Analysis Of Different Aluminum-Induced MRNS In Clones Of Eucalyptus Using DDRT-PCR Method

Eucalyptus is an important fast-growing tree, which was usually used to cultivated pure forest in southern China. Aluminum-rich soil, which was induced by acidic soil, acid rain, continuous cropping, fertilizer and other reasons, is one of the most important factors for forest recession. Analysis the molecular mechanism in response to aluminum toxicity would be useful for selective breeding resistance species of Eucalyptus.In this study, we optimized the mRNA difference display technology and screened the gene fragment with different transcript level in Eucalyptus clones G9（insensitive to aluminium toxity） and W4（sensitive to aluminium toxicity） under aluminum treatment. A series of differential gene fragments were sequenced analyzed. The main results were shown as follows:1. The RNA extraction kit was a reliable method for Total RNA extraction from Eucalyptus leaves. The purified RNA has a highly quality and quantity, which was suitable for further analysis using DDRT-PCR method.2. Using orthogonal experiment method, the main factors in PCR system mainly including TaqDNA polymerase, dNTP and annealing temperature, were optimized. The optimal reaction system was consisted of0.2μl TaqDNA polymerase,1μl dNTP concentration and28℃annealing temperature.3. Combination with the three anchor primers （SP1, SP2and SP3） and7random primers （RP1, RP2, RP3, RP4, RP5RP6and S16）, we found that the combination of SP2anchor primers and7random primer were optional for DDRT-PCR analysis, the ampilified bands exhibited obvious polymorphism and the clarity of bands was more clear. The results indicated that the gene expression files were differently in the two clones under the stress of aluminium.4. From the results of DDRT-PCR for the two Eucalyptus clones,35differential bands were obtained in G9and22bands were obtained. The differential bands in G9was most obtained from samples treated with aluminum, however, bands in W4were mostly obtained from control seedlings.5. By blast against the Nr database, five bands from G9showed highly homology with known functional genes. Among them, the G9₂1₁gene fragment was100%homology to chloroplast gene of Eucalyptus. G9₂4₁sequence was77%homology to Poplar drought-tolerant gene. G9₉₄was85%homology to peroxidase in Cocoa. G9₃5₁was90%homology to heat shock protein in pennisetum and91%homology to heat shock protein in Salicornia. G9₁5₂sequence was82%homology to Cocoa glyoxalase gene. All the five genes were obtained from seedlings treated with aluminum, and the expression level was upregulated associated with the increasing of treating time. In addition, G9₁6₁sequence, which was inhibited in seedlings undergone aluminum stress, was85%homology to Periwinkle iridoid glycosides glucose transferase gene.All varied sequences identified from W4were used to blast against NCBI database, results showed that W4₁0₂was100%homology to dynein gene in Cacao,W4₁6₃was88%homology to GDSL lipase gene in Alfalfa, W4₁9₂was88%homology to U-box gene in Arabidopsis. Transcriptions of the above three genes were inhibited after aluminum treatment. In addition, W4₂2₁was100%homology to the chloroplast gene in Eucalyptus deglupta, which was induced by aluminum treatment.