Studies On Chloroplast Whole Genome Of Chlorella Vulgaris SAG211-12

With people’s living standards rising in the21st century, more energy should be provided to improve their lives. However, energy reserves on the Earth are limited, and the deterioration of ecological environment and the shortage of energy threaten the sustainable development of mankind. Therefore it is urgent to find an eco-sustainable energy. Bio-diesel would be the optimal alternative because it is a renewable clean energy. It is extracted and purified from green algae oil, crops oil and food waste oil. This green energy is non-toxic, non-polluting, easily biodegradable and renewable, which has become important and popular energy alternative. Micro algae with characteristics of high photosynthetic efficiency, fast growth and high oil content have become a kind of potential biomass energy plants. Research on algal chloroplast genome is one of the means to resolve its high photosynthetic efficiency.In this paper, the mode specie of Chlorella vulgaris algae SAG211-12was used as experimental material and an efficient method for extracting total genome DNA from single-cell green algae with rigid cell wall was established. The whole genomic DNA obtained was high purity and structural integrity, which absolutely met high-throughput genome sequencing. Percoll density gradient centrifugation was used to isolate a lot of chlorella intact chloroplasts which were verified by microscope observation. High quality cpDNA was then isolated from the chloroplasts with urea-sarkosyl method, including digesting with proteinase K and purification by phenol/chloroform/isoamyl alcohol extration. Gel-analysis showed that the cpDNA molecular length was approximately22kb. Photospectrometric analysis showed that the A260:A280value of the cpDNA was1.87±0.01, and the yield was (2.52±0.01) μg·g-1(DW). The chloroplast encoded16S rDNA was amplified from the isolated cpDNA, while the nuclear-encoded18S rDNA was not amplified, suggesting that the cpDNA was pure and was not contaminated by nuclear DNA. Thus the cpDNA isolated by urea-sarkosyl method meets the requirements of high-throughput sequencing. This method may also be used to isolate total DNA and cpDNA from other microalgae with similar cell wall components.Based on screening cpDNA associated with sequences from the data library of the whole-genome sequencing, cpDNA contains50unknown areaes:44vacancies (gaps), three indeterminate part of the (uncertain parts), two inserts (insertions) and a missing fragment (deletion). Dozens of mated primers were designed from two wings of all gaps after the initial splicing of cpDNA by Apple’s Mac Vector software. High-purity cpDNA extracted with percoll separation and urea method was used as the template for PCR specific amplification and sequencing. Then all gaps were filled, and a perfect orbicular physical chloroplast genomic map of algal strain SAG211-12was sketched out, summarizing all123genes. In additon, comparison of chloroplast genomes between SAG211-12and C-27was carried out, and some strain-specific and missing genes of SAG211-12were found. The results showed that the fatty acid-synthesis genes accD on cpDNA was also one of oil-producing genes. The data of cpDNA released might provide a platform of genetic engineering research, improving the breeding of high-yielding oil micro algae, and accelerate bio-diesel utilization.