Cloning And Analysis Of Differentially Expression Genes Of Prorocentrum Donghaiense In Response To Inorganic Phosphate–stress

Prorocentrum donghaiense is one of the most important species that causes red tides in the East China Sea. Red tide caused by Prorocentrum donghaiense has raised extensive concern of the researchers because of its high frequency, large scale and long duration, but most of the research mainly concentrated on its physiological ecology, life history and morphological classification, and relevant formation/dissipation mechanism of its molecular biology research is inadequate. Dissolved inorganic phosphorus is known as the phosphorus source of the Prorocentrum donghaiense, however, the data of the red tide outbreak reveals that the total amount of dissolved inorganic phosphorus in the sea is not that large when a red tide took place. Therefore, this research is aimed to study the molecular mechanism in Prorocentrum donghaiense of its response to inorganic phosphorus suppression, by simulating the environment of inorganic phosphorus suppression.This suppression-subtractive-hybridization based research is aiming at construction of the c DNA library of differential expression gene in Prorocentrum donghaiense under phosphorus starvation. We managed to find out how phosphorus starvation influence Prorocentrum donghaiense on the molecular level. We cultured Prorocentrum donghaiense in two seperated mediums, F/2-Si and the phosphorus-freed F/2-Si, then extracted total RNA after 8h, 12 h, 24 h, 48 h and constructed SSH-c DNA library. We completed the total m RNA reverse transcription into c DNA and then jointed two rounds of subtractive and two rounds of hybridization and inhibition of nested PCR after added a cap on it. After transforming the library-combined p MD® 18-T Vector into escherichia coli(DH5α), we received the gene sequences of the library in a total amount of 1865 t.Those homologous sequences can be divided into 11 classes after processing and analyzing with multiple tools according to their biological process and molecular function, namely:(1) gene and cell defense in steady state(3.73%);(2) protein metabolism related genes(20.15%);(3) stress response genes(5.22%);(4) cytoskeletal and structural proteins(6.72%);(5) DNA metabolism and repair related genes(0.75%);(6) unclassified or location function for(26.87%);(7) in signal pathway genes(6.72%);(8) cell metabolism related genes(20.90%);(9) the transcription and translation ofgenes(2.24%);(10) phosphorus metabolism related genes(3%);(11) cell cycle related genes(3.73%).In order to verify, we screened 5 typical genes based on their discrepancy in function, namely NAD-dependent deacetylase, Extracellular sulfatase Sulf-1, Heat shock protein Heat shock 90, protein 70 and Heat shock protein 40 as the object of study, using fluorescence quantitative PCR study on control group and the experimental group and analyzed their differential gene expression, the results showed that: 5 genes are differentially expressed in different degrees, which in addition to the Heat shock protein 70, the other 4 genes by inorganic phosphorus stress expression was up-regulated, before and after the detection of NAD-dependent deacetylase Extracellular, paired T- sulfatase and Heat Sulf-1 shock protein 40 expression the number was statistically significant(P < 0.05). Thus, it is a valid prove that the SSH-c DNA library constructed in this experiment basically represents the differentially expressed genes.