番木瓜果糖-2,6-二磷酸酶CpF2KP基因克隆和蛋白表达纯化

Cloning, expression and purification of fructose-2,6-bisphosphatase gene CpF2KP in papaya

摘要:

番木瓜是岭南四大名果之一，在我国东南部地区广泛种植，因其具有食用和药用双重价值，因此深受人们的青睐。果糖-6-磷酸，2-激酶/果糖-2，6-二磷酸酯酶（fructose-6-phosphate，2-kinase/fructose-2，6-bisphosphatase，F2KP）是一个独特的双功能酶，具有激酶功能域和酯酶功能域，能催化生物体内糖代谢的重要调节物果糖-2，6-二磷酸（Fru-2，6-P₂）的合成和降解。为了研究番木瓜中编码该酶的基因CpF2KP的功能，得到目的蛋白尤为重要。本研究从番木瓜基因组中提取到CpF2KP基因的编码序列（coding sequence，CDS）序列，该基因CDS全长2 274 bp。将该基因CDS全长扩增之后选用pGEX-4T-1载体进行原核表达。对载体pGEX-4T-1用EcoR Ⅰ和BamH Ⅰ进行双酶切，利用基因重组的方式将扩增序列构建到原核表达载体上。经过诱导条件探索，SDS-PAGE结果显示GST-CpF2KP重组蛋白的大小约为110 kDa，诱导CpF2KP蛋白表达的最适条件为：异丙基β-d-硫代半乳糖苷（isopropyl beta-d-thiogalactopyranoside，IPTG）浓度为0.5 mmol/L，温度28℃。对诱导后的CpF2KP蛋白进行纯化，得到了纯化的单一目的蛋白。此外，检测了该基因的组织表达特性发现该基因在种子中表达量最高，在果肉中表达量最低。该研究为进一步深入揭示番木瓜CpF2KP蛋白的功能及研究该基因参与的生物学过程提供了重要基础。

Abstract:

Papaya, which is mainly cultivated in the southeastern region of China, is one of the four famous fruits in Lingnan. It is favored by people because of its edible and medicinal value. Fructose-6-phosphate,2-kinase/fructose-2,6-bisphosphatase (F2KP) is a unique bifunctional enzyme with a kinase domain and an esterase domain that catalyzes the synthesis and degradation of fructose-2,6-bisphosphate (Fru-2,6-P₂), an important regulator of glucose metabolism in organisms. In order to study the function of the gene CpF2KP encoding the enzyme in papaya, it is particularly important to obtain the target protein. In this study, the coding sequence (CDS) of CpF2KP, with a full-length of 2 274 bp, was got from the papaya genome. The amplified sequence of full-length CDS was cloned into the vector PGEX-4T-1 which was double digested with EcoR I and BamH I. The amplified sequence was constructed into a prokaryotic expression vector by genetic recombination. After exploring the induction conditions, the results of SDS-PAGE showed that the size of the recombinant GST-CpF2KP protein was about 110 kDa. The optimum IPTG concentration and temperature for CpF2KP induction were 0.5 mmol/L and 28℃, respectively. The purified sin[A28] gle target protein was obtained after purifying the induced CpF2KP protein. In addition, the expression level of this gene was detected in different tissues, and showed that the gene was expressed at the highest level in seeds and the lowest in pulp. This study provides an important basis for further revealing the function of CpF2KP protein and studying the involved biological processes of this gene in papaya.