黄河裸裂尻鱼MSTN基因RNA干扰研究

Inhibitory effect of RNA interference of MSTN gene expression on the downstream genes in Schizopygopsis pylzovi

目的利用反义寡合甘酸技术抑制肌肉生长抑制素(myostatin,MSTN)的表达,评估MSTN基因的沉默效果,并检测RNA干扰后对下游基因的影响。方法通过构建黄河裸裂尻鱼(Schizopygopsis pylzovi)MSTN基因RNA干扰(RNAi)重组腺病毒载体1P3(DSP MSTN 273+250+1737)和1P2(DSP MSTN 195+1670),并将其注射黄河裸裂尻鱼肌肉组织,进行活体RNA干扰;利用real-time PCR和Western blotting评估MSTN基因的沉默效果,并检测MSTN基因RNA干扰后肌肉肌酸激酶(muscle-type creatine kinase,M-CK)基因转录水平的调控作用。结果real-time PCR分析结果表明,与HK组(病毒通用阴性对照组)和N组(空白对照组)相比,重组腺病毒载体1P3对黄河裸裂尻鱼肌肉MSTN基因的转录具有明显的干扰作用(P<0.05),抑制率达53.5%;而重组腺病毒载体1P2对MSTN基因的转录无明显干扰作用(P>0.05)。Western-blotting分析结果与real-time PCR结果相一致。同时,经1P3干扰后随着MSTN基因转录水平的下降,其肌肉肌酸激酶M-CK基因表达水平显著上升。结论通过RNAi技术能够有效的抑制MSTN基因的表达,并能够上调M-CK基因的表达量。因此,证明了在黄河裸裂尻鱼中MSTN能够抑制M-CK的转录。揭示高原土著鱼类MSTN基因的对肌肉的生长发育起到调控作用。

Objective To investigate the silencing effect of RNA interference on MSTN gene（ myostatin,MSTN）expression,and detect the effects on the downstream genes in Schizopygopsis pylzovi. Methods To construct the recombinant adenovirus vector 1P3（ DSP MSTN 273 ＋ 250 ＋ 1737） and 1P2（ DSP MSTN 195 ＋ 1670） for RNA interference of the MSTN gene in Schizopygopsis pylzovi,and to conduct the RNA interference in vivo experiment by injecting the vector into the muscle tissue of Schizopygopsis pylzovi. Real-time PCR and Western blotting were used to evaluate the silencing effects on MSTN gene expression,and to detect the regulatory function of M-CK at gene transcription level after RNA interference of the MSTN gene. Results The result of real-time PCR showed that compared with the HK team（ Virus general negative control group） and N team（ blank control group）,the 1P3 had significant interference effect on the MSTN gene transcription in Schizopygopsis pylzovi（ P 〈 0. 05）,with an inhibition rate of 53. 5%,but the 1P2 had no significant interference effect on the MSTN gene transcription. The result of Western blotting was consistent with the results of real-time PCR. At the same time,after the 1P3 interference,the level of MSTN gene transcription was declined,and the level of M-CK gene expression was significantly increased. Conclusions Our results demonstrate that the expression of MSTN gene can be effectively suppressed,and the expression of M-CK gene can be up-regulated through the RNA interference. Therefore,it proves that MSTN gene can inhibit the transcription of M-CK gene in Schizopygopsis pylzovi,and reveals the regulatory role of MSTN gene in the muscle growth and development in the plateau fish Schizopygopsis pylzovi.