【摘要】：鳥氨酸脫羧酶抗酶(ornithine decarboxylase antizymes,OAZs)是多胺代謝過程中的關(guān)鍵調(diào)控酶,能抑制多胺的生成,維持多胺的穩(wěn)態(tài)。研究發(fā)現(xiàn)多胺合成受到抑制時小鼠卵巢出現(xiàn)萎縮,卵泡生長停滯甚至閉鎖。OAZ1在產(chǎn)蛋期籽鵝卵巢組織中的表達(dá)量顯著高于產(chǎn)蛋前期,提示OAZ1可能通過調(diào)控卵巢組織中多胺濃度,進(jìn)而參與調(diào)控籽鵝的繁殖。然而,關(guān)于OAZs在卵泡發(fā)育過程中的作用研究還未見報道,其機(jī)制尚不清楚。因此,本研究克隆了OAZ1和OAZ2的全長編碼區(qū)序列,定量檢測了 OAZ1和OAZ2在四川白鵝組織及不同等級卵泡組織中的表達(dá)量,并通過定點(diǎn)突變技術(shù)缺失了OAZ1基因的+1移碼位點(diǎn)T堿基,構(gòu)建突變型OAZ1基因的表達(dá)載體pEGFP/N1-OAZ1-mutation,轉(zhuǎn)染至原代培養(yǎng)的四川白鵝顆粒細(xì)胞中,研究過表達(dá)OAZ1對鵝卵泡顆粒細(xì)胞的影響。結(jié)果表明:四川白鵝OAZ1和OAZ2的全長編碼區(qū)長度分別為652 bp和574 bp,其+1移碼位點(diǎn)分別位于第175位和第97位T堿基。OAZ1和OAZ2基因的表達(dá)具有組織特異性,OAZ1和OAZ2在下丘腦、垂體和卵巢中的表達(dá)趨勢相同,都在垂體中的表達(dá)量最高,在卵巢中的表達(dá)量最低,除胸肌和腿肌外其余各組織中OAZ1表達(dá)均占優(yōu)勢。卵泡組織中,OAZ1表達(dá)量在F5級卵泡中表達(dá)量最低,在F1和POF卵泡中表達(dá)量最高(P0.05),在等級前卵泡和等級卵泡中OAZ1的表達(dá)量均呈遞增趨勢。OAZ2在SWF和F2級卵泡中的表達(dá)量顯著高于卵巢基質(zhì),其余各組織中OAZ2的表達(dá)量均無顯著差異(P0.05)。將轉(zhuǎn)染pEGFP/N1-OAZ1-mutation至鵝卵泡顆粒細(xì)胞48 h后,OAZ1表達(dá)量顯著升高,是對照組(轉(zhuǎn)染pEGFP/N1組)的2.60倍(P0.01)。隨著OAZ1基因表達(dá)量的上調(diào),多胺代謝基因Amd1、AZIN1、OAZ2、ODC、SMOX以及SMS的表達(dá)量顯著升高(P0.05),分別是對照組的1.62倍、1.47倍、1.58倍、1.58倍、1.50倍和1.60倍;細(xì)胞增殖、凋亡關(guān)鍵調(diào)控基因Bax、Bcl-2、Caspase3、Cyclin-D1和PARP的表達(dá)量顯著升高(P0.05),分別是對照組的1.40、1.95、1.59、1.51和1.26倍;細(xì)胞培養(yǎng)液中雌激素顯著升高1.4倍,雌激素受體表達(dá)量顯著升高1.98倍(P0.05)。主要的研究結(jié)論如下:(1)與OAZ2相比,OAZ1在調(diào)控鵝卵泡發(fā)育及維持組織多胺穩(wěn)態(tài)的過程中起主要作用。(2)OAZ1過表達(dá)可誘導(dǎo)鵝卵泡顆粒細(xì)胞內(nèi)多胺穩(wěn)態(tài)的負(fù)反饋調(diào)節(jié),即通過上調(diào)AZIN1來減少過表達(dá)的OAZ1濃度,同時通過上調(diào)ODC表達(dá)來代償過表達(dá)OAZ1對ODC活性的抑制效應(yīng)。(3)OAZ1可通過Bax和Caspase 3途徑參與調(diào)控鵝卵泡顆粒細(xì)胞的凋亡。(4)過表達(dá)OAZ1能夠上調(diào)鵝顆粒細(xì)胞雌激素受體基因的表達(dá),促進(jìn)雌激素的分泌,進(jìn)而參與調(diào)控鵝的繁殖功能。
[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.
【學(xué)位授予單位】：四川農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S835

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