Screening Of Proteins That Interact With Ring-box L From Dunaliella Salina And Preliminary Analysis Of Its Function

Ring-box l(RBX1) is Ubiquitin-protein ligase complex ring subunits, which involved in regulating a variety of cell biology process through targeted degradation of different protein substrates. Ubiquitin proteasome system is an important approach to selective protein degradation in cells, it has the very vital significance when an organism facing environmental stress to maintain steady state. When the ubiquitin-proteasome system fails to target protein degradation, it can cause tumor-suppressor protein degradation, cancer protein aggregation, proliferation acceleration block of mutate cell apoptosis and so on, thus leading to the occurrence and development of tumor. RBX1, as an important part of SCF ubiquitin protein ligase E3, gets increasing attention of the tumor scholars. RBX1 exists in the cytoplasm and nucleus from animals to plants and is relatively conservative. The RBX1 family from different organisms have 108~121 amino acids with molecular weight about 12~16k D. Human RBX1 contains a RING-H2 finger domain, which is required for zinc ion binding and ubiquitin ligation. RBX1, as an essential subunit of ubiquitin ligase E3, its abnormal expressions were observed during tumorigenesis, resulting in loss of regulation effect. It fails to induce tumor cell aging and apoptosis and it plays an important role in the process of cancer development, but the specific molecular mechanism is unclear.Flagellum/cilium, as motor driver organelle, are evolutionarily conserved. This organelle can transmit signal to cell nucleus and triggers cell reactions by accepting environmental, physical and chemical signal. Abnormal cilium structure and function can cause a series of related disease. Understanding of assembly and disassembly of cilia and related diseases were mostly based on model organisms, because it is very difficult to study the cilia of human cells directly. Dunaliella salina is a eukaryotic unicellular algae, highly resistant to salt, without cell wall, short growth cycle, easy to cultivate. There are a pair of equal length double flagellum on the cell. Therefore, D. salina can be used as a model organism to study the structure and assembly of flagellum. The transcriptome sequencing results of my lab showd that the expression of RBX1 transcribed fragment decreased significantly in the flagellum regeneration process, but the specific molecular mechanism is unclear.To preliminary study the molecular regulation mechansim of RBX1 in D. salina flagellum regeneration process, this study will screen the proteins that can interact with RBX1 in D. salina from c DNA yeast library of flagellum regeneration by yeast two-hybrid technique. A bait vector p GBKT7-rbx1 for RBX1 from D. salina was constructed, and the proteins which can interact with RBX1 were screened and vertified. Then the role of the screened protein in flagellum regulation was preliminarily studied. This study will lay the foundation for further study of RBX1 in D. salina flagellum regulation. Methods 1. Screen and verify proteins that can interact with RBX1 in D. salina by yeasttwo-hybrid system.The ORF of rbx1 from D. salina was obtained by PCR amplification and confirmed by sequencing, and then inserted into the yeast expression plasmid p GBKT7 to construct a recombinant bait vector p GBKT7-rbx1. After identified by double digestion, the constructed bait vector was transformed into the yeast strains Y187 and AH109 using PEG/Li Ac method respectively. Subsequently, the transcriptional activation and toxicity of the bait plasmid p GBKT7-rbx1 were tested to identify if the bait protein was suitable to be used in the yeast two-hybrid system. Y187 transformed with bait vector was hybridized with AH109 transformed with the D. salina c DNA expression library. After the formation of the clover shaped zygote, positive clones were screened in auxotrophic medium by the active experiment of alpha galactosidase. Then the screened clones were sequenced. And then the yeast prey vector was extracted, and cotransformed to AH109 with bait plasmids to confirming their interaction in yeast strain. 2. Study the role of screened protein in D. salina flagellum regulation.Double flagellum of logarithmic phase cells of D. salina were removed by mechanical method. During flagellum regeneration, cells at different time points were collected. Normal algal cells were used as control. The transcription level of screened protein during flagellum regeneration was analysed by q PCR using GAPDH as control. Results Screen proteins that can interact with RBX1 in D. salina by yeast two-hybrid methods.The ORF of rbx1 was obtained and the bait carrier p GBKT7-rbx1 was constructed successfully. The fusion plasmid p GBKT7-rbx1 was transformed into yeast strains Y187 and AH109 respectively. After being tested by the transcriptional activation and toxicity assay, the bait protein was both inactive and nontoxic. Therefore, the constructed plasmid can be used in the yeast two-hybrid experiment.In D. salina yeast two-hybrid screen, 298 positive clones were initially selected. Then a gene fragment of 603 bp was screened from D. salina c DNA yeast library. The gene fragment was homology with ferredoxin thioredoxin reductase subunit of Rhine chlamydomonas(38%) and Arabidopsis thaliana(39%) respectively. So the gene fragment was named Ds FTR subunit. The change of m RNA level of Ds FTR subunit during flagellum regeneration was detected by q RT-PCR(P﹤0.001). The results showed that the m RNA relative expression was raised at first and reached, maximum value at 3 h, then the expression decreased. Prey plasmid p GADT7-Ds FTR was extracted and transformed into the yeast strain AH109 with the bait carrier p GBKT7-rbx1. The cotransformation could survive on the auxotrophic medium with blue phenotype, which indicated that RBX1 can interact with Ds FTR subunit in the yeast strains. ConclusionThe protein(Ds FTR subunit) that can interact with RBX1 in D. salina was screened out by yeast two-hybrid methods. The intereaction was vetified by cotransformation experiment. In the process of D. salina flagella regeneration, the expression of the Ds FTR subunit m RNA increased at first, then decreased. RBX1 may influence the expression of the Ds FTR subunit by mediating the degradation of some proteins.