| Natural rubber (cis 1,4-polyisoprene), one of secondary metabolites of Hevea brasiliensis, is a very important industrial raw material among with coal, oil and iron ore, and also one of the most fundamental materials for national economy development. Hevea brasiliensis as a main source of production of natural rubber is a very important tropical economic and energy crops.Hevea brasiliensis self-rooting juvenile-type clone is defined as an anther somatic cell plant via inducing from Hevea brasiliensis anther with the process of dedifferentitation and redifferentitation. The facts that the current research on high-yield of Hevea brasiliensis self-rooting juvenile-type clone only focus on morphologic and physiologic parameters without direct molecular biology evidences effect their production applications to a certain extent, and are also the main reasons that some researcher doubt their potential and actual use value. Therefore, it is important as well as necessary to discover the molecular mechanism of their high yield.We employed the approach of methylation-sensitive amplification polymorphism (MSAP) to study the development of somatic embryo in Hevea brasiliensis self-rooting juvenile-type clone and the pattern of cytosine methylation in genomic DNA from senior-type clone apical meristem. The results are as following:1. Various tissues from each stage of somatic embryogenesis including anther, callus, somatic embryo, regenerated plants bud, regenerated leaf and shoots from regenerated plants were used to further analysis. Using 35 pairs of primers,1549 of clear bands, which was the highest one, was amplified in genomic DNA from anther explants, the lowest one with 1522 bands was obtained from somatic embryo. The level of methylation in genomic DNA from anther is the highest one (methylated sites were 352), while The level of methylation in genomic DNA from somatic embryo is the lowest one (Methylated sites were 330). Methylated sites of two types were 330 in somatic embryo, while methylated sites were 352 in anther.2. Three different methylation-specific bands were observed in all tissues, however, they displayed 17 differential band patterns, which were likely to associate with tissue-specific marks.3. The changes of different type of bands were quiet often observed in the different tissues, such as the presence or absence of three types of bands together, and the interchanges between these types of bands. During the somatic embryogenesis, every phase changed coupling with the changes of band type.4. Fourteen tissue-specific bands have been selected for sequencing. Blast results showed that just 6 sequences had homologous sequences in NCBI. The suppression subtractive hybridization (SSH) cDNA library has been constructed from Hevea brasiliensis self-rooting juvenile-type clone and senior-type clone. After eliminating false positive cDNA clones using reverse northern blot, some positive clones have been selected randomly for sequencing. Blast results indicated that total 33 clones in cDNA library of senior-type clone, including 8 clones related to metabolism,8 clones related to Transcription,8 clones to signal transduction and 2 clones to protein kinase while total 30 clones in cDNA library of self-rooting juvenile-type clone, including 5 clones related to metabolism,5 clones related to Transcription,11 clones to signal transduction and so on.Five positive clones (D5、T744、T227、D24、D267) were selected randomly for further RT-PCR analysis.The results is that(D5、D24、D267) were expressed highly in senior-type clone and (T744、T227) were expressed highly in self-rooting juvenile-type. These results were consistent with reverse northern blot results. From four clones labeled as D267, T744, D24 and D5 were named as HbHDTl, HbSKP1, HbCC and Hb14-3-3a, respectively. Four full-length cDNA were obtained using RACE method. Sequence analyses indicated that HbHDTl, HbSKP1, HbCC and Hbl4-3-3a encoded 304,179,84, and 263 amino acid residues, respectively. Using different tissues including flower, callus, somatic embryo culturing for 12 days,21 days,40 days, and 53 days respectively, leave, bud, and latex, expression pattern of these genes were characterized. Jasmonic acid and ethylene are involved in regulation of nature rubber biosynthesis. RT-PCR results revealed that neither jasmonic acid nor ethylene has effect on the expression of HbHDT1 and Hb14-3-3. The expression level of HbSKPl was increased significantly by jasmonic acid, and inhibited by ethylene. HbCC expression level was also increased significantly by jasmonic acid, and did not change by ethylene. Taking together, these data suggested that these genes were likely to be involved in different pathway of nature rubber biosynthesis. |
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