本文關(guān)鍵詞： 豬 ADAM基因 克隆 剪接體 組織表達(dá)　出處：《畜牧獸醫(yī)學(xué)報(bào)》2017年09期 　論文類型：期刊論文

【摘要】：旨在克隆豬ADAM10基因及其剪接體的編碼區(qū)(Coding sequence,CDS),利用生物信息學(xué)方法分析其序列結(jié)構(gòu)和生物學(xué)功能,并揭示ADAM10基因及其剪接體mRNA在豬組織中的表達(dá)特征。根據(jù)GenBank中公布的豬ADAM10基因的序列信息,設(shè)計(jì)特異性引物,運(yùn)用反轉(zhuǎn)錄PCR(Reverse transcription-PCR,RT-PCR)方法結(jié)合T/A克隆技術(shù)進(jìn)行ADAM10基因及其剪接體編碼區(qū)的克隆,利用生物信息學(xué)方法分析ADAM10及其剪接體蛋白的結(jié)構(gòu),同時(shí)用半定量PCR(Semi-quantitative PCR)分析豬ADAM10基因完整型及其剪接體mRNA在豬不同組織中的表達(dá)譜。結(jié)果顯示,克隆和測序獲得兩種ADAM10CDS序列,其中完整型CDS長2 247bp,編碼748個(gè)氨基酸;剪接型CDS缺失外顯子2~7和部分外顯子8,其長度為1 344bp,編碼447個(gè)氨基酸。和完整型蛋白相比,豬ADAM10剪接體編碼的蛋白不存在前引導(dǎo)序列,但存在完整的信號(hào)肽序列和跨膜結(jié)構(gòu)域,并含有金屬蛋白酶域和解聚素結(jié)構(gòu)域,其三級(jí)結(jié)構(gòu)也和完整型一致。序列比對(duì)和進(jìn)化樹分析表明,豬ADAM10完整型蛋白在物種間具有較高的保守性。半定量PCR分析表明,ADAM10基因完整型mRNA在脾中表達(dá)量較高,在腎、乳腺、腿肌、輸卵管、卵巢、子宮、小腸等組織中低水平表達(dá);ADAM10基因剪接體mRNA在肺中表達(dá)量較高,在脾和胃中也有低水平表達(dá)。本研究為進(jìn)一步探究豬ADAM10基因的結(jié)構(gòu)和生物學(xué)功能提供了基礎(chǔ)資料。
[Abstract]:To clone the coding region of porcine ADAM10 gene and its splicing sequence sequence, the sequence structure and biological function of porcine ADAM10 gene were analyzed by bioinformatics. According to the sequence information of porcine ADAM10 gene published in GenBank, specific primers were designed. Reverse transcription PCR(Reverse transcription-PCRR-RT-PCR method and T / A cloning technique were used to clone the coding region of ADAM10 gene and splice. Bioinformatics was used to analyze the structure of ADAM10 and its splice protein. At the same time, semi-quantitative PCR(Semi-quantitative PCR was used to analyze the expression profiles of porcine ADAM10 gene intact type and splice mRNA in different tissues. The results showed that two kinds of ADAM10CDS sequences were obtained by cloning and sequencing, among which the complete CDS was 2247bplong, encoding 748 amino acids. Splicing type CDS deletion exon 2, exon 7 and partial exon 8, with a length of 1 344 BP, encode 447 amino acids. Compared with the complete protein, porcine ADAM10 splice encoding protein does not contain a preleading sequence, but has a complete signal peptide sequence and a transmembrane domain. It also contains metalloproteinase domain and depolymerization domain, and its tertiary structure is consistent with the complete type. Sequence alignment and evolutionary tree analysis show that, The semi-quantitative PCR analysis showed that the intact type mRNA of ADAM10 gene was highly expressed in spleen, including kidney, mammary gland, leg muscle, fallopian tube, ovary, uterus, oviduct, ovary, uterus, oviduct, ovaries, uterus, kidney, mammary gland, leg muscle, oviduct, ovary and uterus. The expression of ADAM10 gene splicing mRNA in small intestine and other tissues was higher in lung and lower in spleen and stomach. This study provided basic data for further study on the structure and biological function of porcine ADAM10 gene.
【作者單位】： 浙江農(nóng)林大學(xué)動(dòng)物科技學(xué)院;
【基金】：國家自然科學(xué)基金(31501921) 浙江省自然科學(xué)基金(LQ15C170001) 浙江省農(nóng)業(yè)(畜禽)新品種選育重大科技專項(xiàng)(2016C02054-3)
【分類號(hào)】：Q78;S828

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本文編號(hào)：1508010