Evolutional Analysis Of Anopheles Sinensis Chromosomes And Establishment Of Transformation System And Functional Analysis Of Genes In Pythium Guiyangense

There are more than 3,500 mosquito species distributed all over the world which can transmiting more than 20 types of diseases including malaria,dengue fever,yellow fever,West Nile fever,filariasis,Japanese encephalitis and others.According to the previous data,approximately 700 million people have been suffering from these infectious diseases each year posing great threat to people's physical and mental health.Malaria is a deadly disease eaused by Plasmodium with wide geographic distribution around the world.It mainly transmitted by femal Anopheles mosquitoes.In recent years,with the strong support of the goverment,the prevention and control of malaria in China have achieved remarkable results;however the malaria situations in Yunnan,Jiangsu,Sichuan,Henan,and Zhejiang are still serious.Anopheles sinensis is the main vector of vivax malaria transmission in the vast plains of northern China and accounts for more than 83%of the msoquito population in central China.Therefore,the cytogenetic study and chromosomal evolution analysis for An.sinensis will provide valuable information for malaria control in China.The X chromosome in An.sinensis had the shortest synteny blocks and the fastest evolution among the five chromosomes.Here,we used 52 scaffolds on the polytene chromosomes of An.sinensis and previously published genome sequcnence of An.gambiae to identify 3955 orthologous genes and plotted the locations onto five chromosome arms for investigating the synteny blocks between two species.The study of number and distribution of synteny blocks between An.sinensis and An.gambiae indicated that the X chromosome had the shortest synteny blocks among the five chromosomes.To further analyse fixed inversions between An.sinensis and An.gambiae,we input the synteny blocks into the GRIMM program for calculating the number of inversions on each arm and analyzed the inversion rates by caleulating the number of inversionszMb/MY.All results revealed that the sex exhibited higher density of fixed inversions than aotusomes indicating the faster evolution of X chromosome in An.sinensis and An.gambiae.Our analysis in this study provided additional details for understanding chromosome evolution in malaria vectors.We established a transformation method for Py.guiyangense and studying the funetion of the PgTKL by using this genetic transformation approaeh.Pythium guiyangense Su is a promising mosquito control agent first identified by Dr.Xiaoqing Su from Guiyang of China which can effectively kill more than 14 species of mosquitoes within the Genus Culex,Aedes and Anopheles including 6 important infectious disease vectors.However,the molecular meachnisms of pathogenicity of Py.guiyangense in the larvae of mosquitoes are still not clear,so there is an urgent need to construct a stable genetic transformation system to investigate the funcational genes and to further genetic manipulation of this strain for future application.Therefore,in this research,we established a transformation method for Py.guiyangense by modifying suitable medium,rotation speedthe best mycelial age.and the optimal hydrolysis time.By using this genetic transformation approach,the gene expression of PgTKL,a member of the tyrosine kinase-like(TKL)family,was specifically silenced to evaluate the biological function of this gene.Attenuated strain of Py.guiyangense displayed morphology change of hyphae,reduced mycelium growth rate and low zoospore production in comparison with wild type strain.Silencing PgTKL in Py.guiyangense also resulted in decreased virulence in the larvaes of mosquitoes.Our date strongly suggested that PgTKL played important roles in cell growth,biotie stress response and pathogenicity of Py.guiyangense.The crinkling and necrosis protein(CRN)are effectors found from oomycetes and can induce host cell death by unknown mechanisms.A total of 22 CRN effectors were identified from Py.guiyangense by bioinformatic analysis.To investigate the biological functions of these effectors in Py.guiyangense e,we detected the expression patterns of 6 CRN effectors in infected mosquito larvae.Our results showed that the relative expression levels of the CRN 44 effector ware up-regulated during the early atages of infections and other five CRN effectors showed different patterns.However,all the data strongly supported that CRN effectors in Py.guiyangense involved in the virulence in the mosquito pathogen Py.guiyangense.