Aha1基因?qū)Π唏R魚肌小節(jié)組裝調(diào)控作用的初步研究
發(fā)布時間：2019-01-30 19:45
【摘要】：目的:利用分子生物學(xué)技術(shù),干擾Aha1的正常表達(dá),分析干預(yù)前后斑馬魚運(yùn)動能力、肌小節(jié)組分蛋白、肌球蛋白分子伴侶熱激蛋白90(Hsp90α)和Unc45b的變化,探尋Aha1對肌小節(jié)組裝的調(diào)控作用及其與Hsp90α、Unc45b的相互關(guān)系,以期為肌肉組裝異常相關(guān)疾病的診斷治療以及藥物開發(fā)提供理論和實驗證據(jù)。方法:運(yùn)用分子生物學(xué)手段克隆Aha1基因,采用原位雜交及實時定量PCR技術(shù)檢測Aha1基因在斑馬魚不同發(fā)育時期的表達(dá)和分布。采用TALEN、CRISPR-Cas9、si RNA、構(gòu)建過表達(dá)載體等技術(shù),通過顯微注射的方法干擾斑馬魚Aha1的正常表達(dá),采用測序及實時定量PCR等技術(shù)篩選Aha1基因受到干擾的斑馬魚,通過免疫組織化學(xué)染色的方法檢測干預(yù)前后肌小節(jié)組分蛋白actinin的表達(dá)。行為學(xué)實驗分析干預(yù)前后斑馬魚的運(yùn)動能力改變。實時定量PCR法檢測肌小節(jié)組裝相關(guān)分子伴侶Hsp90α和Unc45b的變化,用以分析Aha1與Hsp90α、Unc45b的相關(guān)性。結(jié)果:通過原位雜交及實時定量PCR的方法確定:Aha1基因主要在斑馬魚骨骼肌及心肌中特異性表達(dá),24 hpf時Aha1的兩個同源基因ahsa1a和ahsa1b表達(dá)水平較高,故基本選取發(fā)育至24 hpf的斑馬魚完成之后的系列實驗。利用TALEN基因敲除技術(shù)獲得3個Aha1基因突變體品系,目標(biāo)序列五個不連續(xù)堿基被敲除,且RT-PCR法證明TALEN突變體Aha1基因表達(dá)量降低;CRISPR-Cas9實驗注射CRISPR ahsa1a、ahsa1a+ahsa1b-3、ahsa1a+ahsa1b-4組成功敲除掉ahsa1a基因目標(biāo)序列的五個連續(xù)堿基;si RNA實驗僅顯微注射ahsa1b749組使ahsa1b的表達(dá)量降低;表達(dá)載體ahsa1a和ahsa1b注射斑馬魚實驗中,兩基因在斑馬魚骨骼肌及心肌中均發(fā)生過表達(dá)現(xiàn)象。免疫組化實驗表明采用CRISPR-Cas9及si RNA技術(shù)干預(yù)斑馬魚Aha1基因后肌小節(jié)組裝蛋白輔肌動蛋白actinin未發(fā)生明顯變化。斑馬魚行為學(xué)實驗表明Aha1基因發(fā)生突變或表達(dá)量減少會降低斑馬魚的運(yùn)動能力及發(fā)育水平。分子伴侶表達(dá)量實驗顯示,TALEN突變體斑馬魚Hsp90α和Unc45b的表達(dá)水平均明顯降低,注射si RNA 1b749和1a438+1b462組Unc45b的基因表達(dá)量降低,注射si RNA后Hsp90α表達(dá)水平未降低,結(jié)果表明Aha1作為一個輔助分子伴侶對肌小節(jié)的組裝具有十分重要的作用。結(jié)論:本實驗成功克隆了斑馬魚Aha1基因,檢測出Aha1在斑馬魚肌肉特異性表達(dá)。實驗成功篩選出Aha1基因突變體,且證實Aha1基因發(fā)生突變不僅會影響斑馬魚的發(fā)育及運(yùn)動,也會使分子伴侶Hsp90α和Unc45b的表達(dá)水平降低,從而影響斑馬魚肌小節(jié)的組裝。綜上,Aha1基因作為一種肌小節(jié)組裝的輔助分子伴侶,在肌無力和肌肉萎縮疾病防治中發(fā)揮重要作用。
[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 偽 and Unc45b of zebrafish before and after intervention, and to analyze the changes of myosin molecular chaperone heat shock protein 90 (Hsp90 偽) 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 偽 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 偽 and Unc45b were detected by real-time quantitative PCR to analyze the correlation between Aha1 and Hsp90 偽, 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 偽 and Unc45b in TALEN mutant zebrafish was significantly decreased, Unc45b gene expression in si RNA 1b749 and 1a438 1b462 group was decreased, Hsp90 偽 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 偽 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.
【學(xué)位授予單位】：山西醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R3416
本文編號：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 偽 and Unc45b of zebrafish before and after intervention, and to analyze the changes of myosin molecular chaperone heat shock protein 90 (Hsp90 偽) 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 偽 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 偽 and Unc45b were detected by real-time quantitative PCR to analyze the correlation between Aha1 and Hsp90 偽, 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 偽 and Unc45b in TALEN mutant zebrafish was significantly decreased, Unc45b gene expression in si RNA 1b749 and 1a438 1b462 group was decreased, Hsp90 偽 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 偽 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.
【學(xué)位授予單位】：山西醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R3416
【參考文獻(xiàn)】
相關(guān)期刊論文 前8條
1 馬蘭芳;楊書紅;王世宣;;肌動蛋白相關(guān)蛋白2/3復(fù)合體的研究進(jìn)展[J];中國婦幼保健;2014年34期
2 柳睿殊;劉榮;劉書霞;張輝;耿軼釗;紀(jì)青;;肌肉力量的微觀起源[J];生命科學(xué)研究;2014年05期
3 馬元武;馬婧;路迎冬;陳煒;張旭;于磊;張連峰;;利用CRISPR/Cas9敲除大鼠胰島素受體底物1(Irs1)基因[J];中國比較醫(yī)學(xué)雜志;2014年03期
4 李伯江;李平華;吳望軍;李齊發(fā);黃瑞華;劉紅林;;骨骼肌肌纖維形成機(jī)制的研究進(jìn)展[J];中國農(nóng)業(yè)科學(xué);2014年06期
5 Andrew R.Bassett;Ji-Long Liu;;CRISPR/Cas9 and Genome Editing in Drosophila[J];Journal of Genetics and Genomics;2014年01期
6 沈延;黃鵬;張博;;TALEN構(gòu)建與斑馬魚基因組定點突變的實驗方法與流程[J];遺傳;2013年04期
7 張震華;薛良義;;魚類肌節(jié)的組成及裝配[J];中國細(xì)胞生物學(xué)學(xué)報;2012年12期
8 邱華玲;于建興;陳宏權(quán);;MyoG基因結(jié)構(gòu)與功能研究進(jìn)展[J];生物信息學(xué);2007年04期
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