Identification Of MicroRNAs In Nannochloropsis Oceanica By Small RNA High Throughput Sequencing And Establishment Of A Trangene System In Nannochloropsis Oceanica

microRNAs are endogenous single-strand RNA, with the length of~22nt,generated from stem-loop structure. microRNAs are involved in the regulation ofgene expression through the degradation of complementary target mRNA base pairingto target mRNA or inhibit translation process. Until now, microRNAs are widelyfound in human, animals, plants and viruses. It is proved that microRNAs are closelyrelated to the process of human diseases, animal growth and plants development andresist environmental stress. Compared to microRNA research in plants and animals,the research in the single cell microalgae is quite a little, only reported inChlamydomonas reinhardtii and Phaeodactylum tricornutum. Nannochloropsisoceanica belongs to the Eustigmatophyceae. The algae are rich in EPA, which isexpected to become EPA production of biological resources. In addition, the fatcontent of the algae is very high, reaching more than30%of the dry weight.Nannochloropsis oceanica have been gradually recognized as a good candidate of thebiodiesel microalgae species.This study firstly demonstrated the existence of microRNAs in Nannochloropsisoceanica by Solexa high-throughput sequencing, and established a genetictransformation system in Nannochloropsis oceanica.The total RNA of Nannochloropsis oceanica was extracted and the small RNAs,whose length were less than200bp, was seprated. The cDNA library of the smallRNAs was set up by adding adapters to both ends of them. Millions of small RNAswere obtained by Solexa high-throughput sequencing. After getting rid of tRNA, rRNA and mRNA fragments, nearly2000candidate miRNAs sequence,76%of whichranged20-22nt. The sequences were measured by the reported microRNAsequences and Nannochloropsis oceanica genome.37sequences are similar toreported microRNAs in other species; two sequences can be known inpre-miRNA and matched the genome with a predicted hairpin structure. Andthese sequences are very likely to form new miRNAs;1074sequence did not matchthe reported miRNA or miRNA precursor, but can match with the genome witha predicted hairpin structure. These sequences are thought to be specificallyexpressed miRNA in Nannochloropsis oceanica. Further analysis showed that there isa preference for U base at the position of5’end in the miRNA sequences,which is the same as the miRNAs reported in other species.Genetic transformation System is the platform and foundation gene silencingusing artificial design of miRNA. The second part of this study constructed thetransgenic system in Nannochloropsis oceanica. The new vector pCJ901wassuccessfully constructed with the resistance gene ble gene and multiple cloning sites(MCS) using the vectors pMS188and pCB801. Then the reporter gene eGFP wasinserted into pCJ901, getting pCJ901-eGFP. pCJ901-eGFP was transformed intoNannochloropsis oceanica, and the the resistance gene and reporter gene weresuccessfully expressed.This work laid the foundation for artificial design of microRNAs inNannochloropsis oceanica and supplied a tool for gene silencing in it. In thefollowing work, some key enzymes gene in the he lipid metabolism can be silencedby artificial design of microRNAs in order to achieve the purpose of improving the fatcontent.