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

1. 通过差异筛选变水藻M3.2基因组DNA文库，获得低温诱导基因lti2、lti46、lti39等。测定了lti2和lti46的核苷酸序列，并通过核酸酶S1作图定位了转录区。lti2基因转录为2.0 knt RNA，编码一个由552个氨基酸残基组成的多肽，该多肽与α -淀粉酶同源。lti46基因是一个复杂的操纵子，包含3个开放阅读框，大小分别为119,179和215个氨基酸残基。在0.7 knt的转录本中发现了第一个转录本，而在1.6 knt的转录本中发现了其他转录本。同时还发现了一个包含这三个基因的2.5 knt转录本。lti46的第二种形式与一个表面抗原同源。lti2和lti46基因转录本在低温转换后1小时内迅速升高。该转录本在38和22个温度下的表达是短暂的，稳态水平很低。lti2启动子被融合到细菌荧光素酶编码区，然后转化到水藻细胞中。低温变换后荧光素酶的活性增加了10倍。在适应低温的细胞中，这种活性相当高。内源性1ti2基因与嵌合基因在22℃下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.