Expression Of IPP Isomerase Gene From Escherichia Coli In Cholrella Vulgaris

Lutein is an important caroteniod which has broad activities including antioxidant,preventing cancer and coloring.Chlorella can produce a large number of lutein.It has been found that Chlorella has a simple structure,grows quickly and can be easily cultured.Therefore,biosynthesis of lutein in Chlorella has attracted diverse attentions.Firstly,we optimized the system of protoplast preparation method in Chlorella vulgaris to obtain a high yield of protoplasts.Then,the electroporation condition and system for Chlorella protoplasts were optimized and established by using gus gene as a reporter gene.Subsequently,the IPP isomerase gene?idi?was transferred into Chlorella vulgaris to obtain transgenic algae strains.The details are as follows:Firstly,we prepared protoplasts of Chlorella vulgaris using enzymatic method and studied the effect of several factors including pretreatment time,type of enzyme,hydrolysis temperature,hydrolysis pH,enzymatic hydrolysis time and hydrolysis speed on protoplast formation.The results showed that the rate of protoplast formation increased5.43-17.68 times after EDTA and DTT pretreatment on Chlorella vulgaris.In addition,the protoplast formation rate reached to 49.17%under the condition of 2%cellulase,pectinase 2%and 2%snail enzyme together at hydrolysis temperature 30?,pH5.8,hydrolysis speed 120rpm and hydrolysis time 14h.Secondly,the electro-transformation method for Chlorella vulgaris was established.The results showed that the maximal GUS activity was obtained under a condition of electroporation voltage 1800V,carrier DNA 125?g/mL and plasmid DNA fragment 8?g/mL.Adding of Ca²⁺into shock buffer can greatly increase the transformation efficiency of Chlorella vulgaris.Before or after the shocking,the ice bath treatment of protoplasts and plasmid DNA mixture decreased the transformation efficiency.Thirdly,we constructed the recombinant vector pBS-18S1-nptII-idi-18S2 carrying Chlorella vulgaris 18S rDNA gene as fragment homologous arms,nptII gene as resistance selective maker and IPP isomerase gene?idi?from E.coli.Then,under the optimal eletroporation condition,the recombinant vector pBS-18S1-nptII-idi-18S2 was transferred into Chlorella vulgaris by electroporation.PCR analysis indicated that idi gene was integrated into Chlorella vulgaris genome.It was found that the majority of transformants had similar biomass as the wild type strain.On the other hand,the lutein content in transformants increased 38.27%compared with the one in the wild type.In addition,the lutein yield in transformants increased 42.16%compared with the one in the wild type.Above results demonstrated that idi gene from E.coli can be successfully expressed in C.vulgaris.