Cloning And Analysis Of Zep Gene Encoding Zeaxanthin Epoxidase In Chlorella Protothecoides

Carotenoids are a category of natural pigment, which exist in many organisms, such as plants, photosynthetic or non-photosynthetic bacteria, alga and fungi. They have important physiological function of anti-oxidation, prevention of cancer, macular eye disease and cardiovascular disease. Chlorella has aroused great attention due to its high cellular carotenoids content, especially Chlorella protothecoides. It can produce a large amount of lutein, which is a potential resource for lutein production. It will be a commercial application prospect to enhance carotenoids biosynthesis and production with C. protothecoides by transforming the carotenoids biosynthesis pathway.Using the green alga, C. protothecoides CS-41, this research was focused on the development methods of biomass determination, stimulation effect on algal growth with plant phytohormone; cloning and analysis of zeaxanthin epoxidase gene. The main research contents and results are as follows:1. For the sake of satisfying the requirements for the precise determination of cell dry weight for industrial application purpose, a good linear relationship between the absorbance and microscopic counting/ dry weight was established, providing a convenient method of biomass determination by optical density measurement.2. The growth rate of C. protothecoides CS-41 was increased through the treatments with phytohormones of appropriate concentration. 0.5mg/L IBA was the most effective. On the contrary, ABA treatment had little effect on the growth.3. The degenerate primer sets were designed with the conservative sequence of zep gene found in NCBI database and a core fragments of 972 bp was obtained. RACE (rapid-amplification of cDNA ends) essay and RT-PCR were used to isolate the full-length cDNA of zep from C. protothecoides CS-41. A cDNA of 2616 bp was cloned with an open reading frame of 1680 bp, which encoded a putative ZEP from C. protothecoides CS-41. The phylogenetic tree established with MEGE 4.1 software revealed that the genetic relationship between C. protothecoides CS-41 and Chlamydomonas reinhardtii is close.4. With the aim of exploring the functions of zep gene under the stress-environments and the function in ABA biosynthesis, fluorescence quantitative PCR was performed to detect the expression of zep gene under NaCl and ABA treatments. The results revealed that zep gene expression was up-regulated under salt stress treatment,which clarified that zep gene played an important role in stress response. Under ABA treatment, zep gene was down-regulated, which revealed that zep gene took part in ABA biosynthesis and had an important function.