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Three thousand and ninety Unigenes were obtained from 10 778 Hevea brasiliensis ESTs. Four hundred and thirty SSRs were distributed in 353 Unigenes, which accounts for 11.42% of the total number of Unigenes. The frequency of SSRs was 1/3.93 kb. Dinucleotide and trinucleotide repeats were the dominant types among the obtained unigenes, accounting for 63.49% and 32.09%, respectively. TC/AG, CT/GA and CTT/GAA, AAG/TTC, and AGA/TCT were the most abundant motifs for dinucleotide and trinucleotide motifs. One hundred and forty-eight primer pairs were designed by PRIMER5.0 and 21 primer pairs were synthesized. Among them, 15 primer pairs can produce clear and stable bands, and the PCR products were screened in denaturing polyacrylamide gel following silver staining. Genetic diversity of 44 rubber clones were investigated with these primer pairs, and a dendrogram of 44 rubber clones was built. The results indicated that it is an effective and feasible way to develop EST-SSR markers from H. brasiliensis EST sequences, and the primers designed in this study can be used in genetic study of rubber tree.

In order to enrich gene encoding region of Hevea brasiliensis, a methylation filtration library was constructed using Escherichia coli McrBC restriction-modification system. The titers of the non-amplified library and the amplified library were 2.6×106 pfu/ml and 9.0×109, respectively. The rate of positive clones was 86.4%. The lengths of inserted DNA sequence ranged from 1 kb to 2.5 kb and the average size of inserts was 1.2 kb. One hundred clones were selected randomly for sequencing, resulting in splicing out of 81 non-redundant sequences, including 6 contigs and 75 singlets. The redundancy was 17.35%. Blast analysis showed that 39.5% of non-redundant sequences were homologous with the Nr database, 14.81% with the EST database, and 32.1% were unknown sequences. Some sequences were related genes for flowering, insect and disease resistance. Therefore, the rubber tree methylation library is helpful for discovery and cloning of functional genes.

CBF pathway is the most important pathway during the process of cold acclimation in plants. In previous study, we cloned a CBF like gene HbCBF1 from Hevea brasiliensis, which implied that CBF pathway might exist in this kind of tropical oriented tree. However, it was still unknown if this gene functioned as C-repeat binding factor. Therefore it was very important to analyze the C-repeat binding activity of HbCBF1. Using a prokaryotic expression system, the HbCBF1 was expressed and the soluble fusion protein was extracted under the optimized conditions. The C-repeat binding activity of HbCBF1 protein was analyzed by gel shift assay, which indicated that the fusion protein could bind to the COR15a probe efficiently, and this binding activity could be com-peted with by overloading non-labeled COR15a probe. An unlabeled mutated probe M1, which did not include the C-repeat sequence, could not compete with the binding activity of HbCBF1 to COR15a probe. These results indicated HbCBF1 fusion protein could bind to the C-repeat containing DNA fragment specifically in vitro.