| Oocystis borgei is a common dominant species in the middle and late stages of high-prawn culture ponds.Its population has stable adaptability and can effectively reduce harmful substances such as ammonia nitrogen and nitrite nitrogen in pond water.Due to its significant role in aquaculture,the laboratory for more than ten years has carried out the research and application of the culture biology of O.borgei,and its application value has been reflected in shrimp culture.However,at present,its molecular biology research is almost blank and at the same time lacking of the corresponding research tools limits the molecular researches.Therefore,this paper firstly tried to construct a genetic transformation system by genetic engineering techniques.Meanwhile,transcriptome sequencing and analysis of O.borgei was conducted.The results will provide a tool and basis for the molecular biology research of O.borgei.The research content and results are as follows:（1）The sensitivity of O.borgei to 10 antibiotics was comprehensively evaluated by measuring the biomass and chlorophyll content by UV spectrophotometry and the color change of algae was recorded.The results showed that:（1）O.borgei were very sensitive to bleomycin.Bleomycin at a concentration of 25μg/mL could completely inhibit the growth of algae,the chlorophyll content was significantly lower than the control group（P<0.01）and the color of algae turned yellow and white.Bleomycin with a concentration of 25μg/mL can be used as a screening reagent in the genetic transformation of O.borgei.;（2）O.borgei were sensitive to erythromycin and chloramphenicol,however,ethanol solvent could also inhibit the growth of algae which made these two kinds of antibiotics not suitable as screening reagents for genetic transformation of O.borgei;（3）O.borgei were not sensitive to ampicillin,geneticin,gentamicin,carbenicillin,penicillin,and kanamycin,which even at certain concentrations could also promote algae biomass and/or chlorophyll content.In addition,low concentration of streptomycin could also promote the chlorophyll content of algae.（4）Under the solid culture conditions,3μg/mL bleomycin can inhibit the growth of O.borgei,and can be used as a genetic transformation screening reagent for O.borgei.（2）After removing the mold contamination in the O.borgei by centrifugal washing,pretreatment with lysozyme combined with antibiotics（400μg/mL ampicillin,400μg/mL gentamicin,400μg/mL kanamycin and 200μg/mL streptomycin）to remove algae bacteria.Through microscopic examination and bacterial and fungal plate detection,no bacteria were observed.The axenic algal was successfully established after five times subcultures and examinations.（3）The SYTOXTM Green fluorescence staining method was used to evaluate the permeability and integrity of the cytoplasmic membrane of O.borgei,and the dose of SG staining was determined to be 20μM.It can be used as the best staining condition for O.borgei with 2×107 cells/mL cell density in the dark for more than 20 min.By using flow cytometry combined with SG staining,the intersection of permeability and survival rate of O.borgei was determined to be around 550V,and 550V voltage was applied to the plasmid electrotransformation experiment.In this paper,using O.borgei as a recipient cell,the vector pOb-EGFP-2A containing the CaMV35S promoter,Omega enhancer,EGFP reporter gene and a ble selectable marker gene was introduced by electroporation with the above-mentioned shock conditions in order to construct a stable genetic transformation system of O.borgei,which provided an important reference for the construction of stable genetic transformation system of O.borgei.（4）An Illumina Hiseq high-throughput sequencing platform was used for transcriptome sequencing of O.borgei.By removing low-quality raw reads and obtaining 6.05Gb Clean Data,Trinity software was used to the de novo assembly of transcriptome data,resulting in a total of 35,873 Unigenes.There are 7,837 Unigenes with a length of 1 kb or more.The BLAST similarity alignment method was used to compare these sequences with the NR,Swiss-Prot,KEGG,COG,KOG,GO,and Pfam databases.A total of 11,032 Unigenes were annotated.There were 5,201 Unigenes who received GO annotations and classified them into 46 functional categories.There were 5018 Unigenes classified into 26 functional classifications of COG,9327 Unigenes were summarized into 25 functional classifications of eggNOG,and 6276 Unigenes were classified.Summarized into 25 functional categories of KOG.There are 4,945 Unigenes annotated by KEGG,8331 Unigenes are annotated by Pfam,5,844 Unigenes are annotated by Swissprot,and 9,860 Unigenes are annotated by nr.A total of 2617 SSR loci were found from 11032 Unigenes.The transcriptome information of O.borgei provides abundant resources and clues for related research and application in the future. |
