【摘要】：為了克隆與表達(dá)深黃被孢霉(Mortierella isabellina)M6-22蘋果酸酶基因,根據(jù)深黃被孢霉M6-22轉(zhuǎn)錄組測(cè)序結(jié)果,以其c DNA為模板PCR擴(kuò)增蘋果酸酶基因編碼序列,經(jīng)測(cè)序驗(yàn)證分析后將擴(kuò)增片段連接到表達(dá)載體pET-32a(+)中構(gòu)建重組表達(dá)質(zhì)粒pET32a MIME2并進(jìn)一步轉(zhuǎn)化入大腸桿菌BL21中進(jìn)行誘導(dǎo)表達(dá),經(jīng)鎳柱親和純化目的蛋白和酶活分析確定其為蘋果酸酶.PCR擴(kuò)增得到全長(zhǎng)為1 815 bp的cDNA序列,序列分析表明該序列具有一個(gè)編碼604個(gè)氨基酸的開放閱讀框,預(yù)測(cè)編碼蛋白分子量(Mr)為66.4×10~3.預(yù)測(cè)的氨基酸序列與已報(bào)道的蘋果酸酶同樣具有保守的2個(gè)二核苷酸結(jié)合結(jié)構(gòu)域和1個(gè)二價(jià)金屬離子結(jié)合結(jié)構(gòu)域,因此是一個(gè)新的蘋果酸酶基因序列,命名為MIME2,Gen Bank序列號(hào)為KU097323.將該序列轉(zhuǎn)化大腸桿菌BL21中進(jìn)行誘導(dǎo)表達(dá),SDS-PAGE電泳檢測(cè)到1條約67×10~3的蛋白條帶表達(dá),經(jīng)鎳柱純化和酶活分析表明所純化蛋白能催化蘋果酸脫氫生成丙酮酸,同時(shí)生成NADPH,具有蘋果酸酶的特性,其酶活大小為177.46 U/mg.以上結(jié)果證明所克隆的cDNA序列MIME2為1個(gè)新的蘋果酸酶基因,基因編碼蛋白具有蘋果酸酶活性,可為深入研究蘋果酸酶的結(jié)構(gòu)和功能關(guān)系以及進(jìn)一步應(yīng)用奠定基礎(chǔ).
[Abstract]:In order to clone and express (Mortierella isabellina) M6-22 malase gene, the coding sequence of Malinase gene was amplified by PCR using c DNA as template, according to the results of transcriptome sequencing of M6-22. After sequencing, the amplified fragment was ligated into the expression vector pET-32a () to construct the recombinant expression plasmid pET32a MIME2, which was further transformed into Escherichia coli BL21 for induction expression. A 1 815 BP cDNA sequence was amplified by nickel column affinity purification and enzyme activity analysis. The result showed that the sequence had an open reading frame encoding 604 amino acids. The predicted molecular weight of the encoded protein (Mr) was 66.4 脳 10 ~ (-3). The predicted amino acid sequence has two conserved dinucleotide binding domains and a divalent metal ion-binding domain as well as the reported malonidase, so it is a new malic enzyme gene sequence named MIME2G Bank sequence number KU097323. The purified protein was transformed into Escherichia coli BL21 by SDS-PAGE, and the protein band of 67 脳 10 ~ 3 was detected by SDS-PAGE. The purified protein could catalyze the dehydrogenation of malate to pyruvate by nickel column purification and enzyme activity analysis, and the results showed that the purified protein could catalyze the dehydrogenation of malic acid to pyruvate. At the same time, NADPH, which has the characteristic of malic enzyme, has the activity of 177.46 U / mg. These results suggest that the cloned cDNA sequence MIME2 is a new malonase gene, and the gene encoding protein has malonase activity, which may lay a foundation for further study on the structure and functional relationship of malic enzyme and its further application.
【作者單位】： 昆明理工大學(xué)生命科學(xué)與技術(shù)學(xué)院;
【基金】：國(guó)家自然科學(xué)基金項(xiàng)目(31160016,31660454) 云南省應(yīng)用基礎(chǔ)研究基金資助項(xiàng)目(KKSA201126005) 教育部回國(guó)人員科研啟動(dòng)基金項(xiàng)目(KKQA201226003)資助~~
【分類號(hào)】：Q936;Q78

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