Seperation Of CDNA Fragments Related To Waterlogging Tolerance In Maize Roots Using Suppression Subtractive Hybridization And CDNA Microarray

Two functional technology, SSH (Suppression Subtractive Hybridization) and cDNA microarry, was used to reveal the expression pattern of early response genes when the growth condition changed from normal oxygen supply to waterlogging. Mo17 and Hz32 are two maize inbred lines with differential tolerance to hypoxia. Comparing the two inbred lines, we tried to throw light on the molecular basis of waterlogging tolerance and identify new related genes in maize. Seedlings with two leaves were submerged in hypoxia buffer. SSH libraries were constructed with cDNA samples taken from roots, then were screened with subtracted probes. Mol7 normalized cDNA library of our lab was also screened, using the treated and control mRNA to hybrid with the cDNA microarray previously prepared.105 positive clones from SSH library and 57 differentially expressed clones due to 2h submergence on cDNA microarry were all sequenced. Bioinformatics analysis pointed out that these 162 clones represent 85 TUGs (tentative unique genes), including 41 EST with unknown function as well as many genes involved in several biochemistry pathways, such as glycolysis, protection, signal transduction, cell construction and energy metabolism.Hypoxic metabolism related genes identified by our work are mainly enzymes of glycosis and nitrogen metabolism; and energy metabolism related genes include plasma membrane ATPase vacuole membrane ATPase and so on. These two classes of genes we mentioned above have already been identified by several similar research projects, thus it is sure that they are related to the adaptation to waterlogging in higher plants. We also obtained some proteins, such as heat shock protein ubiquitin-conjugating protein glutathione S-transferase, known to be induced in many stress conditions, which have wide-range physiological effects. Some genes involved in cell cycle were detected by SSH, whereas some genes related to cell structure were detected by cDNA microarray. Furthermore two signal transduction related genes, receptor-like protein kinase and calcium-binding protein, appeared in Mo 17 SSH library. It is very interesting that receptor-like protein kinase gene wasobtained from forward subtracted library but calcium-binding protein gene was obtained from reverse subtracted library. This result indicates that waterlogging may influence signal transduction of Maize roots in different ways.We identified 51 TUGs in Mol7 (waterlogging untolerant) and 54 TUGs in Hz32 (waterlogging tolerant). 20 TUGs were found in both plant materials, including 5 unknown genes. 65 TUGs , 76.5% of the total TUGs, appeared in Hz32 or Mol7. Genes encoding enzymes of glycosis play an important role in plant response to hypoxia. Almost all of these genes we identified appeared in both material. Furthermore, among the 20 TUGs we identified in both Hz32 and Mol7, there are 8 TUGs having different expression pattern. A case in point is that Cholesterol esterase was induced in Mol7 but repressed in Hz32 while aldolase was induced in Hz32 but repressed in Mol7. This result shows that different plant material have different expression pattern under waterlogging.Comparing 105 clones form SSH library and 57 clones on cDNA microarray, we found that several genes with similar function were identified by both technology. This kind of genes mainly included enzymes of sugar metabolism nitrogen metabolism and energy metabolism. Among these genes there were two genes involved in signal transduction. 105 clones form SSH library represented 44 TUGs while 57 clones on cDNA microarray represented 47 TUGs. Thus we can make a conclusion that the efficiency of cDNA microarray is higher than SSH. This result is due to the lack of normalization in SSH method, so a few genes identified by SSH have several copies.Northern and RT-PCR were used to analyze the expression pattern of some positive clones. One clone related to sterol metabolism, which was proved to be induced by hypoxia, have different expression pattern in two plant materials. Bioinformatics analysis shows this clone is homologous to CYP51 cDNA of Sorghum bicolor Oryza sativa Triticum aestivum and Arabidopsis thaliana, the identities are 93% 86%, 86% and 79%. This clone is also highly homologous to Maize EST AY112504 (96%), but no Maize CYP51 gene was found by homologous search using both DNA sequence and protein sequence derived from cDNA sequence. We infer that this gene may be the putative Maize CYP51 gene submergence-induced, and named it SicypSl (Submergence-inducible CYP51) .