[Objectives] This study was conducted to investigate the function of Dunaliella salina calmodulin kinase(CaM K) gene.[Methods] The sense and antisense gene fragments(223 bp) and spacer sequence(129 bp) of D.salina calmodulin kinase gene were cloned and inserted into the downstream part of the35 S promoter of the eukaryotic expression vector pM DCMGN-Cat.The siRNA expression system of CaM K gene was successfully constructed.The p CaM K-RNAi expression vector was transformed into D.salina cells by the LiA c/PEG-mediated method,giving transgenic D.salina.The expression of CaM K gene was then analyzed by real-time fluorescence quantitative PCR.[Results]The expression of CaM K gene in the transgenic D.salina was significantly reduced,by 70% compared with the control group,suggesting that the expression of CaM K gene was significantly inhibited.The examination of the growth status of D.salina showed that D.salina cell division and proliferation were also affected.It is proved that CaM K gene has a positive regulation effect on the division and proliferation of D.salina cells.[Conclusions] The study provides important information for further elucidating the function and action mechanism of D.salina calmodulin kinase gene.
In order to investigate the role of MAPK gene in adaptation of Dunaliella salina to hypersaline environment, the Ds MAPK gene of D. salina was amplified by PCR. After inverted insertion of the open reading flame ofDs MAPK gene into downstream sequence of 35S promoter of plant expression vector, an antisense expression vector of Ds MAPK gene was successfully constructed and introduced into D. salina cells by LiAc/PEG-mediated method. The expression of Ds MAPK gene in transgenic D. salina was analyzed by semi-quantitative RT-PCR method. The results showed that the expression of Ds MAPK gene in D. salina was significantly inhibited at the transcriptional level. The study laid the foundation for further identification of the function of Ds MAPK gene.
