Construction Of A Genomic DNA Library Of Dunaliella Salina

Dunaliella salina(D. salina) is a photoautotrophic unicellular eukaryotic green alga, belonging to Chlorophyta, Chlorophyceae, Volvocales. It is one of the most extremely halotolerant eukaryotes and can live in a variety of salt concentrations ranging from 0.05 to 5M solution of sodium chloride. It has a thin cellular membrane, a single, large cup-shaped chloroplast with photosynthetic thylakoid membranes, pyreniod, starch and abundant β-carotene globules, but lack of rigid cell wall. The simple and cheap culture of D. salina make it a desirable host for increasing production of natural compounds by genetic engineering or for exploitation as biological factories for the synthesis of novel high-value compounds.The genomic DNA library is the basic tool for the genome research. It is important for the genome physical mapping, genome sequencing and map-based gene cloning.The studies of the structure, function, expression and regulation of genes have deepened into the level of molecular with the rapid development of the genetic engineering techniques, so it is possible to isolate and obtain the specific gene fragments. Genomic library is an aggregation of recombinant DNA clones containing the genomic DNA fragments that are obtained by partially digesting the high-quality genomic DNA with restriction enzymes. Each DNA sequence of the genome of interest should be presented proportionally in the recombinant cloning library in theory,but this never comes true in reality. On the contrary, some sequences will be losing totally, while some will be presented completely in the genomic library. This result cannot be avoided when creating the genomic library through different means.The coverage is partially deviated because there was no one-way to really cut the genome complex randomly without interference of genomic sequence or base pairs component. And sometimes it is hard to avoid the deviation, because the library is only a statistical sample of the restricted fragment population. More pitied the replication ability of some genomic clones was stronger than others, therefore, the deviation of gene presentation will happen with the replication and amplification of the library. Though the genomic library is not prefect , we were still confidented, that it can make the interested gene cloned more conveniently.The type of the genomic library was determined by the chosen vector. When constructing a genomic library, there are many factors affecting the selection of vector, such as: the size of the target genomic DNA, the capacity of the vector, the stableness of the cloned sequences and if the design of the vector fits to the chromosome walking. In this experiment, we constructed a genomic library of Dunaliella salina with phage lambda as vector.Methods and results:The genomic DNA of Dunaliella salina was extracted by SDS lysis. And detected by agarose gel electrophoresis, the result shows that the length of the DNA fragment obtained is more than 50Kb. The value of the DNA A260/A280 is 1.8 through UV detection, and the concentration is about 1 μg/μL. A number of small-scale restriction digestion using various amounts of Sau3AI were performed to optimize the conditions for obtaining a large amount of genomic DNA fragments of the desired size (9~23kb) .Amass of DNA were then partially digested with Sau3AI under optimized conditions. After further purification the fragments were ligated to Lambda EMBL3 vector, and the ligation products were incubated with packaging extracts , then infected into E. coli LE392 . As a result , we constructed a genomic DNA library which obtained about 2.2 X 10° recombinants, which provides sufficient clones to cover all Dunaliella salina genes .Randomly select clones to determine the fragment , we found that the inserted DNA fragments were approximately 12.6kb.Using RbcS cDNA as probe, the library is screened and verified by means of hybridization of bacteriophage plaques. The result shows that the recombinant clones are earring Rbcs gene .Conclusion:The genomic library of Dunaliella salina was successfully constructed in this experiment.