λN基因前导序列对下游基因表达的控制作用

λN Gene Expression Regulated by the Leader Sequence

已有研究表明，几Ｎ基因上游的ＴＩＲ（Ｔｒａｎｓｌａｔｉｏｎａｌ ｉｎｉｔｉａｔｉｏｎ ｒｅｇｉｏｎ）的改变可提高ＡＮ基因的表达，而且表达量的差异是在翻译水平上形成的．构建了 ３类λＮ基因上游 ＴＩＲ的突变体质粒，并通过测定它们在大肠杆菌转化体中表达的β－半乳糖苷酶的活性和进行ＲＮＡ－ＤＮＡ圆点印迹杂交实验证明：由于λＮ因上游有一个翻译效率很低的编码１９个氨基酸的短肽的可读框（ＯＲＦλＮ），因而使 因的表达也较低。如果使ＯＲＦλＮ前终止或改变ＯＲＦλＮ的ＴＩＲ序列可提高翻译效率，能在翻译水平上有效地提高下游λＮ基因的表达。

It was reported recently that changing the TIR (translational initiation region) of λN gene resulted in the increasing expression of λN gene and it was regulated at translational level. According to the alignment the leader sequence of λN gene had fore parts: a code region for ORFλN,the upstream sequence of ORFλm and 17 bp of spacer between ORFλN and downstream of λN gene. ORFλN is an open reading flame located at upstream of λN gene coding a peptide of 19 amino acids. To study the mechanism of regulation of λN gene expression, three serials of plasmids with mutant leader region of λN gene were constructed. (1) pMC1403-XT, in which the start codon or the partial code of ORFλN was connected with lacZ to obtain the ORFλN-lacZ fusion gene and in which the ORFλn-lacZ expression was under the control of a strong trp/alc promoter; (2) The ORFλN mutants in which the termination codon TAA was introduced into ORFλN at different positions by site-directed mutagenesis to preterminate the ORFλN on plasmid pMC1403N; (3) Mutants in which a deletion was located at upstream ORFλN in the ORFλN mutants above. The results obtained from. determination of the β-galactosidase activity in the transformants harboring the different plasmids showed that the ORFλn-lacZ expression was suppressed by the ORFλN code region and the AN expression was increased in both the second and third serials of mutants. At the same time the results from RNA-DNA dot hybridization showed that the λN gene expression was regulated at translational level. Therefore it was predicted that the reason of relatively low expression for λN gene in pMC1403N was due to the low efficiency of ORFλN translation There are two ways to increase the expression of λN gene. One is to preterminate the tanslation of ORFλN at a suitable position to decrease the ORFλN effects on downstream λN gene translation; the other is to change the upstream sequence of ORFλN to improve its translation efficiency,