Aha1基因对斑马鱼肌小节组装调控作用的初步研究
发布时间：2019-01-30 19:45
【摘要】：目的:利用分子生物学技术,干扰Aha1的正常表达,分析干预前后斑马鱼运动能力、肌小节组分蛋白、肌球蛋白分子伴侣热激蛋白90(Hsp90α)和Unc45b的变化,探寻Aha1对肌小节组装的调控作用及其与Hsp90α、Unc45b的相互关系,以期为肌肉组装异常相关疾病的诊断治疗以及药物开发提供理论和实验证据。方法:运用分子生物学手段克隆Aha1基因,采用原位杂交及实时定量PCR技术检测Aha1基因在斑马鱼不同发育时期的表达和分布。采用TALEN、CRISPR-Cas9、si RNA、构建过表达载体等技术,通过显微注射的方法干扰斑马鱼Aha1的正常表达,采用测序及实时定量PCR等技术筛选Aha1基因受到干扰的斑马鱼,通过免疫组织化学染色的方法检测干预前后肌小节组分蛋白actinin的表达。行为学实验分析干预前后斑马鱼的运动能力改变。实时定量PCR法检测肌小节组装相关分子伴侣Hsp90α和Unc45b的变化,用以分析Aha1与Hsp90α、Unc45b的相关性。结果:通过原位杂交及实时定量PCR的方法确定:Aha1基因主要在斑马鱼骨骼肌及心肌中特异性表达,24 hpf时Aha1的两个同源基因ahsa1a和ahsa1b表达水平较高,故基本选取发育至24 hpf的斑马鱼完成之后的系列实验。利用TALEN基因敲除技术获得3个Aha1基因突变体品系,目标序列五个不连续碱基被敲除,且RT-PCR法证明TALEN突变体Aha1基因表达量降低;CRISPR-Cas9实验注射CRISPR ahsa1a、ahsa1a+ahsa1b-3、ahsa1a+ahsa1b-4组成功敲除掉ahsa1a基因目标序列的五个连续碱基;si RNA实验仅显微注射ahsa1b749组使ahsa1b的表达量降低;表达载体ahsa1a和ahsa1b注射斑马鱼实验中,两基因在斑马鱼骨骼肌及心肌中均发生过表达现象。免疫组化实验表明采用CRISPR-Cas9及si RNA技术干预斑马鱼Aha1基因后肌小节组装蛋白辅肌动蛋白actinin未发生明显变化。斑马鱼行为学实验表明Aha1基因发生突变或表达量减少会降低斑马鱼的运动能力及发育水平。分子伴侣表达量实验显示,TALEN突变体斑马鱼Hsp90α和Unc45b的表达水平均明显降低,注射si RNA 1b749和1a438+1b462组Unc45b的基因表达量降低,注射si RNA后Hsp90α表达水平未降低,结果表明Aha1作为一个辅助分子伴侣对肌小节的组装具有十分重要的作用。结论:本实验成功克隆了斑马鱼Aha1基因,检测出Aha1在斑马鱼肌肉特异性表达。实验成功筛选出Aha1基因突变体,且证实Aha1基因发生突变不仅会影响斑马鱼的发育及运动,也会使分子伴侣Hsp90α和Unc45b的表达水平降低,从而影响斑马鱼肌小节的组装。综上,Aha1基因作为一种肌小节组装的辅助分子伴侣,在肌无力和肌肉萎缩疾病防治中发挥重要作用。
[Abstract]:Objective: to interfere with the normal expression of Aha1 by molecular biology technique, and to analyze the changes of motor ability, muscle component protein, Hsp90 伪 and Unc45b of zebrafish before and after intervention, and to analyze the changes of myosin molecular chaperone heat shock protein 90 (Hsp90 伪) and Unc45b in zebrafish before and after intervention. In order to provide theoretical and experimental evidence for the diagnosis and treatment of muscular dysplasia and the development of drugs, the regulation of Aha1 on muscle assembly and its relationship with Hsp90 伪 and Unc45b were explored. Methods: Aha1 gene was cloned by molecular biology, and the expression and distribution of Aha1 gene in zebrafish at different developmental stages were detected by in situ hybridization and real-time quantitative PCR. The overexpression vector was constructed by TALEN,CRISPR-Cas9,si RNA, and the normal expression of Aha1 in zebrafish was interfered by microinjection. The zebrafish whose Aha1 gene was interfered was screened by sequencing and real-time quantitative PCR. Immunohistochemical staining was used to detect the expression of sarcoprotein actinin before and after intervention. Behavioral experiment was used to analyze the change of zebrafish's motor ability before and after intervention. The changes of Hsp90 伪 and Unc45b were detected by real-time quantitative PCR to analyze the correlation between Aha1 and Hsp90 伪, Unc45b. Results: by in situ hybridization and real-time quantitative PCR, the expression of Aha1 gene was found to be specific in skeletal muscle and myocardium of zebrafish, and the expression level of two homologous genes ahsa1a and ahsa1b of Aha1 was high at 24 hpf. Therefore, the zebrafish developed to 24 hpf after the completion of a series of experiments. Three Aha1 gene mutants were obtained by using TALEN gene knockout technique. Five discontinuous bases were knocked out from the target sequence, and the expression of Aha1 gene in TALEN mutants was reduced by RT-PCR method. The expression of ahsa1b in ahsa1b749 group was reduced only by microinjection of ahsa1b749 in CRISPR-Cas9 experiment. The five consecutive base; si RNA experiments in which the target sequence of ahsa1a gene was successfully knocked out in CRISPR ahsa1a,ahsa1a ahsa1b-3,ahsa1a ahsa1b-4 group were only microinjected into ahsa1b749 group. The expression vector ahsa1a and ahsa1b were injected into zebrafish. The two genes were expressed in skeletal muscle and myocardium of zebrafish. The immunohistochemical results showed that there was no significant change in actinin in the posterior segment of Aha1 gene of zebrafish treated with CRISPR-Cas9 and si RNA. Behavioral experiments of zebrafish showed that mutation or decrease of Aha1 gene expression decreased the motor ability and developmental level of zebrafish. The expression level of Hsp90 伪 and Unc45b in TALEN mutant zebrafish was significantly decreased, Unc45b gene expression in si RNA 1b749 and 1a438 1b462 group was decreased, Hsp90 伪 expression level was not decreased after si RNA injection. The results show that Aha1, as an accessory molecular chaperone, plays a very important role in the assembly of muscle bars. Conclusion: the Aha1 gene of zebrafish was cloned successfully and the specific expression of Aha1 in zebrafish muscle was detected. The Aha1 gene mutants were successfully screened, and it was proved that the mutation of Aha1 gene not only affected the development and movement of zebrafish, but also decreased the expression levels of Hsp90 伪 and Unc45b in molecular chaperones, thus affecting the assembly of muscle sections of zebrafish. In conclusion, Aha1 gene plays an important role in the prevention and treatment of myasthenia and muscular atrophy.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R3416
本文编号：2418437
[Abstract]:Objective: to interfere with the normal expression of Aha1 by molecular biology technique, and to analyze the changes of motor ability, muscle component protein, Hsp90 伪 and Unc45b of zebrafish before and after intervention, and to analyze the changes of myosin molecular chaperone heat shock protein 90 (Hsp90 伪) and Unc45b in zebrafish before and after intervention. In order to provide theoretical and experimental evidence for the diagnosis and treatment of muscular dysplasia and the development of drugs, the regulation of Aha1 on muscle assembly and its relationship with Hsp90 伪 and Unc45b were explored. Methods: Aha1 gene was cloned by molecular biology, and the expression and distribution of Aha1 gene in zebrafish at different developmental stages were detected by in situ hybridization and real-time quantitative PCR. The overexpression vector was constructed by TALEN,CRISPR-Cas9,si RNA, and the normal expression of Aha1 in zebrafish was interfered by microinjection. The zebrafish whose Aha1 gene was interfered was screened by sequencing and real-time quantitative PCR. Immunohistochemical staining was used to detect the expression of sarcoprotein actinin before and after intervention. Behavioral experiment was used to analyze the change of zebrafish's motor ability before and after intervention. The changes of Hsp90 伪 and Unc45b were detected by real-time quantitative PCR to analyze the correlation between Aha1 and Hsp90 伪, Unc45b. Results: by in situ hybridization and real-time quantitative PCR, the expression of Aha1 gene was found to be specific in skeletal muscle and myocardium of zebrafish, and the expression level of two homologous genes ahsa1a and ahsa1b of Aha1 was high at 24 hpf. Therefore, the zebrafish developed to 24 hpf after the completion of a series of experiments. Three Aha1 gene mutants were obtained by using TALEN gene knockout technique. Five discontinuous bases were knocked out from the target sequence, and the expression of Aha1 gene in TALEN mutants was reduced by RT-PCR method. The expression of ahsa1b in ahsa1b749 group was reduced only by microinjection of ahsa1b749 in CRISPR-Cas9 experiment. The five consecutive base; si RNA experiments in which the target sequence of ahsa1a gene was successfully knocked out in CRISPR ahsa1a,ahsa1a ahsa1b-3,ahsa1a ahsa1b-4 group were only microinjected into ahsa1b749 group. The expression vector ahsa1a and ahsa1b were injected into zebrafish. The two genes were expressed in skeletal muscle and myocardium of zebrafish. The immunohistochemical results showed that there was no significant change in actinin in the posterior segment of Aha1 gene of zebrafish treated with CRISPR-Cas9 and si RNA. Behavioral experiments of zebrafish showed that mutation or decrease of Aha1 gene expression decreased the motor ability and developmental level of zebrafish. The expression level of Hsp90 伪 and Unc45b in TALEN mutant zebrafish was significantly decreased, Unc45b gene expression in si RNA 1b749 and 1a438 1b462 group was decreased, Hsp90 伪 expression level was not decreased after si RNA injection. The results show that Aha1, as an accessory molecular chaperone, plays a very important role in the assembly of muscle bars. Conclusion: the Aha1 gene of zebrafish was cloned successfully and the specific expression of Aha1 in zebrafish muscle was detected. The Aha1 gene mutants were successfully screened, and it was proved that the mutation of Aha1 gene not only affected the development and movement of zebrafish, but also decreased the expression levels of Hsp90 伪 and Unc45b in molecular chaperones, thus affecting the assembly of muscle sections of zebrafish. In conclusion, Aha1 gene plays an important role in the prevention and treatment of myasthenia and muscular atrophy.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R3416
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