Preliminary Study On The Regulation Of Aha1 On Sarcomere Assembly Using Zebrafish Model

Objective:In order to provide theoretical and experimental evidence for the diagnosis and treatment of diseases and drug development related to abnormal assembly for muscle,the normal expression of Aha1 gene was disturbed by applying molecular biological technique,then analysis of zebrafish movement ability,sarcomere proteins,changes in myosin molecular chaperones Heat shock protein 90α(Hsp90α)and Unc45b before and after the intervention,to explore the role of Aha1 in regulation of sarcomere assembly and its relationship with Hsp90α and Unc45b.Methods:Using molecular biology methods to clone Aha1 gene,in situ hybridization and real-time quantitative PCR were used to detect Aha1 gene expression and distribution in different developmental stages of zebrafish.The technology of TALEN,CRISPR-Cas9,siRNA and construction of expression vector was used to disturb the normal expression of Aha1 in zebrafish by method of micro-injection,screening of the Aha1 gene disturbed zebrafish by method of sequencing and real-time PCR.The expression of sarcomeric components actinin was detected by immunohistochemical technology before and after the intervention.The exercise ability of zebrafish before and after the intervention was analyzed by behavioral experiment.The method of real-time quantitative PCR was used to detect the change of sarcomere assembly related molecular chaperone Hsp90α and Unc45b in order to analyze the relationship among Aha1 and Hsp90α and Unc45b.Results:Determined by in situ hybridization and real-time PCR,Aha1 gene specifically expressed in zebrafish skeletal muscle and cardiac muscle.Two Aha1 homologous gene ahsa1 a and ahsa1 b expression level was higher at 24 hpf(hour post fertilization).So the 24 hpf zebrafish was selected to complete a series of experiments.The experiment of TALENhad screened three Aha1 gene mutation zebrafish,the target sequence of five discrete bases was knocked out,and the RT-PCR method proved the expression of TALEN mutant Aha1 gene decreased.The target sequence of five continuity bases in ahsa1 a gene was knocked out by injecting CRISPR ahsa1 a,ahsa1a+ahsa1b-3,ahsa1a+ahsa1b-4 group in CRISPR-Cas9 experiment.The expression of ahsa1 b was decreased in only micro-injected siRNA ahsa1b749 group of siRNA experiment.The experiment of expression vector ahsa1 a and ahsa1 b showed that both two genes in zebrafish skeletal muscle and myocardium occured overexpression phenomenon.Immunohistochemical results indicated that there was no significant change of sarcomere assembly proteins-actinin after disturbing Aha1 gene by technology of CRISPR-Cas9 and siRNA.Zebrafish behavioral experiments showed that Aha1 gene mutation or lowly expressed reduced the zebrafish’s exercise ability and development level.The experimental of expression of molecular chaperone displayed that the expression level of Hsp90α and Unc45b was obviously reduced on TALEN mutant zebrafish.The gene expression of Unc45b was decreased in injecting siRNA 1b749 and 1a438+1b462.The expression of Hsp90α was not decreased after injection of siRNA.The results showed that,Aha1,as an auxiliary molecular chaperone,played an important role in the assembly of sarcomere.Conclusion:This experiment successfully cloned the Aha1 gene of zebrafish and detected specific expression of Aha1 in zebrafish muscle.The experiment successfully screened the mutant Aha1 gene,and confirmed that Aha1 gene mutation not only affected the zebrafish development and movement,also reduced the expression of molecular chaperone Hsp90αand Unc45b,thus affecting the sarcomere assembly in zebrafish.In summary,Aha1,as an auxiliary molecular chaperone,played an important role in the onset and development of myasthenia and muscular atrophy.