Studies On The Separation, Identification And Mutation Breeding Of Botryococcus Braunii

1. The germplasm separation and identification of B. brauniiAccording to the physiological ecological and morphological properties, uniaglal cultures of B. braunii were obtained from natural water through spread plate method and capillary microscopic operation, then the molecular identification was studied based on18S rDNA and ITS sequences. The main results are as follows:(1) Three unialgal strains were obtained, whose morphologies present the typical morphological characteristics of B. braunii. They numbers are ZJU3001, ZJU3008and ZJU3013successively. The morphologies, like the colony size, cell size, color, etc, of these strains are obviously different.(2) The three unialgal strains indeed belong to B. braunii according to the molecular systematic analysis based on18S rDNA and ITS sequences. The results showed that the sequence of B. braunii ZJU3001is consistent with that of B. braunii CCAP807/1. The sequence of ZJU3008is consistent with that of introduced strain ZJU3005, and it has the highest similarity (99%) with B. braunii AGB-Bb02, only existing three difference sites. The sequence of B. braunii ZJU3013has the highest sequence similarity (99%) with B. braunii Ayame, seven difference sites existing.(3) According to the comprehensive analysis of morphology and molecular systematics, etc, B. braunii ZJU3001, ZJU3008and ZJU3013successively belongs to chemical race A, L and B.2. Preparation of B. braunii single cells and the improvement of lipid content determination methodThe colonies of B. braunii make it an obstacle to conduct studies such as spectrometry and mutation breeding. Thus, the preparation method of single cells of B. braunii was established based on the ultrasound technology. The fluorescence spectrometric determination method of the lipid content in B. braunii was improved by optimizing Nile red dyeing conditions. Utilizing the established methods, the growth and lipid accumulations of three separated chemical races, B. braunii ZJU3001, ZJU3008and ZJU3013in six common mediums of microalgae were investigated. The main results are as follows:(1) The details of preparation method of single cell are: Transfer4mL of algal cultures in logarithmic phase to a5mL centrifuge tube and placed in ice-water bath, treated with ultrasonic for20s at20kHz frequency,100W transmitting power and with1s on/1s off intermittent cycle. In this case,82.3%of total cells were prepared to single cells, and98.3%were prepared to cells that formed colonies of four cells or less, meanwhile the morphological structure and regeneration rate were kept well. After being single-celled, the cell biomass measuring sensitivity of spectroscopy was increased by52.7%and the correlation coefficient was increased from0.988to0.998.(2) The details of improved fluorescence spectrum detection method of B. braunii are:After the colonial algal sample was single-celled, equivoluminal15%(v/v) dimethyl sulfoxide and3μg/mL Nile red were added into the algal sample successively and mixed evenly, then the staining system was incubated in dark at40℃for10min, and conducted fluorescence spectroscopy detection with an excitation wavelength of490nm subsequently. Compared with the traditional method, the improved one not only had remarkably higher detection sensitivity, which was increased by196.6%. but also had obviously better detection repeatability, whose characteristic parameter——relative standard deviation (RSD) was decreased from10.91%to1.84%.(3) All of the three strains, B. braunii ZJU3001, ZJU3008and ZJU3013had the highest growth rate and lipid productivity in Chu10medium. The growth rate of these strains in Chu10medium was B. braunii ZJU3001>B. braunii ZJU3008>B. braunii ZJU3013; the lipid productivity was B. braunii ZJU3013>B. braunii ZJU3001>B. braunii ZJU3008.3. Study of the mutation breeding of B. brauniiThe effects of three mutagens,60Coγ-rays, UV and EMS on the survival rate and morphology of B. braunii ZJU3001belonging to chemical race A were studied, and subsequently mutants were selected and identified. The main results are as follows:(1) The dosage effects of three mutagens to B. braunii respectively cultured in liquid and solid cultures after the treatment were ascertained. For60Coγ-rays (dose rate=2.5Gy/min), LD50=100Gy and LD=280Gy in liquid culture, LD50=51Gy and LD=200in solid culture; For UV (dose rate=180μJ/cm2), LD50=64.8mJ/cm2and LD=345.6 mJ/cm2in liquid culture, LD50=10.8mJ/cm2and LD=172.6mJ/cm2in solid culture; For3%EMS, LT50=4h and LT=15h in liquid culture, LT50=2h and LT=12h in solid culture.(2) The effects of three mutagens on the morphology of B. braunii were similar, which were reflected in the diminishment of the colonies, cells scattered, indistinction of outline, out-of-shape. cell matrix disorder and the vacuolization of the cell top or even the whole cell with the cellular contents spilling over.(3)A clone whose growth rate was significant enhanced in solid culture was selected in the group treated by80Gy of60Co γ-rays, and was named Clone-107. Compared with B. braunii ZJU3001, the growth rate of Clone-107in liquid culture was enhanced by46%, liquid content remained nearly unchanged, lipid productivity was enhanced by47%. The colonies of Clone-107were diminished significantly which were generally consisted of2-4cells and most of cells were single-celled. Meanwhile, the18S rDNA sequence of Clone-107is consistent with that of B. braunii ZJU3001, however, the analysis result of the RAPD showed that the genetic background between Clone-107and B. braunii ZJU3001were high-homologous, but they still had obvious polymorphism and their genetic similarity coefficient was0.954. Therefore, Clone-107. with high growth rate and lipid productivity, is a mutant of B. braunii ZJU3001. And it was named as B. braunii ZJU3001Ms.