Cloning Of Genes Involved In Agar Synthesis And The Analysis Of Its Expressions And Regulations In Gracilaria Lemaneiformis (Rhodophyta)

Gracilaria lemaneiformis is a red macroalga which belongs to Rhodophyta, Rhodophyceae, Gigartinales, Gracilariaceae. The most economical significance of G. lemaneiformis is the agar. The metabolism of agar is a major feature of red-algal physiology. As a main agarophyte, the research on the agar correlation genes of G. lemaneiformis is very important to the theoretic study and economic values.UDP-glucose pyrophosphorylase (UGPase) is a key enzyme in-agar metobiolism in red algae.Reverse transcription and PCR amplification were employed to obtain the part sequence of UGPase gene(glugp) of G.leimaneiformis. PCR amplification of the 3'and 5'end were performed using RACE. Putative glugp gene was characterized at the genomic DNA and cDNA levels. The gene has 2200 bp of cDNA, coding for 494 amino acids of ORF, involving 1485 bp. The deduced amino acid sequence showed significant similarity to the UGPase sequences of other species. RpsBLAST analysis indicated that UGPase has the conserved active sites between 105 and 400 amino acid, and it has five identified conserved lysine residues, that are at, or close to, the subtrate binding site.The gene have a 5'flanking region of 330 bp and a 3'flanking region of 385 bp. The 5'flanking sequence (GCTATG) conforms to the canonical sequence, RCYATG, at translation initiation sites in red algal genes so far characterized. The gene has its own special polyA signal, ATTATT, found 261 bp downstream of the stop codon site.The glugp devoid of introns and Southern blotting indicated that it has only one copy in G. lemaneiformis genome.The relationship between gene expression level of glugp and the agar content in G.leimaneiformis were investigated. The same algae strains which divided into two parts from the holdfast were cultured in normal and low salinity conditions respectively. RNA of each sample was extracted and revere-transcribed into cDNA. Real time quantitative PCR technique was used to detect the gene expression of each sample.2'ΔΔCT data denoted for the comparative expression. The results showed that there were no statistically significant differences in the agar content of the two parts of algal samples, and glugp gene expressions of the two parts have no significant differences two. There are distinctnesses in agar content of wild type algal strains in G. lemaneiformis at the same sea area. The different algal strains with the biggest discrepancy on agar content were divided into two groups. RNA of each sample was extracted and revere-transcribed into cDNA. Real time quantitative PCR technique was used to detect the glugp expression of algal strains with different agar contents. 2-ΔΔCT data denoted for the comparative expression. The result revealed a highly correlation between glugp expression and agar contents. The expression level of glugp in high agar content strains is significantly higher than those in low, which may be applicable in indication of agar content and further algal cultivation.The effect of some environmental factors and culture conditions on agar content and biochemical properties of G. leimaneiformis were studied in the laboratory. The selection of the high agar yielding culture condition is the foundation to screen differentially expressed genes involved in agar synthesis. The agar content of female gametophyte and tetrasporophyte of G. lemaneiformis were measured. The result shows that the agar content of algae is higer in tetrasporophyte than in female gametophyte. Agar content in Gracilaria and Gelidium has been shown to increase in nitrogen-limited cultures, phosphate-limited cultures and low salinity cultures. To study the differences in agar content, the same strain of G. leimaneiformis was treated in four conditions, including nitrogen-limited, phosphate-limited, low salinity and normal cultures. The normal culture condition is rich in nutrients. The contents of chlorophyll a and phycoerythrin in algae with different culture conditions were also detected. There were no dissimilarity in agar content after one week of experiments from nitrogen-limited, low salinity and normal culture conditions, but lower in phosphate-limited culture conditions. Increased agar productivity was observed in low salinity treatment after three weeks experiments. It was found that the most algae from the nitrogen limited condition contained higher levels of agar, but it not significantly different from the normal culture conditions. The contents of chlorophyll a and phycoerythrin were low nitrogen limited condition. Agar yielded was less dependent on the phosphate concentration after three weeks, no statistical differences in the content of chlorophyll a and phycoerythrin were observed between phosphate-limited condition and the normal.The result indicated that low salinity lead to an increase in agar content. The differentially expressed genes which induced by low salinity may be the key genes involved in the agar synthesis. The low salinity condition should be investigated as a favorable condition to construct suppressive subtractive hybridization library and obtain the related genes in agar synthesis.