本文選題：牦牛 + CCND　； 參考：《中國(guó)農(nóng)業(yè)科學(xué)》2017年13期

【摘要】：【目的】研究牦牛細(xì)胞周期蛋白D2(Cyclin D2,CCND2)基因序列特征及其在牦牛不同發(fā)情時(shí)期卵巢中的表達(dá)�！痉椒ā恳躁笈檠芯繉�(duì)象,提取卵巢的總RNA進(jìn)行反轉(zhuǎn)錄為第一鏈c DNA,根據(jù)Gen Bank中黃牛CCND2的m RNA序列(Gen Bank登錄號(hào):NM_001076372.1),使用Primer 5.0軟件設(shè)計(jì)引物。應(yīng)用PCR方法對(duì)CCND2基因擴(kuò)增克隆,并測(cè)序獲得CCND2基因完整的CDS區(qū)序列及部分5′端和3′端UTR區(qū)。使用Protparam、SOPMA、SWISS-MODEL及SMART分別對(duì)牦牛CCND2蛋白的理化性質(zhì)、二級(jí)結(jié)構(gòu)、三級(jí)結(jié)構(gòu)及結(jié)構(gòu)域進(jìn)行預(yù)測(cè)分析,并進(jìn)行同源性分析和系統(tǒng)進(jìn)化樹構(gòu)建,利用半定量PCR方法對(duì)CCND2基因在牦牛各組織中的表達(dá)進(jìn)行檢測(cè);同時(shí)采用實(shí)時(shí)熒光定量PCR技術(shù)分析CCND2基因在牦牛不同發(fā)情時(shí)期(卵泡期、紅體期、黃體期)卵巢中的相對(duì)表達(dá)量。【結(jié)果】克隆獲得牦牛CCND2基因序列為909 bp,其中包含可編碼289個(gè)氨基酸殘基的長(zhǎng)為870 bp的CDS區(qū)。與黃牛相比,牦牛CCND2的CDS區(qū)存在5個(gè)堿基突變,導(dǎo)致其中一個(gè)氨基酸改變。牦牛CCND2基因CDs序列與野牦牛、黃牛、印度水牛、綿羊、野豬、馬、家犬、貓、人類的進(jìn)行比對(duì)同源性分別是99.54%、99.31%、99.31%、98.16%、94.81%、90.46%、90.80%、91.49%、88.62%,物種之間同源性較高,與進(jìn)化樹分析顯示的親緣關(guān)系遠(yuǎn)近一致,表明CCND2基因編碼區(qū)在進(jìn)化中較為保守。對(duì)CCND2蛋白預(yù)測(cè)分析知牦牛CCND2基因編碼蛋白的分子式為C1445H2315N377O440S18,相對(duì)分子質(zhì)量約為32.59k D,理論等電點(diǎn)為4.75,總平均親水性為-0.107,不穩(wěn)定指數(shù)為44.56,屬親水不穩(wěn)定的酸性蛋白,該蛋白無(wú)跨膜區(qū)和信號(hào)肽;二級(jí)結(jié)構(gòu)主要包含α-螺旋和無(wú)規(guī)卷曲,且與三級(jí)結(jié)構(gòu)分析結(jié)果一致。蛋白含位于第26—152位氨基酸處的Cyclin_N和位于第154—257位氨基酸處的Cyclin_C兩個(gè)結(jié)構(gòu)域。CCND2基因在牦牛各組織中均有表達(dá),其中在胃、卵巢和腦中表達(dá)相對(duì)較高;q RT-PCR結(jié)果顯示在牦牛不同發(fā)情時(shí)期卵巢中CCND2 m RNA均有表達(dá),且卵泡期卵巢中的表達(dá)水平最高(P0.01),在紅體期和黃體期卵巢中表達(dá)水平無(wú)差異(P0.05)�！窘Y(jié)論】成功獲得了牦牛CCND2基因的CDS區(qū)全長(zhǎng)序列和組織表達(dá)譜,對(duì)其進(jìn)行生物信息分析及蛋白功能結(jié)構(gòu)分析為該基因在牦牛繁殖調(diào)控中的進(jìn)一步研究提供理論依據(jù);在牦牛不同發(fā)情時(shí)期卵巢中CCND2m RNA表達(dá)水平存在差異,可能與卵泡發(fā)育過程中顆粒細(xì)胞增殖分化、卵泡發(fā)生波及卵巢內(nèi)分泌有關(guān),為進(jìn)一步研究牦牛卵巢發(fā)育機(jī)制及改善牦牛繁殖效率提供了基礎(chǔ)資料。
[Abstract]:[objective] to study the sequence characteristics of yak cell cycle protein D2(Cyclin D2 (CCND2) gene and its expression in the ovaries of yak at different estrous stages. [methods] Yak was studied. The total RNA extracted from the ovary was reverse transcribed into the first strand of c-DNA. According to the m RNA sequence of Gen Bank, the Bank accession number of Gen Bank was: NM00776372.1, and the primers were designed with Primer 5.0 software. The CCND2 gene was amplified by PCR and sequenced to obtain the complete CDS region of the CCND2 gene and part of the 5 'end and 3' end UTR region of the CCND2 gene. The physicochemical properties, secondary structure, tertiary structure and domain of CCND2 protein in yak were predicted and analyzed by using the SWISS-MODEL and SMART, and the homology analysis and phylogenetic tree construction were carried out. The expression of CCND2 gene in yak tissues was detected by semi-quantitative PCR, and real-time fluorescence quantitative PCR was used to analyze the expression of CCND2 gene in different estrous stages (follicle stage, red body stage) of yak. [results] the sequence of yak CCND2 gene was 909 BP, which contained 870 BP CDS region encoding 289 amino acid residues. Compared with yellow cattle, there were 5 base mutations in the CDS region of yak CCND2, which resulted in one amino acid change. The homology of CDs sequence of yak CCND2 gene with wild yak, yellow cattle, Indian buffalo, sheep, wild boar, horse, dog, cat and human were 99.5454, 99.31, 99.31, 98.16, 90.46, 90.80 and 91.4988.62, respectively. The results showed that the coding region of CCND2 gene was conserved in evolution. The prediction and analysis of CCND2 protein showed that the molecular formula of yak CCND2 gene encoding protein was C1445H2315N377O440S18, the relative molecular weight was about 32.59kD, the theoretical isoelectric point was 4.75, the total mean hydrophilicity was -0.107, and the instability index was 44.56.It was a hydrophilic acid protein. The protein has no transmembrane region and signal peptide, and the secondary structure mainly contains 偽 -helix and random crimp, which is consistent with the results of tertiary structure analysis. The protein contains two domains, Cyclin_N located at the 26-152 amino acid and Cyclin_C at the 154-257 amino acid. CCND2 gene is expressed in all yak tissues, including the stomach. The expression of CCND2 m RNA in ovary and brain was higher than that in brain. The results showed that the expression of CCND2 m RNA was found in the ovary of yak at different estrous stages. The highest expression level of P0.01 was found in follicular ovary, but there was no difference in expression level between erythroid and luteal stage. [conclusion] the full-length CDS sequence and tissue expression profile of CCND2 gene in yak were successfully obtained. The biological information analysis and protein functional structure analysis provide theoretical basis for further study of the gene in yak reproductive regulation, and the expression level of CCND2m RNA in the ovaries of yak at different estrus stage is different. It may be related to the proliferation and differentiation of granulosa cells during follicular development, follicle development and ovarian endocrine, which provides basic data for further study on the mechanism of ovarian development and improvement of reproductive efficiency of yak.
【作者單位】： 西南民族大學(xué)生命科學(xué)與技術(shù)學(xué)院;西南民族大學(xué)青藏高原研究院;
【基金】：四川省科技支撐計(jì)劃(2014NZ0114) 西南民族大學(xué)中央高校基本科研業(yè)務(wù)費(fèi)專項(xiàng)資金(2016NZYQN38)
【分類號(hào)】：S823.85

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