鹅OAZ基因克
发布时间：2018-12-16 02:45
【摘要】：鸟氨酸脱羧酶抗酶(ornithine decarboxylase antizymes,OAZs)是多胺代谢过程中的关键调控酶,能抑制多胺的生成,维持多胺的稳态。研究发现多胺合成受到抑制时小鼠卵巢出现萎缩,卵泡生长停滞甚至闭锁。OAZ1在产蛋期籽鹅卵巢组织中的表达量显著高于产蛋前期,提示OAZ1可能通过调控卵巢组织中多胺浓度,进而参与调控籽鹅的繁殖。然而,关于OAZs在卵泡发育过程中的作用研究还未见报道,其机制尚不清楚。因此,本研究克隆了OAZ1和OAZ2的全长编码区序列,定量检测了 OAZ1和OAZ2在四川白鹅组织及不同等级卵泡组织中的表达量,并通过定点突变技术缺失了OAZ1基因的+1移码位点T碱基,构建突变型OAZ1基因的表达载体pEGFP/N1-OAZ1-mutation,转染至原代培养的四川白鹅颗粒细胞中,研究过表达OAZ1对鹅卵泡颗粒细胞的影响。结果表明:四川白鹅OAZ1和OAZ2的全长编码区长度分别为652 bp和574 bp,其+1移码位点分别位于第175位和第97位T碱基。OAZ1和OAZ2基因的表达具有组织特异性,OAZ1和OAZ2在下丘脑、垂体和卵巢中的表达趋势相同,都在垂体中的表达量最高,在卵巢中的表达量最低,除胸肌和腿肌外其余各组织中OAZ1表达均占优势。卵泡组织中,OAZ1表达量在F5级卵泡中表达量最低,在F1和POF卵泡中表达量最高(P0.05),在等级前卵泡和等级卵泡中OAZ1的表达量均呈递增趋势。OAZ2在SWF和F2级卵泡中的表达量显著高于卵巢基质,其余各组织中OAZ2的表达量均无显著差异(P0.05)。将转染pEGFP/N1-OAZ1-mutation至鹅卵泡颗粒细胞48 h后,OAZ1表达量显著升高,是对照组(转染pEGFP/N1组)的2.60倍(P0.01)。随着OAZ1基因表达量的上调,多胺代谢基因Amd1、AZIN1、OAZ2、ODC、SMOX以及SMS的表达量显著升高(P0.05),分别是对照组的1.62倍、1.47倍、1.58倍、1.58倍、1.50倍和1.60倍;细胞增殖、凋亡关键调控基因Bax、Bcl-2、Caspase3、Cyclin-D1和PARP的表达量显著升高(P0.05),分别是对照组的1.40、1.95、1.59、1.51和1.26倍;细胞培养液中雌激素显著升高1.4倍,雌激素受体表达量显著升高1.98倍(P0.05)。主要的研究结论如下:(1)与OAZ2相比,OAZ1在调控鹅卵泡发育及维持组织多胺稳态的过程中起主要作用。(2)OAZ1过表达可诱导鹅卵泡颗粒细胞内多胺稳态的负反馈调节,即通过上调AZIN1来减少过表达的OAZ1浓度,同时通过上调ODC表达来代偿过表达OAZ1对ODC活性的抑制效应。(3)OAZ1可通过Bax和Caspase 3途径参与调控鹅卵泡颗粒细胞的凋亡。(4)过表达OAZ1能够上调鹅颗粒细胞雌激素受体基因的表达,促进雌激素的分泌,进而参与调控鹅的繁殖功能。
[Abstract]:Ornithine decarboxylase (ornithine decarboxylase antizymes,OAZs) is a key regulatory enzyme in the metabolism of polyamines, which can inhibit the formation of polyamines and maintain the stability of polyamines. It was found that the ovarian atrophy, follicular growth arrest and even atresia occurred in mice when the synthesis of polyamines was inhibited. The expression of OAZ1 in egg laying stage was significantly higher than that in egg laying stage, suggesting that OAZ1 might regulate the concentration of polyamines in ovarian tissues. And then participate in the regulation of seed goose reproduction. However, the role of OAZs in follicular development has not been reported, and its mechanism is not clear. Therefore, the full-length coding region sequence of OAZ1 and OAZ2 was cloned, and the expression of OAZ1 and OAZ2 in Sichuan white goose tissues and follicular tissues of different grades were quantitatively detected. The T base of 1 frame shift site of OAZ1 gene was deleted by site-directed mutagenesis. The expression vector pEGFP/N1-OAZ1-mutation, of mutant OAZ1 gene was constructed and transfected into the primary cultured granulosa cells of Sichuan White Goose. The effect of overexpression of OAZ1 on Goose follicular granulosa cells was studied. The results showed that the length of full-length coding region of OAZ1 and OAZ2 of Sichuan White Goose was 652 bp and 574 bp, respectively. The 1 frameshift site was located at the 175th and 97th position respectively. The expression of OAZ1 and OAZ2 genes was tissue specific, and OAZ1 and OAZ2 were found in hypothalamus. The expression of OAZ1 was the highest in pituitary and the lowest in ovary. The expression of OAZ1 was dominant in all tissues except pectoralis and leg muscles. In follicular tissues, the expression of OAZ1 was the lowest in F5 follicles, and the highest in F1 and POF follicles (P0.05). The expression of OAZ1 in pre-grade follicles and grade follicles showed an increasing trend. The expression of OAZ2 in SWF and F2 grade follicles was significantly higher than that in ovarian stroma. There was no significant difference in the expression of OAZ2 in other tissues (P0.05). After transfection of pEGFP/N1-OAZ1-mutation to Goose follicular granulosa cells for 48 h, the expression of OAZ1 increased significantly, which was 2.60 times of that of the control group (P0.01). With the upregulation of OAZ1 gene expression, the expression of polyamine metabolite gene Amd1,AZIN1,OAZ2,ODC,SMOX and SMS increased significantly (P0.05), which were 1.62 times, 1.47 times, 1.58 times and 1.58 times of the control group, respectively. 1.50 times and 1.60 times; The expression of Bax,Bcl-2,Caspase3,Cyclin-D1 and PARP, the key genes of cell proliferation and apoptosis, were significantly increased (P0.05), which were 1.401.95, 1.59 and 1.26 times of those of the control group, respectively. The expression of estrogen receptor was 1.98 times higher than that of the control group (P0.05). The main conclusions are as follows: (1) compared with OAZ2, OAZ1 plays a major role in regulating the development of goose follicles and maintaining the polyamine homeostasis. (2) overexpression of OAZ1 can induce negative feedback regulation of polyamine homeostasis in goose follicular granulosa cells. That is, by upregulating AZIN1 to reduce the overexpression of OAZ1, (3) OAZ1 can regulate the apoptosis of Goose follicular granulosa cells through Bax and Caspase 3 pathway. (4) overexpression of OAZ1 can up-regulate Goose granulosa cells by up-regulation of ODC expression. (3) OAZ1 can regulate the apoptosis of Goose follicular granulosa cells through Bax and Caspase 3 pathway. (4) overexpression of OAZ1 can up-regulate goose granulosa cells. Estrogen receptor gene expression, Promote the secretion of estrogen, and then participate in the regulation of goose reproductive function.
【学位授予单位】：四川农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S835
[Abstract]:Ornithine decarboxylase (ornithine decarboxylase antizymes,OAZs) is a key regulatory enzyme in the metabolism of polyamines, which can inhibit the formation of polyamines and maintain the stability of polyamines. It was found that the ovarian atrophy, follicular growth arrest and even atresia occurred in mice when the synthesis of polyamines was inhibited. The expression of OAZ1 in egg laying stage was significantly higher than that in egg laying stage, suggesting that OAZ1 might regulate the concentration of polyamines in ovarian tissues. And then participate in the regulation of seed goose reproduction. However, the role of OAZs in follicular development has not been reported, and its mechanism is not clear. Therefore, the full-length coding region sequence of OAZ1 and OAZ2 was cloned, and the expression of OAZ1 and OAZ2 in Sichuan white goose tissues and follicular tissues of different grades were quantitatively detected. The T base of 1 frame shift site of OAZ1 gene was deleted by site-directed mutagenesis. The expression vector pEGFP/N1-OAZ1-mutation, of mutant OAZ1 gene was constructed and transfected into the primary cultured granulosa cells of Sichuan White Goose. The effect of overexpression of OAZ1 on Goose follicular granulosa cells was studied. The results showed that the length of full-length coding region of OAZ1 and OAZ2 of Sichuan White Goose was 652 bp and 574 bp, respectively. The 1 frameshift site was located at the 175th and 97th position respectively. The expression of OAZ1 and OAZ2 genes was tissue specific, and OAZ1 and OAZ2 were found in hypothalamus. The expression of OAZ1 was the highest in pituitary and the lowest in ovary. The expression of OAZ1 was dominant in all tissues except pectoralis and leg muscles. In follicular tissues, the expression of OAZ1 was the lowest in F5 follicles, and the highest in F1 and POF follicles (P0.05). The expression of OAZ1 in pre-grade follicles and grade follicles showed an increasing trend. The expression of OAZ2 in SWF and F2 grade follicles was significantly higher than that in ovarian stroma. There was no significant difference in the expression of OAZ2 in other tissues (P0.05). After transfection of pEGFP/N1-OAZ1-mutation to Goose follicular granulosa cells for 48 h, the expression of OAZ1 increased significantly, which was 2.60 times of that of the control group (P0.01). With the upregulation of OAZ1 gene expression, the expression of polyamine metabolite gene Amd1,AZIN1,OAZ2,ODC,SMOX and SMS increased significantly (P0.05), which were 1.62 times, 1.47 times, 1.58 times and 1.58 times of the control group, respectively. 1.50 times and 1.60 times; The expression of Bax,Bcl-2,Caspase3,Cyclin-D1 and PARP, the key genes of cell proliferation and apoptosis, were significantly increased (P0.05), which were 1.401.95, 1.59 and 1.26 times of those of the control group, respectively. The expression of estrogen receptor was 1.98 times higher than that of the control group (P0.05). The main conclusions are as follows: (1) compared with OAZ2, OAZ1 plays a major role in regulating the development of goose follicles and maintaining the polyamine homeostasis. (2) overexpression of OAZ1 can induce negative feedback regulation of polyamine homeostasis in goose follicular granulosa cells. That is, by upregulating AZIN1 to reduce the overexpression of OAZ1, (3) OAZ1 can regulate the apoptosis of Goose follicular granulosa cells through Bax and Caspase 3 pathway. (4) overexpression of OAZ1 can up-regulate Goose granulosa cells by up-regulation of ODC expression. (3) OAZ1 can regulate the apoptosis of Goose follicular granulosa cells through Bax and Caspase 3 pathway. (4) overexpression of OAZ1 can up-regulate goose granulosa cells. Estrogen receptor gene expression, Promote the secretion of estrogen, and then participate in the regulation of goose reproductive function.
【学位授予单位】：四川农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S835
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