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

1.通过对变异鱼腥藻M3.2基因组DNA文库的差异筛选，获得了低温诱导基因lti 2、lti 46、lti 39等。测定lti 2和lti 46的核苷酸序列，并通过核酸酶S1作图定位转录区。基因lti 2转录为2.0 knt的RNA，其编码由552个氨基酸残基组成的多肽，该多肽与α-淀粉酶同源。lti 46基因是一个复杂的操纵子，含有3个开放阅读框，大小分别为119、179和215个氨基酸残基。第一个ORF在0.7 knt的转录本中被发现，而其余的ORF在1.6 knt的转录本中被发现。另外还发现了一个2.5knt的转录本，它包含所有三个开放阅读框，其中lti 46的第二个开放阅读框与一个表面抗原同源. lti 2和lti 46基因的转录水平在低温转换后1小时内迅速增加。表达是短暂的，在38和22 ℃时转录本的稳态水平都很低。将lti 2的启动子与细菌荧光素酶的编码区融合，然后转化鱼腥藻细胞。荧光素酶的活性增加10倍后，低温转换。在适应低温的细胞中，活性相当高。22 ℃时内源1 ti 2基因表达与嵌合基因表达的差异有待解决。

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.