發(fā)布時(shí)間：2018-10-30 21:03

【摘要】：轉(zhuǎn)基因動(dòng)物的外源基因通過使用體細(xì)胞克隆與胚胎移植的方法將表達(dá)載體轉(zhuǎn)入細(xì)胞后獲得,大多以隨機(jī)整合的形式存在于受體動(dòng)物的基因組中,而轉(zhuǎn)基因動(dòng)物中外源基因的整合拷貝數(shù)是影響外源基因表達(dá)水平及遺傳穩(wěn)定性的重要因素之一,作為轉(zhuǎn)基因動(dòng)物遺傳穩(wěn)定性的鑒定指標(biāo),獲得存活的轉(zhuǎn)基因動(dòng)物后便有必要對(duì)其外源基因拷貝數(shù)進(jìn)行檢測(cè)。為了研究轉(zhuǎn)基因阿爾巴斯白絨山羊(Capra hircus)基因組內(nèi)外源�？�紅色熒光蛋白(red fluorescence protein from Discosoma sp.,Ds Red)基因的拷貝數(shù)與其表達(dá)量之間是否存在一定的聯(lián)系,本研究采用較鑒定外源基因拷貝數(shù)的傳統(tǒng)方法DNA印跡雜交(Southern blot)更為高效率、高靈敏度、高準(zhǔn)確性的絕對(duì)定量q RT-PCR法檢測(cè)了6只轉(zhuǎn)基因白絨山羊外源Ds Red基因表達(dá)框中3個(gè)不同片段的拷貝數(shù)。結(jié)果表明,外源基因3個(gè)片段的拷貝數(shù)分別為巨細(xì)胞病毒(Cytomegalovirus,CMV)啟動(dòng)子:2.80±0.38~13.68±0.16;Ds Red基因:1.34±0.04~11.84±0.02;CMV啟動(dòng)子與Ds Red連接處:1.58±0.04~19.22±0.38。通過q RT-PCR中相對(duì)定量的方法檢測(cè)了6只轉(zhuǎn)基因白絨山羊外源Ds Red基因m RNA表達(dá)量,將其與外源基因中3個(gè)不同片段的拷貝數(shù)數(shù)據(jù)分別使用SPSS軟件進(jìn)行相關(guān)性分析后,相關(guān)性系數(shù)P值的結(jié)果為:CMV啟動(dòng)子為P=0.763;Ds Red基因?yàn)镻=0.665;CMV啟動(dòng)子與Ds Red連接處為P=0.803,3組P值均大于0.05,無(wú)顯著性相關(guān)。上述結(jié)果表明,在所檢測(cè)的6只轉(zhuǎn)基因阿爾巴斯白絨山羊中外源CMV啟動(dòng)子啟動(dòng)的Ds Red基因的表達(dá)量與其在轉(zhuǎn)基因白絨山羊基因組內(nèi)的拷貝數(shù)在0.05的水平?jīng)]有顯著相關(guān)性。另外驗(yàn)證了絕對(duì)定量q RT-PCR法在大型轉(zhuǎn)基因家畜的外源基因拷貝數(shù)的研究中的可靠性:其重復(fù)性良好,誤差可控,可以成為檢測(cè)轉(zhuǎn)基因動(dòng)物外源基因拷貝數(shù)的首選方法。
[Abstract]:The foreign genes of transgenic animals were transformed into cells by somatic cell cloning and embryo transfer, and most of them existed in the genome of recipient animals in the form of random integration. The integrative copy number of foreign genes in transgenic animals is one of the important factors that affect the expression level and genetic stability of foreign genes, which is used as the identification index of genetic stability of transgenic animals. After the survival of transgenic animals, it is necessary to detect the copy number of foreign genes. In order to study the relationship between the copy number and the expression level of the (Capra hircus) gene of the transgenic Albus white cashmere goat, the internal and external (red fluorescence protein from Discosoma sp.,Ds Red gene of sea anemone was studied. In this study, DNA blotting (Southern blot) was more efficient and sensitive than the traditional method of identifying the copy number of foreign genes. The copy numbers of three different fragments of exogenous Ds Red gene expression frame of 6 transgenic white cashmere goats were detected by absolute quantitative Q RT-PCR method. The results showed that the copy number of the three fragments of exogenous gene was cytomegalovirus (Cytomegalovirus,CMV) promoter: 2.80 鹵0.38C 13.68 鹵0.16Ds Red gene: 1.34 鹵0.04nb 11.84 鹵0.02; The junction between CMV promoter and Ds Red was 1.58 鹵0.04 鹵19.22 鹵0.38. The expression of exogenous Ds Red gene m RNA in 6 transgenic white cashmere goats was detected by using the relative quantitative method in Q RT-PCR. The correlation between the expression of m RNA and the copy number data of three different fragments of exogenous gene was analyzed by SPSS software. The correlation coefficient P value was: the CMV promoter was P0. 763; There was no significant correlation between the P value of Ds Red group 0.803 and P0. 803 because of the connection between Ds Red promoter and P0. 665CMV promoter, P value was higher than 0. 05, there was no significant correlation between P0. 665 and P0. 803 group. The results showed that there was no significant correlation between the expression of Ds Red gene and the number of copies in the genomes of the transgenic white cashmere goats at the level of 0.05, which was initiated by the foreign CMV promoter of 6 transgenic Albus white cashmere goats. In addition, the reliability of the absolute quantitative Q RT-PCR method in the study of foreign gene copy number of large transgenic domestic animals was verified: its reproducibility was good, the error was controllable, and it could be used as the preferred method to detect the foreign gene copy number of transgenic animals.
【作者單位】： 內(nèi)蒙古大學(xué)生命科學(xué)學(xué)院/哺乳動(dòng)物生殖生物學(xué)及生物技術(shù)教育部重點(diǎn)實(shí)驗(yàn)室;
【基金】：國(guó)家轉(zhuǎn)基因生物新品種培育重大專項(xiàng)(No.2014ZX08008-002)
【分類號(hào)】：S827

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