CDNA Cloning And Initial Expression Analyzing Of Alcohol Dehydrogenase(ADH1) And Pyruvate Decarboxylase(PDC1) Genes Of Coix Lacryma-jobi L. Under Waterlogging Stress

The cDNA coding sequence of Alcohol dehydrogenase(ADH1)and Pyruvate decarboxylase(PDC1)gene of Coix lacryma-jobi L. were amplified by RT-PCR on the base of two pairs of specificities primers which were designed according to the conservative sequence of ADH1 and PDCl from maize,rice,and wheat We also studied the ADH1 and the PDC1 expression identity with semi-quantitative RT-PCR as well as the time schedule correspondence of the enzyme activity of ADH1 and PDC1 genes under waterlogging stress. The main result are as follows:1. Total RNA of root-tip and leaves were extracted integrally, the RNA band can be observed obviously on the electrophoresis gel; As for the leaves, the brightness of 28sRNA was two times of 18sRNA and the 5sRNA without trail-tag; root RNA was slightly smear , but 28sRNA and 18sRNA still could be separated. The ultraviolet absorption value at the beam of OD260/OD280 was about 2.0, indicated that the RNA with high quality and purity.2. 743bp and 498bp segments of cDNA sequence of ADH1,PDC1 gene was amplified respectively by RT-PCR on the base of the two pairs of specificities primers ,.the two segment both contain partial coding area, which code mature multi-peptide with 247 and 162 amino acids respectively. Bioinformatics analysis found :The homologous of ADH1 gene nucleotide sequence of Coix lacryma-jobi L. comparing with barley, rice, and maize, the identical rate is 87% and 89%, 90% respectively .the identical rate of amino acid were 94% and 94%, 95% to the barley, rice, and maize respectively; PDC1 gene nucleotide sequence of Coix lacryma-jobi L .compared with maize, rice the identical rate is as high as 95%, 91%.the amino acid also have the identical rate of 97%,95% respectively. All the multi-peptides are hydrophilic and have the trans- membrane sequence as well as post-translation modification, the PI and molecular weight of multi-peptide of ADH1gcne are 6.14% 26.1KD respectively. Phylogenetic tree analysis shown that: the origin of the proteins coded by PDC1 and ADH1 gene is closest to gramineous , both contain some conservative active sites, it was ght evolvedpresumed that these mi from a common ancestor. The two sequence of the ADH1 and the PDC1 which we had cloned from Coix had already obtained the landing number are DQ455071 and DQ455583, respectively in the genebank3 ADH1, PDC1 expression pattern of root-tip and leaves were analysised by RT-PCR semi-quantitative methods under waterlogging stress. The results revealed that : ADH1, PDC1 gene in root-tip organizations have high expressed abundance, but both in the leaves are lower, expressed quantity of ADH1 in root-tip will reach peaks after 4h treatment, PDC1 slightly behind, but after 8h treatment, the expression quantity also reached peak. In the leaves, ADH1, PDC1 expression is not obvious, whether in root or in leaves, ADH1 abundance is higher than PDC1 in the schedule time, ADH enzyme in root-tip reached peaks after 6h with 179.21U/mg protein,but PDC enzyme get its highest after 10h with 150.11U/mg protein; The activity of these two enzyme ascended slightly, but not obviously in leaves. The quantity of ADH1,PDC1 mRNA is not parallel with the enzyme activity, it hinted that there were some post-transcriptional processes among the two gene.