[關(guān)鍵詞]
[摘要]
目的 克隆茅蒼術(shù)Atractylodes lancea細(xì)胞分裂素羥化酶基因(AlCYP735A,GenBank:PP839072.1),對(duì)其進(jìn)行生物信息學(xué)分析、組織特異性表達(dá)分析以及構(gòu)建原核表達(dá)載體對(duì)該蛋白進(jìn)行重組表達(dá)。方法 從茅蒼術(shù)cDNA中克隆AlCYP735A序列全長(zhǎng),利用生物信息學(xué)方法分析其編碼蛋白的理化性質(zhì),qRT-PCR檢測(cè)其組織特異性表達(dá)情況,連接pET28a-mbp表達(dá)載體轉(zhuǎn)入大腸桿菌BL21進(jìn)行重組蛋白的表達(dá),并在煙草葉片中對(duì)其進(jìn)行了亞細(xì)胞定位。結(jié)果 AlCYP735A基因的開放閱讀框(open reading frame,ORF)長(zhǎng)度為1 566 bp,編碼521個(gè)氨基酸;擁有1個(gè)P450基因保守域,相對(duì)分子質(zhì)量為59 328.6,等電點(diǎn)為9.13,有信號(hào)肽,屬于分泌蛋白。qRT-PCR結(jié)果表明其根莖中表達(dá)量最高,與其他組織器官具有較明顯差異。重組蛋白mbp-AlCYP735A獲得可溶性表達(dá),大小符合106 700的預(yù)期。煙草亞細(xì)胞定位顯示其定位在葉綠體。結(jié)論 成功克隆AlCYP735A的基因,完成生物信息學(xué)分析、探明組織特異性表達(dá)情況和在煙草中的亞細(xì)胞定位,重組蛋白在原核表達(dá)系統(tǒng)中獲得了可溶性表達(dá),為后續(xù)該基因的功能驗(yàn)證提供了條件。
[Key word]
[Abstract]
Objective The gene AlCYP735A (GenBank: PP839072.1), coding for cytokinin hydroxylase in Atractylodes lancea was cloned for bioinformatic analysis, tissue-specific expression analysis, and recombinant expression using a prokaryotic expression vector. Method The full length of AlCYP735A was cloned from the cDNA of A. lancea, and its physicochemical properties were analyzed using bioinformatics tools. The tissue-specific expression of this gene was examined using qRT-PCR, and it was ligated into the pET28a-mbp expression vector and transformed into E. coli BL21 for expression of the recombinant protein. The subcellular localization prediction was carried out in tobacco leaves. Result The open reading frame of AlCYP735A is 1 566 bp, encoding 521 amino acids. It has a conserved P450 domain, with a relative molecular weight of 59 328.6 and an isoelectric point of 9.13. It contains a signal peptide and is classified as a secretory protein. The qRT-PCR results indicated that its expression is highest in the rhizome, significantly different from other tissues and organs. The recombinant protein mbp-AlCYP735A was solubly expressed, with a size consistent with the expected 106 700. Tobacco subcellular localization shows its localization in chloroplasts. Conclusion The successful cloning of the AlCYP735A gene, completion of bioinformatic analysis, determination of tissue-specific expression, soand subcellular localization in tobaccoluble and expression of the recombinant protein in the prokaryotic system provide the groundwork for subsequent functional validation of this gene.
[中圖分類號(hào)]
R286.12
[基金項(xiàng)目]
湖北省教育廳青年項(xiàng)目(Q20222013);湖北省科學(xué)技術(shù)廳青年項(xiàng)目(2022CFB576)