中文摘要：

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。为筛选出高酯酶酶活性的改造酶分子，本研究通过定点突变与片段缺失方法改造蔗叶F2KP基因，将蔗叶F2KP基因的Asp442、HisS08和血g446分别突变为Gly、Ala和Pro，并且将分别缺失F2KP基因的酯酶的大部分和激酶的大部分构建F2KP激酶重组片段和酯酶重组片段，将突变后的基因与表达载体pPICgK双酶切后进行连接，构建重组质粒。经过菌落PCR和双酶切重组质粒均证明已将目的片段转到酵母表达载体pPIC9K内。

英文摘要：

Fructose-6-phosphate, 2-kinase/fructose-2,6-bisphosphatase （F2KP）, which is cow.firmed to be a hifunctional enzyme, is a key regulatory enzyme involved in carbon accumulation and partitioning processes. In order to find high activity of ester Enzyme, The study reformed F2P, 2K/F26Bpase genes of sugarcane and rats liver by site-specific mutagenesis and piece missing process. The Asp442, His.508, Arg446 of F2KP protein product are replaced by Gly, Ala, Pro separately, and Asp191, His259, Arg196, Set33 of PFKFB protein product are replaced by Gly, Ala, Ala, Ala separately. Also, The study has also missed most of the kinase and esterase section respectively. Then the recombinant plasmids were transformed into E. coli DH5α. The PCR and restriction en- zyme digestion results showed that the Pichia pastoris expression vectors contained our mutant genes.