枸杞木糖异构酶基因LbxylA的克隆、原核表达及多克隆抗体的制备

Cloning and Prokaryotic Expression of the Xylose Isomerase Gene(LbxylA) from Chinese Wolfberry(Lycium barbarum Linn.) Fruit and Preparation of Polycloal Antibody against the Fused Protein

以‘宁杞1号’枸杞果实为材料,利用逆转录-聚合酶链反应(reverse transcription-polymerase chain reaction,RT-PCR)技术,克隆出枸杞木糖异构酶基因(Lycium barbarum Linn. xylose isomerase,LbxylA)的全长,开放阅读框为1 446 bp,编码481个氨基酸,蛋白质分子质量为53.54 kDa,理论等电点5.37;对其进行生物信息学分析,发现LbxylA与烟草和马铃薯的木糖异构酶基因序列相似性较高,达到95%;构建重组质粒pGEX4T-1-LbxylA,转化到大肠杆菌Rosetta(DE3)中进行原核表达,通过异丙基-β-D-硫代半乳糖苷(isopropyl-β-Dthiogalactoside,IPTG)诱导培养,筛选出重组蛋白的适宜表达条件为:在18℃条件下100 μmol/L IPTG诱导10 h蛋白量最大,该蛋白为融合表达的且部分可溶;进一步纯化该蛋白并将获到的LbxylA融合蛋白为抗原,以日本大耳兔免疫制备Lbxyl A多克隆抗体,采用间接酶联免疫吸附测定法,测定抗体血清的效价高于1∶81 000;随着果实的发育,果实中LbxylA的相对表达量呈现逐渐下降的趋势,且在开花后9 d果实中LbxylA表达丰度最高;利用Western blot检测到LbxylA蛋白的变化与LbxylA相对表达量基本一致,但在开花后9 d果实中蛋白表达量低于开花后15 d,随后逐渐降低,果实成熟时两者的表达量到达最低。这表明枸杞果实发育前期LbxylA蛋白由于翻译后加工可能导致蛋白质表达滞后于基因,其结果有助于进一步研究LbxylA功能及其对果实糖积累的作用。

In this study, the full-length sequence of the xylose isomerase gene(Lbxyl A) was obtained from Chinese wolfberry fruit from the cultivar ‘Ningqi 1’(Lycium barbarum Linn.) by using reverse transcription-polymerase chain reaction(RTPCR). The open reading frame(ORF) of LbxylA was 1 446 bp in length encoding a polypeptide of 481 amino acids. The relative molecular mass was 53.54 kDa and the theoretical isoelectric point(pI) was 5.37. The deduced amino acid sequence of LbxylA shared a high similarity(95%) to those of the tobacco and potato xylose isomerase genes as revealed by bioinformatics analysis. The recombinant plasmid pGEX4 T-1-LbxylA was constructed and transformed into E. coli Rosetta(DE3) for prokaryotic expression induced by isopropyl-β-D-thiogalactoside(IPTG), and the optimum expression conditions were determined as induction at 18 ℃ for 10 h with 100 μmol/L IPTG. The fused protein, partially soluble, was purified and used as an antigen to immunize Japanese big-ear white rabbits for the preparation of polyclonal antibody against the LbxylA protein. As a result, the serum antibody titer was higher than 1:81 000 as determined by indirect ELISA. The relative expression level of LbxylA in the fruit reached its maximum at 9 d after full bloom, and then decreased gradually with fruit development. The changes of LbxylA protein level as detected by Western Blot were basically consistent with the changes of LbxylA relative expression. The protein expression level of LbxylA was lower at 9 d after full bloom than at 15 d, and then decreased gradually from the 15 th day onwards. Both the gene and protein expression levels reached the lowest values at the end of fruit ripening. This study indicated that the protein expression of LbxylA lags behind the gene transcription possibly owning to the post-translation processing at the early stage of fruit development. The results of this study are helpful to gain further insights into LbxylA function and its effect on sugar accumulation during fruit development.