| Taxus, also known as yew, is a genus of gymnosperm and contains at least 14 species. Until now, the nature product- Taxol, extracted from the bark of yew, is a highly effective anticancer drug,but yields are low; approximately 0.01-0.02 % of the dry bark weight. Although the great advance have been acquired to increase the production of taxol by finding alternative sources and methods of synthesis, due to the growing demand for the taxol for medical using, the contradiction of supply and demand in taxol have not been fundamentally solved. According to the sequences of unigenes obtained from the transcriptome database of the regeneration tissues after bark girdling in Taxus chinensis, differentially expressed genes involved in vascular tissue development were found out. The cDNA sequences of 4 LBD(LATERAL ORGAN BOUNDARIES DOMAIN) and 3 APL(ALTERED PHLOEM DEVELOPMENT) genes were isolated using RT-PCR(reverse transcription-polymerase chain reaction) from barks in T. chinensis, these key genes were selected to be useful for the research of gene engineering. Our results demonstrated that important candidate genes had effected on vascular tissue and especially phloem development, studies of the molecular regulation of the tissue regeneration after girdling would provide novel insights into transgenic T. chinensis cultivated with increased secondary phloem production( thicker barks).The main results were as followings:1. The regeneration system of secondary vascular tissue in T. chinensis was builded up. During secondary vascular tissue regeneration after bark girdling in T. chinensis, our experiments showed that phloem or cambium regeneration does not need cell dedifferentiation into a callus state, but from differentiating xylem cells and ray cells. Anatomical studies showed that discontinuous meristematic cell appeared at 24 D after being debarked, and then new cambium was formed,and began to differentiate into phloem and xylem tissue, untile 60 D, the whole vascular system in the structure was formed.2. The Illumina Genome Analyzer technology(Illumina HiSeq?2000)was carried out to obtain massively parallel sequencing. RNA of different types of six samples from bark regeneration was pooled to provide a broad gene library. A total of 165 557 061 clean reads with the total nucleotides(nt) of 16.55 G were obtained in the transcriptome sequencing and 224 010 contigs with length not less than 200 nt were obtained. Through trinity assembly, we acquired 156,713 unigenes. These unigenes provided important genetic resources to the regulation of vascular tissue development in Taxus chinensis.3. According to the sequences of TcLBDs genes obtained from the transcriptome database of the regeneration tissues after bark girdling in T. chinensis, specific primers were designed. The 4 TcLBDs genes were isolated using RT-PCR. The genes showed highest homology of LBD from Arabidopsis and were therefore named TcLBsD1, TcLBD6, TcLBD11 and TcLBD15, respectively.The results showed that the open reading frame(ORF) cDNA of TcLBD1 contained 549 bp in length which encoded polypeptide of 182 AA(amino acids residues), and TcLBD6 contained a 687 bp ORF encoding 228 AA, TcLBD11 included 462 bp encoding 153 AA; TcLBD15 with 540 bp encoding 179 AA. The sequences analysis showed that the amino acid sequences of TcLBDs contained one specific conserved LOB motifs in N-terminal.4. The analysis of tissues expression patterns showed that the transcript abundance of TcLBD1 was higher in stems and xylem with cambium than that in roots, leaves, phloem with cambium; while TcLBD6 was mainly expressed in roots and stems. TcLBD11 was higher in the other tissues than that of roots; the transcript abundances of TcLBD15 was mainly expressed in roots and phloem with cambium Through analysis of the expression patterns in regeneration tissues after bark girdling, the mRNA expressions of TcLBD1 and TcLBD11 appeared sharply expression after 6 days bark girdling and showed a continuously upregulated pattern, that of TcLBD6 and TcLBD15 were found to increase notably after 18 days and show a rising trend in the following periods.5. The overexpression vectors of pBI121-TcLBD11 and pBI121-TcLBD11 were constructed based on the character of TcLBD11 and TcLBD15 sequences which were selected according to the expression pattern of TcLBDs. The TcLBD11 and TcLBD15 were transformed into P.alba×P.glandulossa and Arabidopsis thaliana ecotype Columbia by Agrobacterium-mediated method. The phenotype in TcLBD11-oe transgenic poplar showed suppressed phloem development and especially rudimentary phloem ?ber formation. TcLBD11-oe transgenic Arabidopsis exhibited abnormalities both in vegetative and reproductive,including small upward curled leaves and long juvenility.4-week-old transformants displayed clustered cauline leaves suggesting altered SAM development. qRT-PCR analysis revealed that the AtAPL and AtNAC45/86 transcript level was strongly down-regulated in TcLBD11-oe transgenic transgenic Arabidopsis compared with the wild-type plants.The expression of TcLBD15-oe transgenic poplar resulted in opposite phenotypes to those observed for TcLBD11-oe transgenic poplar. The height,stem diameter and secondary phloem production of TcLBD15-oe transgenic poplar was signi?cantly increased. TcLBD15-oe transgenic transgenic Arabidopsis experienced increased main stem diameter.Meanwhile, qRT-PCR analysis revealed that the AtAPL and AtNAC 86 mRNA expression was strongly down-regulated compared with the wild-type plants expect AtNAC45.6. The length three APL genes were isolated using RT-PCR and were named as TcAPL1, TcAPL2 and TcAPL3, respectively. Our results showed that the ORF length cDNA of TcAPL1 was 1650 bp which encoded 549 AA; TcAPL2 included 1341 bp ORF encoding 466 AA; TcAPL3 with 1167 bp ORF encoding 388 amino acid residues, they all contain conserved MYB DNA-binding domains. The analysis of gene expression patterns in different tissues showed that the transcript abundances of TcAPL1 and TcAPL2 were mainly expressed in root, stems, leaf and phloem with cambium; while TcAPL3 was higher in leaf than that in roots and xylem with cambium. Through analysis of the expression patterns in regeneration tissues after bark girdling, the mRNA expressions of TcAPL1 and TcAPL2 showed a up-down trend in the following periods and were found to decrease notably at 36 days after bark girdling, while the expression of TcAPL3 was repressed at all stages after bark girdling.Their expressions were regulated in regeneration processes after bark girdling. |
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