Molecular Cloning And Analysis Of Plant Defence Related Genes From Camptothecaacuminata And The Effect On Camptothecin Content

Plant secondary metabolites have a variety of important biological functions, whichpromise a wide range of applicational prospects in agricultural and industrialproduction and in human's daily life. Plant alkaloids, which constitute one of thelargest group of natural products, provide many pharmacologically active compounds.Camptothecin (CPT) and its derivatives, which belong to a group of monoterpenoidindole alkaloids, exhibit a significant anti-tumor action. CPT is a kind of alkaloids inCamptotheca acuminata, exhibits a certain of role in plant defence reaction. Jasmonicacid (JA) and its methyl ester (MeJA), commonly known as jasmonates, are identifiedas signals of altered gene expression in various plants responding to biotic and abioticstresses as well as in distinct stages of plant development. According to somephysiological characteristic in Camptotheca acuminata and several specific functionsof CPT, allene oxide cyclase, a key enzyme of jasmonites pathway, was isolated fromCamptotheca acuminata (named CaAOC); mealwhile, it was investigated whether themRNA expression of CaAOC was related to the content of CPT in Camptothecaacuminata. Besides, the study of some other aspects was carried out.1) The study of alkoids concentration in Camptotheca acuminata differentorgans and Camptotheca acuminata cellsIn order to facilitate the production of these pharmaceuticals, the contents ofCPT and 10-hydroxycamptothecin (10-HCPT) in different tissues including roots,stems, leaves, young flower buds, opening flowers, fading flowers and seeds fromCamptotheca acuminata, were investigated. The young flower buds were of thehighest in alkaloids concentrations with 2.46 mg g^-1 (Dried Weight) CPT and 1.41 mgg^-1 (Dried Weight) 10-HCPT. Callus was also considered as a potential source of thesepharmaceuticals. In the present study, the growth rate of Camptotheca acuminata cellswas not observed to be correlated with contents of CPT and 10-HCPT. Responses ofthe cells to various treatments including wounding (heavy metal ions and UV-B),methyl-jasmonate (MeJA), abscisic acid (ABA), salicylic acid (SA) and hydrogenperoside (H₂O₂) were examined in the changes of alkaloids accumulation, the mostpotent effects appeared in the cells induced by UV-B light with 11-fold CPTconcentration above the control, and in the cells induced by salicylic acid (SA) with the content of 10-HCPT 25-fold above control. These results are helpful for advancingthe production of the two pharmaceuticals.2) Allene oxide cyclase from Camptotheca acuminateAllene oxide cyclase (AOC, EC 5.3.99.6), an essential enzyme in jasmonic acid(JA) and its methyl ester (MeJA) biosynthesis, was cloned from Camptothecaacuminata (named as CaAOC, Database Accession No. AY863428), a nativemedicinal plant species in China. CaAOC had significant similarity at amino acidlevel with AOCs from various other plant species. Comparison between the sequencesof the full-length cDNA and genomic DNA of CaAOC revealed that the genomicDNA of CaAOC contained an 89-bp intron and a 240-bp intron. Southern blotanalysis indicated that CaAOC was a multiple copy gene and real-time quantitativePCR analysis showed that CaAOC expressed constitutively in all tested organs, withthe highest expression level in leaves. The results from treatment experiments usingdifferent signaling components including methyl-jasmonate (MeJA), abscisic acid(ABA), salicylic acid (SA) and hydrogen peroside (H₂O₂) revealed that expression ofCaAOC had a prominent diversity. The heavy metal (copper) significantly enhancedthe CaAOC expression, whereas wounding (UV-B) was not so effective.3) CaAOC was transformated into Escherichia coli and tobacco byAgrobacterium tumefaciensThe mRNA expression of AOC gene from Camptotheca acuminata (CaAOC,Database Accession No. AY863428) was analyzed by real-time quantitative PCR. Theresults showed that it could be induced by 120 mM NaCl and low temperature (4℃).A 5'-truncated coding region of CaAOC was cloned into the expression vector pET30and then expressed in Escherichia coli strain BL21DE3(pLysS). The positive bacteriacould grow on 2YT agar containing 400 mM NaCl. Simultaneously, CaAOC wasintroduced into tobacco and transgenic plants were generated and confirmed by PCRanalysis. Four transgenic lines were examined for tolerance to salt stress and lowtemperature. The chlorophyll content was higher in transgenic lines thanuntransformed control plants under salt stress. The electrolyte release test showed thattransgenic tobacco plants were resistant to low temperature damage. Our studysuggested that CaAOC could be a potential target gene in the engineering of plants forimproved stress-tolerance. 4) Agrobacterium tumefaciens-mediated CaAOC transformation of CamptothecaacuminateThrough Agrobacterium tumefaciens-mediated transformation, the CaAOC wassuccessfully transformated into Camptotheca acuminate. The result of RT-PCRshowed the CaAOC's expression level of transgenic callus was obviously higher thanthat of untransgenic callus. Besides, CaAOC's expression level of transgenic anduntransgenic callus both increased after MeJA treatment, and in transgenic callus itwas still higher than in untransgenic ones. The results of HPLC analysis indicated thatthe camptothecin content of transgenic callus was higher than untransgenic callus.The highest camptothecin content of transgenic callus was 3.9310 mg/g DW while thelowest was 1.0028 mg/g DW. However, the camptothecin content of untransgeniccallus was too low to be tested. Transgenic and untransgenic calli's camptothecincontent significantly decreased after MeJA's induction, while the content of CPT intransgenic callus was still higher than in untransgenic one. All the results indicatedthat jasmonate's accumulation may be promoted after CaAOC was transformed intoCamptotheca acuminate. Jasmonate can affect second metabolism pathway genes'expression and then the camptothcin content was improved. Perhaps the mechanismof exogenous jasmonate to secondary metabolism of Camptotheca acuminate wasdifferent from that of endogenic jamonate.5) Cytochrome P450 from Camptotheca acuminateIn higher plants, P450 participates in the biosynthesis of many importantsecondary metabolites. Here we reported for the first time the isolation of a newcytochrome P450 cDNA that expressed in a stem-specific manner from Camptothecaacuminata (designated as CaSS, Database Accession No. AY466856), a nativemedicinal plant species in China, using RACE-PCR. The full-length cDAN of CaSSwas 1735 bp long containing a 1530bp open reading frame (ORF) encoding apolypeptide of 509 amino acids. Bioinformatic analysis revealed that CASS containeda heme-binding domain PFGXGRRXCX and showed homology to other plantcytochrome P450 monooxygenases and hydroxylases. Southern blotting analysisrevealed that there was only one copy of the CaSS present in the genome ofCamptotheca acuminata. Northern blotting analysis revealed that CaSS expressed, in atissue-specific manner, highly in stem and lowly in root, leaf and flower. Our studysuggests that CaSS was likely to be involved in the phenylpropanoid pathway.