Somatic Hybridization Of Taxus And Functional Analysis Of The Promoter Of DBTNBT-the Gene Encoding A Key Enzyme Of Taxol Biosynthesis Pathway

The vast majority of efficacious ingredients of Chinese medicinal herbs derived from secondary metabolites, Paclitaxel (taxol), a secondary metabolite of the Taxus species, has been recognized as the highly effective anticancer drug. However, due to the scarce resources of wild yew and the low content of taxol in yew trees, the supply of taxol is seriously limited. Using somatic cell hybridization technology could transfer and increase the effective ingredients of Chinese medicinal materials. In the first part of work, asymmetric protoplast fusion between Bupleurum scoronerifolium and Taxus. chinensis var.mairei was studied.The main results were follows:1. Asymmetric somatic hybridization between B.scoronerifolium and T.chinensis var.mairei1.1 Induction of embryogenic callus of T.chinensis var.mairei and optimization of subculture conditionsPink dense and white loose calli were induced from Taxus embryos, then the calli were transferred to MB2+B medium, yellow granular callus was obtained after subculture for three or four generations. The calli were transferred to MB2+B liquid medium to obtain yellow suspension culture with good dispersion and high growth rate, and used to be the cell fusion material.Growth state and taxol content of taxus calli were compared, which cultured in subclture medium with different hormone combinations and concentrations. Results showed that subculture medium supplemented with 2.0 mg/L 2,4-D and 1.0 mg/L 6-BA was suitable for long-term subculture of callus, and for obtaining higher taxol yields. 1.2 Cell fusion between B.scoronerifolium and T.chinensis var.mairei and the culture of fusion productsSomatic hybrids were obtained by asymmetric protoplast fusion between B.scoronerifolium and T.chinensis var.mairei. T.chinensis var.mairei was the donor and B.scoronerifolium was the receptor. Protoplasts of T.chinensis var.mairei irradiated with ultraviolet light(UV)at an intensity of 380μW/cm2 for 1,2 and 3 min respectively. Sixty regenerated clones were obtained and it was divided into three fusion combinations. Fusion combinationⅠ(Donor irradiated with UV for 1 min)has emerged thirty-two regenerated clones, Fusion combinationⅡ(Donor irradiated with UV for 2 min)has emerged sixteen regenerated clones, and Fusion combinationⅢ(Donor irradiated with UV for 3 min)has emerged twelve regenerated clones. The growth rate and phenotype of regenerated clones were between B.scoronerifolium and T.chinensis var.mairei.1.3 Identification of hybridity of regenerated calliAll the regenerated clones were analyzed for their hybrid nature by Esterase isozyme analysis, Chromosome counting, RAPD analysis and SSR analysis. Nine regenerated clones (Ⅰ-18,Ⅰ-19,Ⅰ-27,Ⅱ-1,Ⅱ-2,Ⅱ-6,Ⅱ-9,Ⅲ-2, andⅢ-3) were recognized as hybrid clones.1.3.1 Esterase isozyme analysisEsterase isozyme analysis showed that regenerated clones had similar bands as B. scoronerifolium, whileⅠ-19,Ⅰ-27 andⅡ-3 had bands characteristic of T.chinensis var.mairei and novel bands, and clonesⅠ-18,Ⅱ-2,Ⅱ-6 contained bands characteristic of T.chinensis var.mairei. This evidence supports the hybridity status of these regenerated clones.1.3.2 Chromosome countingChromosome counting analysis showed that chromosome numbers of regenerated clones were distributing from ten to seventeen, and the chromosome numbers of clonesⅠ-15,Ⅰ-18,Ⅰ-27,Ⅱ-2 andⅡ-3 were all above 12. This evidence supports the hybridity status of these regenerated clones.1.3.3 RAPD analysis RAPD analysis showed that:1) Regenerated clonesⅠ-18/19/27,Ⅱ-1/2/6/9 andⅢ-2/3 had nuclear DNA of both parents and recombined DNA, these clones are hybrid clones.2) Statistics of RAPD pattern showed the hybrids reserved about 82.4%-96.8% bands of B.scorzonerifolium, and 4.62%-13.85% bands of T.chinensis var.mairei, which were partial to recipient B. scorzonerifolium nuclear DNA.3) The UV dosage had effects on the nuclear genome of hybrids, the combinationⅡandⅢirradiated with UV for longer time than combinationⅠ, which made the number of characteristic bands of T.chinensis var.mairei and novel bands accounted for larger proportion. The cloneⅡ-6 had more donor's bands than other clones, the number of characteristic bands of T.chinensis var.mairei and novel bands accounted for 20.77% of its total bands'number.1.3.4 SSR analysisSSR analysis revealed that the most hybrids had whole B. scoronerifolium bands, but no T.chinensis var.mairei band, except clonesⅠ-18 andⅢ-2 carried novel bands.1.4 HPLC of oleanolic acid in B. scoronerifolium and hybrid calliThe efficacious ingredients of hybrid callus lines were analyzed by HPLC. Results showed that hybrid clonesⅡ-6,Ⅱ-9,Ⅲ-2 andⅢ-3 had higher content of Oleanolic acid (OA) than B. scoronerifolium, and III-3 had the highest content of OA, which is 4.6 fold than B. scoronerifolium. Hybrid clonesⅠ-18,Ⅰ-19,Ⅰ-27,Ⅱ-1 andⅡ-2 had lower content of OA than B. scoronerifolium.1.5 Gene expression patterns analysis of Hybrid clonesExpression levels of five upstream genes (HMG-CoA synthase, IPP isomerase, FPP synthase, squalene synthase, squalene epoxidase) and three downstream genes (lupeol synthase, cycloartenol synthase andβ-amyrin synthase) related with the oleanlic acid biosynthesis were analyzed by RT-PCR. Results showed that several related genes were expressed significantly different in B. scoronerifolium and hybrid callus lines. The expression levels of the above eight genes in Hybrid clonesⅡ-1,Ⅱ-2,Ⅱ-6 were higher than B.scoronerifolium, while only the cloneⅡ-6 had the higher content of OA than B.scoronerifolium. The result showed that OA contents weren't had a highly positive correlation with the expression levels of genes related with OA biosynthesis. The expression level of IPP isomerase, squalene synthase, squalene epoxidase andβ-amyrin synthase in hybrid clone-3 was higher than in B. scoronerifolium, while the expression level of cycloartenol synthase (branching enzyme) was lower than in B.scoronerifolium. Therefore we concluded that low expression level of branching gene made the metabolic flow towards the direction ofβ-amyrin biosynthesis, which made higher content of OA in cloneⅢ-3.Most of taxol biosynthesis related genes has been cloned, and their function were confirmed in E. coli and yeast. The research about transcriptional regulation of the taxol biosynthesis pathway has just been started. Taxane C13-side chain-N-benzoyl transferase (DBTNBT), is the enzyme that catalyzed the last step enzyme reaction of taxol biosynthesis pathway, it transform taxol precursor 3'-N-debenzoyltaxol to taxol. The transcription level of DBTNBT was significantly lower than the taxol biosynthesis related genes that in the early pathway. DBTNBT was considered as the rate-limiting enzyme in taxol biosynthesis pathway. DBTNBT gene had been cloned, but its promoter function and its trans-acting elements was not yet been studied. In our laboratory DBTNBT promoter had cloned, and the promer was 1488 bp in length from the translation initiation site. Functional analysis of DBTNBT promoter and isolation of its binding factors were studied in this paper.The main results are as follows:2. Functional analysis of DBTNBT gene promoter2.1 Bioinformatics analysis of DBTNBT promoterDBTNBT gene promoter 1488 bp upstream of the transcription start site was cloned by our lab. There was a 36bp in length overlap region between the promoter 3' and the 5'-untranslated region of cDNA sequence of DBTNBT gene, indicating the reliability of the cloned promoter by blast in NCBI.Sequence analysis by Bioinformatics revealed that several putative cis-elements, such as MYC,WRKY and MeJA responsive elements exist in the promoter.2.2 Functional analysis of DBTNBT promoter2.2.1 Construction of plant expression vector In order to carry out the 5'deletion and 3'deletion analysis of promoter,35S promoter region of GUS expression vector pCAMBIA1304 was replaced by the 5' deletion fragments or 3'deletion fragments of DBTNBT promoter, and thus a series of plant expression vector were constructed, including a full length promoter expression vector pDtA::GUS, five 5'deletion vector (pDtB::GUS, pDtC::GUS, pDtD::GUS, pDtE::GUS, pDtF::GUS), and two 3'deletion vector (pDtG::GUS, pDtH::GUS).2.2.2 Transient expression assay of promoter in onion epidermal cellVector (pDtA::GFP) with full Promoter fragment A fused with GFP gene had been constructed, and it was transferred into onion epidermal cells by particle bombardment. GFP protein was observed in the cytoplasm of onion epidermal by fluorescence microscope, indicated that DBTNBT promoter had a promoter-driven activity.2.2.3 Transient expression assay of promoter in tobacco leavesIn transient expression assays, by histochemical stain analysis and quantitative fluorometric GUS assay of GUS activity, we found that 5'deletion fragment C (-696～-1bp) of DBTNBT promoter had the highest drive capacity, its GUS activity was 7.1-fold than that of D (-406～-1bp),2.2-fold than that of A (-1488～-1bp) and 3.8-fold than that of B (-1013～-1bp), indicated that the region-696～-406bp was its positive regulatory region, while the region-1013～-696bp was its negative regulatory region.2.2.4 Stable expression assays of Promoter in transgenic tobaccosFour types transgenic tobaccos were obtained by transforming the plant expression vectors which contained the A (-1488～-1bp), B (-1013～-1bp), C (-696～-1bp), D (-406～-1bp) region of the DBTNBT promoter. GUS activity analysis showed that the transgenic tobacco of B had the highest GUS activity. GUS activity of type B transgenic tobacco was 7.5-fold than A,283.6-fold than C, and 73.7-fold than D. It was concluded that B-C(-1013～-696bp) was its positive regulatory region. This conclusion was quite different with the conclusion from its transient expression assays.GUS activity analysis was carried out to study tissue specific expression pattern of DBTNBT promoter. Results showed that promoter A and B could drive gus gene express higher level in stems, leaves than roots. The promoter C and D could drive gus gene express higher level in roots than stems and leaves. It was speculated that the A-C region (-1488～-696bp) had positive cis-elements which respond to light.2.3 Isolation binding protein of DBTNBT promoterSeveral binding proteins of P1(-1488～-1005bp), P3(-706～-398bp) and P4(-417～-175bp) regions of DBTNBT promoter were isolated by yeast one-hybrid strategy. Four binding proteins of P1 belongs to superfamilies including Histone, BTK, Ubiquitin family, and three factors were unknown proteins. Four binding proteins of P3 belonged to superfamilies including BetVI-family,Rieske,UEP and Protease inhibitor/seed storage/LTP family, and the function of one protein was unknown. Two binding proteins of P4 belonged to superfamilies including Histone and family with biotin carboxylase C-terminal functional domain, and the function of two proteins were unknown. Factors belonged to BetVI-family and BTK would have a role in transcriptional regulation, and this speculation needed a further study.