在酵母表达过程中,传统的表达载体存在着拷贝数低以及稳定性差的缺点.因此,找到一个可用于构建复制整合型载体的高效酵母自动复制区(Autonomously Replicating Sequence,ARS)是解决问题的关键.研究中,从酿酒酵母基因组中扩增得到4种不同的ARS(ARS304、ARS315、ARS735、ARS1512),然后连接到整合载体pNTS2K中,得到4种复制整合型载体(pNTS2K-ARS304、pNTS2K-ARS315、pNTS2K-ARS735、pNTS2 K-ARS1512).经电转化后,4种转化子(36a(pNTS2 K-ARS304)、36a(pNTS2K-ARS315)、36a(pNTS2K-ARS735)、36a(pNTS2K-ARS1512))都能在含有300 μg/mL G418的YPD平板上生长.然而,只有转化子36a(pNTS2K-ARS315)能在含有500 μg/mL G418的YPD液体培养基里较快生长.经过G418耐受性检测后,36a(pNTS2K-ARS315)仍能在含有1 mg/mL G418的YPD培养基中很好地生长.此外,通过质粒pNTS2 K-A RS315表达荧光蛋白,其表达效果高于普通商业质粒.实验结果证明,4种复制区都能在酿酒酵母中独立自主复制,其中ARS315复制能力最强.因此,ARS315可用于构建高拷贝且稳定的重组质粒,为构建高表达且稳定的工程酵母菌株奠定了基础.%In the case of yeast expression,low-copy number of transformed genes and low stability are disadvantages of the conventional types of expression vectors.Therefore,finding a high effective Saccharomyces cerevisiae autonomously replicative sequence (ARS) that can be used for construction of replicative/integrated plasmid is significantly important.In this study,four different ARSs (ARS304,ARS315,ARS735,and ARS1512) were amplified from Saccharomyces cerevisiae and inserted into integrated yeast vector pNTS2K and four plasmids pNTS2K-ARS304,pNTS2K-ARS315,pNTS2K-ARS735,pNTS2K-ARS1512 were obtained.All the four plasmids were successfully transformed into S.cerevisiae and all transformants were able to grow on YPD plate containing 300 μg/mL of G418.However,only transformant containing plasmid pNTS2K-ARS315 was able to grow well in the YPD medium containing 500 μg/mL of G418.After G418 tolerance detection,36a (pNTS2K-ARS315) still could grow well in YPD medium containing 1 mg/mL of G418.Furthermore,the expression of the fluorescent protein by the plasmid pNTS2K-ARS 315 was higher than that by the ordinary commercial plasmid.It can be concluded that all of the four ARSs functioned and the ARS315 had the highest replicate ability in S.cerevisiae.Consequently,ARS315 can be potentially used for high expression and stable recombinant strain construction and metabolic engineering of S.cerevisiae.
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