改善奶牛的泌乳性能,提高奶牛的泌乳产量,一直以来都是奶牛遗传育种工作者的目标。有研究表明,MEN1基因可以通过影响垂体的功能、抑制催乳素启动子活性、催化ERα激活等方面,而对乳腺发育和性能的改善发挥重要的调节功能。MEN1(multiple endocrine neoplasia type 1)基因,是多发性内分泌肿瘤1型综合征的关键致病基因,其表达的蛋白menin不仅可以与大量关键转录因子相互作用,通过表观遗传方式调控其靶基因的转录和细胞表型,也可与大量的细胞内信号通路因子相互作用,广泛参与机体正常发育和代谢的平衡稳态调节。本研究拟以MEN1基因作为奶牛泌乳重要候选基因,研究其在奶牛乳腺泌乳过程,尤其是在乳蛋白合成过程中的调控作用。PI3K/Akt/mTOR和JAK2-STAT5信号通路作为乳腺中调节乳蛋白合成的重要信号途径,研究MEN1基因对其信号通路内因子的调控作用对于研究该基因对乳蛋白合成的调控具有重要意义。因此,本研究以奶牛乳腺上皮细胞系MAC-T为模型,构建及瞬时转染重组质粒pEGFP-C2-bMEN1,进行MEN1基因过表达实验;应用RNAi的方法瞬时转染细胞后,进行MEN1基因表达抑制实验。后采用qRT-PCR、Western blot技术,在mRNA水平及蛋白水平检测与乳蛋白合成相关信号通路基因的表达变化,在mRNA水平检测主要酪蛋白之一κ-酪蛋白基因CSNK的表达变化;同时,选取干奶期及泌乳峰期的奶牛乳腺组织,qRT-PCR技术检测不同泌乳时期乳腺组织内MEN1基因及乳蛋白合成相关信号通路基因在mRNA水平的表达关系。通过体外试验和体内验证的联合,寻求MEN1基因的表达对奶牛乳腺内乳蛋白合成的调控关系,从而为奶牛乳腺泌乳调节机制的研究提供一定的理论基础,为遗传改良奶牛乳品质和乳产量奠定理论基础。研究结果表明:(1)构建的重组质粒pEGFP-C2-bMEN1在MAC-T细胞内成功表达,并在转染24h后检测发现MEN1基因在mRNA及蛋白水平的表达量均极显著提高(P0.01);(2)qRT-PCR检测发现,MEN1基因的过表达可在mRNA水平显著降低包括Akt、mTOR、S6K1、4E-BP1及STAT5在内的与乳蛋白合成相关基因的表达(P0.01);Western Blot检测发现MEN1基因过表达也同时有下调包括Akt、mTOR、S6K1在内的蛋白表达(P0.05)的趋势;而MEN1基因低表达则表现出相反的调节作用。(3)在MEN1基因过表达条件下检测发现,作为主要酪蛋白亚型之一的κ-酪蛋白基因CSNK在mRNA水平表达显著降低(P0.05),而MEN1基因的低表达则能显著上调CSNK基因在mRNA水平的表达(P0.01)。(4)在体内奶牛乳腺组织的研究结果显示,泌乳高峰期乳腺组织内MEN1基因的表达量低于干奶期乳腺组织内的表达;而与干奶期相比,泌乳高峰期的乳腺组织在mRNA水平不仅存在较高水平的酪蛋白基因表达,包括CSNB(P0.05)、CSNAS1、CSNAS2、CSNK(P0.05),同时也有更高水平的信号通路内基因包括Akt(P0.05)、mTOR、S6K1、4E-BP1及STAT5(P0.05)的表达,这与我们在奶牛乳腺上皮细胞中所研究的结果相一致。综上所述,MEN1基因可通过负向调控包括PI3K/Akt/mTOR信号通路及JAK2-STAT5信号通路在内的与乳蛋白合成相关因子的表达,进而参与负向调控奶牛乳腺上皮细胞内乳蛋白的合成。
[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.
【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S823
本文编号：2171860
[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.
【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S823
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