毕赤酵母中tRNAPro CCG基因的表达及其作用效果

Expression of Pichia pastoris tRNAPro CCG and its function

摘要:

转运核糖核酸 (tRNA) 是蛋白质合成过程中重要参与成分之一，为了探索稀有密码子对应的tRNA (稀少tRNA) 丰度改变对外源基因表达量的影响，文中构建了毕赤酵母稀少tRNA基因与外源基因共表达体系。首先在GFP基因中添加由4个连续脯氨酸稀有密码子CCG组成的阻遏区，结果显示该GFP基因的表达量明显降低。然后将带有阻遏区的GFP基因和tRNAPro CCG基因顺次连接于pPIC9K载体上，在毕赤酵母GS115中共表达，结果使GFP表达量提高了4.9%；另将带有阻遏区的GFP基因和tRNAPro CCG基因分别连接于pPIC9K和pFLDα载体，在毕赤酵母GS115中共表达，GFP表达量最高提高了12.5%；应用同样方式将tRNAPro CCG基因与NFATc3T-GFP融合基因共表达，其表达量提高了21.3%。可见，tRNAPro CCG在毕赤酵母GS115中确为稀少tRNA，通过共表达tRNAPro CCG基因可显著提高带有连续该密码子的外源基因表达量，并且，文中构建的共表达体系将同样适用于其他稀少tRNA基因的筛选和验证。

Abstract:

Translocation ribonucleic acid (tRNA) is one of the important components in protein synthesis. In order to explore the effect of the changes of tRNAs corresponding to rare codons (rarity tRNAs) on the expression of exogenous genes, the co-expression system of rare tRNA gene and exogenous gene in Pichia pastoris was constructed. The expression of GFP in P. pastoris can be greatly reduced when a repressor region composed of four continuous proline rare codon CCG was added into the GFP gene. The expression amount of the repressed GFP could be increased about 4.9% when tRNAPro CCG gene was cointegrated to the 3′ of the repressed GFP gene through pPIC9K to the genome of P. pastoris GS115. Meanwhile, the expression amount of the repressed GFP increased about 12.5% by integrating the repressed GFP gene and tRNAPro CCG gene to the genome of P. pastoris GS115 through pPIC9K and pFLDα, respectively. Using the same method, NFATc3T-GFP fusion gene and tRNAPro CCG gene were co-expressed in P. pastoris GS115 resulting in 21.3% increased of the expression amount of NFATc3T-GFP fusion protein. In conclusion, tRNAPro CCG gene has been confirmed to be a kind of rare tRNAs in P. pastoris GS115. Through co-expression of tRNAPro CCG gene and heterologous genes which containing the continuous rare codon CCG, the expression of the repressed heterologous genes could be increased significantly. Furthermore, this co-expression system would contribute to screening and determining the other rare tRNAs.