Preliminary Structure And Function Analysis Of GCα Protein During The Intraerythrocytic Stage In Plasmodium Yoelii

Malaria is a kind of disease that caused by Plasmodium,which belongs to the Apicomplexa and survives in humans or animals,and the Plasmodium is transmitted by the bite of Anopheles mosquitoes.Malaria is actually caused by asexual multiplication of the Plasmodium in the blood of the host and is one of the three most major infections in the world which causes almost 500 thousands deaths last year.Plasmodium itself has two types of multiplication:asexual multiplication and sexual multiplication.Plasmodium mainly undergoes asexual multiplication in the host,and some cells will develop sexually into male and female gametophytes when they are sucked into the midgut by mosquitoes.And the cells will fertilize to become zygotes which will continue undergoes asexual proliferation in the body of Anopheles mosquitoes.As Apicomplexa,the important physiological processes of malaria parasite as egress,invasion and movements are strictly regulated by the second messengers such as cGMP.In the asexual phase of Plasmodium,GCa protein which has an independent P4 type ATPase domain(ALD)and a Guanylate Cyclase Domain(GCD)is the only source of second messenger cGMP,but no studies have yet fully explained how GCa protein participates in these processes and how they are regulated.In this paper,the Plasmodium yoelii 17XNL strain was used as the model,and the PyGCa protein was endogenously labeled and modified by the CRISPR/Cas9 gene editing system.First,we constructed an endogenous bilateral tagged strain of gca gene,which was added with a 4Myc short peptide at its N-terminus,and a 6HA short peptide at its C-terminus.WB results with other unilateral tag-modified strains indicate that the N-terminus and C-terminus of GCa protein may be cleaved at the protein level.Further,we constructed an overexpressed peptide fragment based on the linker portion of GCa protein to investigate whether the cut can be achieved in a non-endogenous situation.The experimental results show that in Plasmodium,the GCa protein has natural shear and the cleavage site is at the N-terminus of the linker part.To determine if this shear is necessary for Plasmodium,we designed a protein truncation experiment with three different deletion modifications to the gca gene.Finally,the first 50 amino acids of the gca gene linker parter could not be deleted,indicating that this amino acid sequence is necessary for the asexual phase of Plasmodium yoelii 17XNL.We also performed a point mutation experiment on the conserved phosphorylation site of the ALD domain,and found that D at the 720 site could not be missensely mutagenized,indicating that the ALD part is functionally conserved and may have the function of phosphorylation.Moreover,the co-IP experiment with DTS strain shows that although ALD and GCD are separated at the protein level,they are functionally related.The work of this thesis mainly used the CRISPR/Cas9 gene editing system to systematically reveal that the GCa protein of Plasmodium yoelii 17XNL is different from other Apicomplexa sub-gate GC proteins in the asexual period,and studied the mechanism of GCa protein.The research may provides new ideas for studying the development of Plasmodium in the asexual period and provides a new target for the antimalarial drugs.