论文部分内容阅读
硫氧还蛋白(thioredoxin,Trx)是一类高度保守的低分子量蛋白,广泛存在于植物、动物、细菌、酵母中,具有维持生物体内氧化还原平衡和调控生物信号传导等多种功能。本研究克隆了葡萄(Vitis vinifera)Vv Trx基因989 bp的g DNA序列(Gen Bank登录号:EU280166)和561 bp的c DNA序列(Gen Bank登录号:EU280164),开放阅读框(open reading frame,ORF)为381 bp,编码126个氨基酸。氨基酸多序列比对和进化树分析发现,无核白(Vitis vinifera cv.Thompson Seedless)Vv Trx基因的活性功能位点为WCIPS,属于h型硫氧还蛋白的第四亚族。实时荧光定量PCR(quantitative Real-time PCR,q RT-PCR)分析表明,在无核白和黑比诺(Vitis vinifera cv.Pinot Noir)的胚珠发育不同时期中,Vv Trx基因在花后40 d表达量均达到整个胚珠发育期的最高水平,二者差异极其显著(P<0.001),且无核白的表达量约为黑比诺的5倍。将Vv Trx克隆到原核表达载体p ET-32a(+)构建p ET-32a(+)/Vv Trx并转入大肠杆菌(Escherichia coli)BL21中,经异丙基硫代半乳糖苷(isopropylβ-D-1-thiogalactopyranoside,IPTG)诱导后,获得30 k D的可溶性融合蛋白。研究表明,Vv Trx基因与无核葡萄的胚败育存在一定关系,研究结果为进一步揭示Vv Trx基因在无核葡萄胚珠败育过程中的分子机理和无核葡萄育种提供研究方向。
Thioredoxin (Trx) is a highly conserved, low-molecular-weight protein widely found in plants, animals, bacteria and yeast. It has the functions of maintaining the redox balance and regulating the biological signal transduction. In this study, we cloned the 989 bp V DNA of Vitis vinifera (Gen Bank Accession No. EU280166) and 561 bp of c DNA sequence (Gen Bank Accession No .: EU280164), open reading frame ORF) is 381 bp, encoding 126 amino acids. Multiple amino acid sequence alignment and phylogenetic tree analysis revealed that Vitis vinifera cv.Thompson Seedless Vv Trx gene is a functional subfamily of WCIPS and belongs to the fourth subgroup of h-thioredoxin. Quantitative real-time PCR (q RT-PCR) analysis showed that at different stages of ovule development of Vitis vinifera cv. Pinot Noir, The expression level reached the highest level during the whole ovule development stage. The difference between the two was extremely significant (P <0.001), and the expression level of non-nuclear white was about 5 times that of pinot noir. Vv Trx was cloned into the prokaryotic expression vector p ET-32a (+) to construct p ET-32a (+) / Vv Trx and transformed into Escherichia coli BL21. The recombinant plasmid was induced by isopropylβ- D-1-thiogalactopyranoside, IPTG), a 30 kD soluble fusion protein was obtained. The results showed that the Vv Trx gene is related to the embryo abortion in seedless grapes. The results provide the research direction for further revealing the molecular mechanism of Vv Trx gene abortion in seedless grapes ovule and the seedless grape breeding.