Isolation and analysis of structure and expression of low-temperature-induced genes in the cyanobacterium

1.通过对多变鱼腥藻M3.2基因组DNA文库的差异筛选，获得了低温诱导基因lti2、lti46、lti39等。测定了Lti2和Lti46的核苷酸序列，并通过核酸酶S1定位了转录区。Lti2基因转录为2.0kNT的RNA，编码552个氨基酸残基的多肽，与α-淀粉酶同源。Lti46基因是一个复杂的操纵子，由3个开放阅读框组成，长度分别为119、179和215个氨基酸残基。第一个ORF在0.7kNT的转录本中被发现，而其余的ORF在1.6kNT的转录本中被发现。还发现了一个2.5knt的转录本，其中包含所有三个ORF。Lti46的第二个ORF与一个表面抗原同源。Lti2和Lti46基因的转录水平在低温转换后1小时内迅速升高。在38和22ﾟC.4中，表达是短暂的，并且转录本的稳态水平很低。将lti2启动子与细菌荧光素酶的编码区融合，转化鱼腥藻细胞。低温漂移后，荧光素酶活性提高了10倍。在适应低温的细胞中，该酶活性较高。内源性1ti2基因和嵌合基因在22ﾟC的表达差异有待解决。

1. Low-temperature-induced genes, lti2, lti46, lti39 etc, were obtained by differential screening of the genomic DNA library of Anabaena variabilis M3.2. The nucleotide sequences of lti2 and lti46 were determined and the transcribed regions were located by nuclease S1 mapping. The gene lti2 was transcribed as a 2.0 knt RNA that encoded a polypeptide consisting of 552 amino acid residues which was homologous to alpha-amylases. The gene lti46 was a complex operon containing 3 open reading frames having sizes of 119,179 and 215 amino acid residues, respectively. The first orf was found in a 0.7 knt transcript, while the remaining orfs were found in a 1.6 knt transcript. A 2.5 knt transcript which contained all three orfs were also found. The second orf of lti46 was homologous to a surface antigen.3. The level of the transcripts of lti2 and lti46 genes increased rapidly within 1 hr after a low-temperature shift. The expression was transient and the steady-state level of the transcripts were quite low at both 38 and 22ﾟC.4. The promoter of lti2 was fused to the coding region of bacterial luciferase, and then transformed into Anabaena cells. The activity of luciferase increased 10-fold after a low-temperature shift. The activity was quite high in the cells adapted to a low temperature. The discrepancy between the expression of endogenous 1ti2 gene and that of the chimeric gene at 22ﾟC remains to be solved.