基于rhaSR嵌合操纵子的穿梭表达载体及其在鱼腥藻细胞中表达的初步研究

Shuttle Expression Vectors Based on rhaSR Chimeric Operon and Expression in Anabaena sp.PCC 7120

通过PCR等基因重组技术,以pKT-215(起源于RSF1010)为载体,构建了带有PR序列(含rhaSR操纵子表达调控元件、rhaR基因、报告基因gst的嵌合操纵子)和PT序列(含rhaSR操纵子表达调控元件及rhaT基因)的二种穿梭表达载体pKT-PTR、pKT-PR。其中,在RhaR基因上游设计和插入了增强的SD序列。首先将pKT-PTR、pKT-PR分别转化入大肠杆菌BL21(DE3)中,在不同培养基条件下,即含鼠李糖与不含鼠李糖的培养基中进行表达,紫外分光光度计测定GST蛋白(谷胱甘肽-S-转移酶)的活性。结果表明,含嵌合操纵子的穿梭表达载体能在L-鼠李糖的诱导下表达,gst基因的表达量诱导条件下比非诱导条件下高3倍左右,且表达载体上的rhaT基因并不能提高gst的表达;其次,利用三亲结合的方法将2种表达质粒转入鱼腥藻Anabaena sp.PCC7120中,抗生素筛选后得到能稳定遗传的转基因藻,并以转基因藻的质粒DNA为模板进行PCR检测。最后,转基因藻GST酶活力分析结果表明,嵌合操纵子PR在蓝藻细胞中的诱导表达效率极低,表达条件还需进一步优化。尝试将诱导型的操纵子转入蓝藻,具有潜在的应用价值。

In this study,one chimeric operon containing rhaSR promoter （Psr）,rhaR gene,reporter gene gst was got from the plasmid pALEX-PR by using PCR technology and inserted into pKT-215 （stem from RSF1010）. The chimeric operon was designed to make the upstream of rhaR with an enhanced SD （Shine-Dalgarno）sequence.Then rhaT gene （containing rhaSR promoter）was also constructed and inserted into above plasmid. Firsdy,two expression vectors were transformed into Escherichia coil BL21 （DE3）and expressed in different growth mediums,and the expression of gst was positively regulated by the RahaR coded by the same chimeric operon. Enzyme activity analysis showed that the GST expressed by reporter gene gst of the chimeric operon was 3 fold more in existence of L-rhanmose than in absence of L-rhamnose,and the expression level of gst gene has no improvement in existence of additional rhaT gene. Secondly,two expression vectors were transformed into Anabaena sp. PCC 7120,a kind of cyanobacterium strain by using triparental conjugation. When cells were cultivated without antibiotics formany generations,the transgenic cyanobacteria remained resistant to Chloranaphenicol. Moreover,the transgenic Anabaena cells was tested by PCR. Enzyme activity analysis showed that the GST expressed by reporter gene gst of the chimeric operon was low, and some ways have been presented to optimize the expression conditions.This was the first report of expression induced operon in cyanobacteria cells.