Cloning and Function Analysis of the Fructose-6-phosphate,2-kinase/ fructose-2,6-bisphosphatase Gene (F2KP) from Sugarcane(Saccharum officinarum L.) | 甘蔗果糖-6-磷酸,2-激酶/果糖-2,6-二磷酸酯酶基因(F2KP) 的克隆及其功能研究

Chino. 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)是影响生物碳积累和分配的调控关键酶，同时也是一个具有激酶和酯酶两种催化活性的双功能酶。本研究在已获得甘蔗(Saccharum officinarum)蔗叶 F2KP(命名为SoF2KP-L)的基础上，以蔗茎 cDNA 为模板克隆获得不同长度的同源片段，分别命名为 SoF2KP-S1、SoF2KP-S2和SoF2KP-S3。生物信息学分析结果显示，SoF2KP-L 翻译的蛋白具有完整的激酶和酯酶结构域，SoF2KP-S1、SoF2KP-S2和SoF2KP-S3 所含开放阅读框长度均小于 SoF2KP-L，其中 SoF2KP-S1 翻译的蛋白只具残缺的激酶结构域；SoF2KP-S2 翻译的蛋白具完整激酶结构域但缺失酯酶结构域；SoF2KP-S3 只能翻译数个氨基酸即终止翻译。选择双功能域完整的 SoF2KP-L构建表达载体，农杆菌(Agrobacterium tumefaciens)介导转化烟草(Nicotiana tabacum)。RT-PCR证实， SoF2KP-L 在转基因植株成熟叶中转录表达。碳水化合物等生理指标测定结果表明，转基因植株成熟叶片中可溶性总糖/淀粉、还原糖/淀粉、蔗糖/淀粉比值均有不同程度的升高，碳水化合物含量和相关分配比率发生改变。研究结果提示，甘蔗中可能存在不同的 F2KP 转录产物，并初步证实甘蔗 SoF2KP-L 在光合组织中对蔗糖和淀粉的分配起到一定的调控作用，为进一步研究甘蔗 F2KP 基因的功能及为甘蔗分子育种提供参考依据。

Inglés. Fructose-6-phosphate,2-kinase/fructose-2,6-bisphosphatase(F2KP), which is comfirmed to be a bifunctional enzyme, is a key regulatory enzyme involved in carbon accumulation and partitioning processes. In the previous study, F2KP with both kinase and phosphatase domain named SoF2KP-L was obtained from the leaf of sugarcane. In this research, different sizes of F2KP homologous fragments from the stem of sugarcane(Saccharum officinarum L.) were amplified and named SoF2KP-S1, SoF2KP-S2 and SoF2KP-S3, respectively. Based on the bioinfomatical analysis, the putative protein SoF2KP-S1, SoF2KP-S2 and SoF2KP-S3 were incomplete respectively with part of kinase domain, phosphatase domain and only several amino acid. Therefore, SoF2KP-L with complete kinase/phosphatase catalytic domains was inserted into plant expression plasmid under the Cauliflower mosaic virus 35S promoter, then transferred into tobacco(Nicotiana tabacum) plants by Agrobacterium-mediated transformation. RT-PCR (Reverse transcription PCR) analysis confirmed that SoF2KP-L was successfully transcripted in mature leaf. The changing rates of soluble sugar/starch, reducing sugar/starch and sucrose/starch indicated that carbohydrate level and carbohydrate partitioning of the transgenic plants had been changed. These results indicate that there may be different transcripts existing in sugarcane and SoF2KP-L regulates the carbon portioning in sugarcane as F2KP in other plants. This stuty will provide theory basis for further study on the biological function of F2KP from sugarcane and for its potential application in sugarcane molecular breeding.