集胞藻PCC6803光激活异养生长必需基因s110886的研究

STUDIES ON s110886,A GENE ESSENTIAL TO LIGHT-ACTIVATED HETEROTROPHIC GROWTH IN SYNECHOCYSTIS SP.PCC6803

集胞藻PCC6803能够在微弱、短时光刺激的条件下利用葡萄糖进行异养生长,称为光激活异养生长(LAHG)。从其随机插入诱变文库中,筛选到3个不能进行光激活异养生长的突变株,通过反向PCR和测序确定突变基因全部为s110886。将s110886克隆到表达载体pET21-b,在大肠杆菌BL21(DE3)诱导表达,并对产物进行了纯化。用Western印迹法研究s110886在集胞藻PCC6803的表达情况,发现在完全黑暗和光照情况下,其表达水平几乎没有差异,并证明其编码产物分布于膜上。因此,s110886是一个编码膜蛋白的不受光调控的LAHG基因。

Synechocystis sp. PCC6803 is able to grow on glucose in complete darkness with a daily stimulation of short and weak light, which is called light activated heterotrophic growth （LAHG）. By random insertion of a kanamycin-resistance cassette into Hae m or Alu I sites of the genome, a mutant library of Synechocystis sp. PCC6803 was constructed. From this library, 3 mutants incapable of LAHG but similar to wild type in autotrophic, mixotrophic and phototrophic growth were selected. Inverse PCR and sequence determination showed that all the insertion were at a Hae Ⅲ （GGCC ,666-669Nt.） site of s110886, s110886 without its N-terminal trans-membrane encoding sequence was generated by PCR, cloned into pET21b and expressed in E. coli BL21 （DE3）. The expressed product, found in the soluble fraction, was purified with the assistance of its C-terminal His. Tag and used to prepare the rabbit antiserum against S110886. Western Blot was employed to investigate the presence of S110886 in membrane or soluble protein fraction of the cyanobacterium, and it was found to be a membrane protein. Using total membrane from cells with different periods of light exposure, the expression of s110886 was examined and found to be non-responsive to illumination. We conclude that s110886 is essential to LAHG and not induced by light.