一個組成型表達的草魚ADAR1基因剪切異構(gòu)體ADAR1a的鑒定與特征
發(fā)布時間：2019-02-19 18:33
【摘要】：作為ADAR家族中的一員,ADAR1(adenosine deaminase acting on RNA)能夠?qū)㈦p鏈RNA的腺嘌呤(A)置換為次黃嘌呤(I)。在哺乳動物中,ADAR1擁有很多剪切異構(gòu)體,包括研究較為普遍的能被干擾素誘導(dǎo)表達的150kD大小的剪切異構(gòu)體ADAR1-p150和組成型表達的110kD大小的剪切異構(gòu)體ADAR1-p110。ADAR1這種結(jié)構(gòu)的多樣性可能反應(yīng)了其不同剪切異構(gòu)體之間功能的多樣及差異。ADAR1不同剪切異構(gòu)體也被發(fā)現(xiàn)存在于魚類之中。在我們之前的研究中,我們已經(jīng)在草魚中克隆得到了ADAR1兩種不同剪切異構(gòu)體Ci ADAR1和CiADAR1-like,它們都可以被干擾素誘導(dǎo)表達。在本文中,我們鑒定了一個新的草魚ADAR1基因剪切異構(gòu)體ADAR1a。ADAR1a基因包含15個外顯子和14個內(nèi)含子,它的全長cDNA序列包括359 bp的5'UTR,229 bp的3'UTR和一個2592 bp的最大ORF,編碼983個氨基酸的多肽。ADAR1a蛋白具有1個Z-DNA結(jié)合域,3個dsRNA結(jié)合域和一個高度保守的水解脫氨酶激酶活性區(qū)。在草魚CIK細胞中,通過western blot分析,草魚ADAR1a蛋白表現(xiàn)出組成型表達特性并不受poly(I:C)刺激影響。在正常狀況下,草魚腎細胞中ADAR1a蛋白主要分布在細胞核,而通過poly(I:C)刺激,隨著時間推移,ADAR1a蛋白由細胞核轉(zhuǎn)移至細胞質(zhì),其具體分子機制尚不清楚。為了進一步研究草魚ADAR1a基因的轉(zhuǎn)錄調(diào)控機制,通過5'full Race技術(shù),我們確定了草魚ADAR1a的轉(zhuǎn)錄起始位點,位于ADAR1基因的第二個外顯子上。然后我們克隆了其包含部分5'UTR及其上游序列在內(nèi)共1303 bp作為ADAR1a基因的啟動子,通過軟件分析,其中包含4個干擾素調(diào)節(jié)因子元件。通過構(gòu)建草魚IRF1、IRF3的真核表達載體pcDNA3.1-IRF1和pcDNA3.1-IRF3與pGL3-Ci ADAR1a啟動子報告載體共轉(zhuǎn)入草魚腎細胞。雙熒光素酶活性分析結(jié)果顯示在草魚腎細胞中無論是草魚IRF1、IRF3蛋白還是Poly I:C都不能影響草魚ADAR1a蛋白的表達。通過其分子與表達機制,細胞內(nèi)亞定位結(jié)果及其轉(zhuǎn)錄調(diào)控機制,我們推測草魚ADAR1a與哺乳動物ADAR1蛋白剪切異構(gòu)體ADAR1-p110具有高度同源性。隨后,通過序列比對推測其核輸出信號(NES)序列,并構(gòu)建pEGFP-C1-ADAR1a,pEGFP-C1-ADAR1N,pEGFP-C1-ADAR1NΔNES表達載體,瞬時轉(zhuǎn)染進CIK細胞,通過熒光顯微鏡觀察,發(fā)現(xiàn)轉(zhuǎn)染pEGFP-C1-ADAR1a質(zhì)粒的細胞熒光集中于細胞核,轉(zhuǎn)染pEGFP-C1-ADAR1N(ADAR1 N端區(qū)域,1-505aa)質(zhì)粒的細胞在細胞質(zhì)與細胞核都有熒光,而轉(zhuǎn)染pEGFP-C1-ADAR1NΔNES載體的細胞熒光主要在細胞核,證實了草魚ADAR1 N端區(qū)域擁有一段NES序列。以上所有結(jié)果都表明,草魚ADAR1a是一個組成型表達的ADAR1基因剪切異構(gòu)體。
[Abstract]:As a member of the ADAR family, ADAR1 (adenosine deaminase acting on RNA) can replace the adenine (A) of double-stranded RNA with Hypoxanthine (I). In mammals, ADAR1 has many shearing isomers, Including the study of the more common shearing isomer ADAR1-p150, which can be induced to express 150kD by interferon, and the shearing isomer ADAR1-p110.ADAR1, which is composed of 110kD, the diversity of structures may reflect their different shearing. The diversity and difference of the function of the isomers. The different shear isomers of ADAR1 are also found in fish. In our previous studies, we have cloned two different shearing isomers of ADAR1, Ci ADAR1 and CiADAR1-like, from grass carp, both of which can be induced by interferon. In this paper, we have identified a new ADAR1 gene shearing isomer ADAR1a.ADAR1a gene containing 15 exons and 14 introns. Its full-length cDNA sequence consists of 5 UTR229 bp 3'UTR of 359 bp and a maximum ORF, of 2592 bp. ADAR1a protein has one Z-DNA binding domain, three dsRNA binding domains and a highly conserved active domain of water deaminase kinase. In grass carp CIK cells, western blot analysis showed that the constitutive expression of ADAR1a protein in grass carp was not affected by poly (I: C) stimulation. Under normal conditions, the ADAR1a protein in the kidney cells of grass carp is mainly distributed in the nucleus, but through the stimulation of poly (I: C), the ADAR1a protein is transferred from the nucleus to the cytoplasm with the passage of time, and its molecular mechanism is not clear. In order to further study the transcriptional regulation mechanism of grass carp ADAR1a gene, we identified the ADAR1a transcription initiation site of grass carp by 5'full Race technique, which is located on the second exon of ADAR1 gene. Then we cloned 1303 bp which contains part of 5'UTR and its upstream sequence as promoter of ADAR1a gene. By software analysis, it contains four interferon regulatory factors. The eukaryotic expression vectors pcDNA3.1-IRF1, pcDNA3.1-IRF3 and pGL3-Ci ADAR1a promoter of grass carp IRF1,IRF3 were co-transferred into the kidney cells of grass carp by constructing the eukaryotic expression vector of grass carp IRF1,IRF3. The results of double luciferase activity analysis showed that neither grass carp IRF1,IRF3 protein nor Poly I: C could affect the expression of ADAR1a protein in grass carp kidney cells. Based on its molecular and expression mechanism, intracellular sublocalization and transcriptional regulation, we speculate that ADAR1a has high homology with mammalian ADAR1 shearing isomer ADAR1-p110. Then, the (NES) sequence of nuclear output signal was deduced by sequence alignment, and pEGFP-C1-ADAR1a,pEGFP-C1-ADAR1N,pEGFP-C1-ADAR1N 螖 NES expression vector was constructed. The expression vector was transiently transfected into CIK cells and observed by fluorescence microscope. It was found that the fluorescence of the transfected pEGFP-C1-ADAR1a plasmid was concentrated in the nucleus, and that the cells transfected with the pEGFP-C1-ADAR1N (ADAR1 N-terminal region, 1-505aa) plasmid had fluorescence in both the cytoplasm and the nucleus. The fluorescence of the transfected pEGFP-C1-ADAR1N 螖 NES vector was mainly in the nucleus, which confirmed that there was a NES sequence in the N-terminal region of grass carp ADAR1. All the above results suggest that ADAR1a is a constitutive expression of ADAR1 gene shearing isomer.
【學(xué)位授予單位】：南昌大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：Q78
本文編號：2426750
[Abstract]:As a member of the ADAR family, ADAR1 (adenosine deaminase acting on RNA) can replace the adenine (A) of double-stranded RNA with Hypoxanthine (I). In mammals, ADAR1 has many shearing isomers, Including the study of the more common shearing isomer ADAR1-p150, which can be induced to express 150kD by interferon, and the shearing isomer ADAR1-p110.ADAR1, which is composed of 110kD, the diversity of structures may reflect their different shearing. The diversity and difference of the function of the isomers. The different shear isomers of ADAR1 are also found in fish. In our previous studies, we have cloned two different shearing isomers of ADAR1, Ci ADAR1 and CiADAR1-like, from grass carp, both of which can be induced by interferon. In this paper, we have identified a new ADAR1 gene shearing isomer ADAR1a.ADAR1a gene containing 15 exons and 14 introns. Its full-length cDNA sequence consists of 5 UTR229 bp 3'UTR of 359 bp and a maximum ORF, of 2592 bp. ADAR1a protein has one Z-DNA binding domain, three dsRNA binding domains and a highly conserved active domain of water deaminase kinase. In grass carp CIK cells, western blot analysis showed that the constitutive expression of ADAR1a protein in grass carp was not affected by poly (I: C) stimulation. Under normal conditions, the ADAR1a protein in the kidney cells of grass carp is mainly distributed in the nucleus, but through the stimulation of poly (I: C), the ADAR1a protein is transferred from the nucleus to the cytoplasm with the passage of time, and its molecular mechanism is not clear. In order to further study the transcriptional regulation mechanism of grass carp ADAR1a gene, we identified the ADAR1a transcription initiation site of grass carp by 5'full Race technique, which is located on the second exon of ADAR1 gene. Then we cloned 1303 bp which contains part of 5'UTR and its upstream sequence as promoter of ADAR1a gene. By software analysis, it contains four interferon regulatory factors. The eukaryotic expression vectors pcDNA3.1-IRF1, pcDNA3.1-IRF3 and pGL3-Ci ADAR1a promoter of grass carp IRF1,IRF3 were co-transferred into the kidney cells of grass carp by constructing the eukaryotic expression vector of grass carp IRF1,IRF3. The results of double luciferase activity analysis showed that neither grass carp IRF1,IRF3 protein nor Poly I: C could affect the expression of ADAR1a protein in grass carp kidney cells. Based on its molecular and expression mechanism, intracellular sublocalization and transcriptional regulation, we speculate that ADAR1a has high homology with mammalian ADAR1 shearing isomer ADAR1-p110. Then, the (NES) sequence of nuclear output signal was deduced by sequence alignment, and pEGFP-C1-ADAR1a,pEGFP-C1-ADAR1N,pEGFP-C1-ADAR1N 螖 NES expression vector was constructed. The expression vector was transiently transfected into CIK cells and observed by fluorescence microscope. It was found that the fluorescence of the transfected pEGFP-C1-ADAR1a plasmid was concentrated in the nucleus, and that the cells transfected with the pEGFP-C1-ADAR1N (ADAR1 N-terminal region, 1-505aa) plasmid had fluorescence in both the cytoplasm and the nucleus. The fluorescence of the transfected pEGFP-C1-ADAR1N 螖 NES vector was mainly in the nucleus, which confirmed that there was a NES sequence in the N-terminal region of grass carp ADAR1. All the above results suggest that ADAR1a is a constitutive expression of ADAR1 gene shearing isomer.
【學(xué)位授予單位】：南昌大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：Q78
【參考文獻】
相關(guān)碩士學(xué)位論文 前1條
1 孫志成;草魚ADAR1(adenosine deaminase acting on RNA 1)及其剪接異構(gòu)體ADAR1-like的轉(zhuǎn)錄調(diào)控分析[D];南昌大學(xué);2016年
，本文編號：2426750
本文鏈接：http://sikaile.net/kejilunwen/jiyingongcheng/2426750.html
最近更新
教材專著
