Construction Of Transgenic Pogostemon Cablin (Blanco) Benth. Plant With Allene Oxide Synthase Gene (AOS) From Arabidopsis Thaliana

1 ObjectJasmonic acid (jasmonates, JAs) is one of plant hormones, and is a vital signaling molecule which plays important roles in plant growth and development, inducing secondary metabolites production, pathogens and adversity resistance. Allene oxide synthase is one of enzymes in the octadecanoid pathway of jasmonates biosynthesis. Aim of the research is to clone the allene oxide synthase gene from Arabidopsis thaliana, and get the transgenic Pogostemon cablin plant produced by Agrobacterium-mediated transformation, which have the ability of pest-resistant and contains higher secondary metabolites.2 Methods2.1 Cloning allene oxide synthase gene from Arabidopsis thaliana2.1.1 Amplification of the gene of allene oxide synthaseSpecific RT-PCR primers were designed, and allene oxide synthase from Arabidopsis thaliana was amplified by RT-PCR technique.2.1.2 Construction of cloning plasmidRT-PCR product was recovered, purified, and then ligated into T vector (such as pGEM-T Easy).Ligations were transformed into Escherichia coli DH5a competent cells. Transformation colonies were selected by using ampicillin (50μg/mL) on LB medium with IPTG and X-gal (Blue and White Screening), then positive recombinant colonies were screened and identified through PCR amplification, restriction enzyme digestion, sequencing.2.2 Construction of the patchouli regeneration system2.2.1 The induction of callusUsing the MS as the based culture medium, the optimum induction medium for patcholi callus was obtained by screening combinations of 2,4-D and KT at different concentrations.2.2.2 The induction of regeneration plant and the differentiation of the callusUsing the MS as the based culture medium, the optimum induction medium for patcholi regeneration plant and the differentiation of the callus was obtained by screening combinations of 6-BA at different concentrations.2.2.3 The determination of the concentration of the antibioticUsing the callus induction medium as the based polarization medium, we add carbenicillin at different concentration then the best Carb choice pressure of transgenic patcholi was screened.2.3 Construction of plant expression vector and transforming to patchouli2.3.1 Construction of plant expression plasmid2.3.1.1 Construction of expression plasmidThe cloning plasmid which contained insertion DNA fragment of allene oxide synthase from Arabidopsis thaliana was digested with suitable restriction enzyme, the target DNA fragment was separated through agarose electrophoresis and purified using silicon columns, and then was ligated into the expression vector which was purified after digestion with the suitable restriction enzyme and dephosphorylated with CIA. Ligations were transformed into Escherichia coli DH5a competent cells. Transformation colonies were selected using Kanamycin (50μg/mL) on LB medium, then positive recombinant colonies were further screened and identified through PCR amplification, restriction enzyme digestion, sequencing.2.3.1.2 Electroperation of Expression Plasmid into Agrobacterium(EHA105)The recombinant expression plasmid and the Agrobacterium tumefaciens competent cells (EHA105) were put into the cuvette, the Agr shock program were used, The mixture were spred onto YEB plate. The recombinant transformants were screened by chloramphenicol and kanamycin, and further identified by PCR.2.3.2 The pre-training of the patchouli leaves for tansformationTo get the best transformation conditions transformation, we set different number of days on the pre-culture of the patchouli leaves upon the differentiation medium of the regeneration plants.2.3.3 transformation of insertion DNAMultiple dilution of the agrobacteriums suspension which carrying expression plasmids, we make different times of treatment on the callus of the patchouli and the pre-cultured leaf. After two days'dark incubation, we transfer them to the antibiotic selection medium to carry a light training.2.3.4 Identification of transgenic regeneration plants.PCR amplifications were conducted using specific primers for AtAOS gene and GUS gene and RT-PCR on the AtAOS gene repectively. And histochemical localization of GUS gene expression was assayed.The confirmation of the transgenic plants were finally made.3 Results3.1 cloning of AtAOS Gene 3.1.1 RT-PCR amplificationAgarose gel electrophoresis of RT-PCR products showed that the complete sequence of allene oxide synthase from Arabidopsis thaliana was successfully amplified with the length 1640 bp.3.1.2 Construction of cloning plasmidPCR amplification of the positive recombinant colonies, agarose electrophoresis analysis and sequence alignment showed that the cloning plasmid pGEM-AtAOS (pA) which containing allene oxide synthase gene fragment was successfully constructed. The nucleotide sequence of AtAOS in pGEM-AtAOS (pA) exhibited 100%(1645/1645) identity to the sequence of AtAOS from Genebank.3.1.3 Construction of express plasmidPCR amplification of the positive recombinant colonies, agarose gel electrophoresis and sequence alignment showed that the expreesion plasmid pGEM-AtAOS-pBI (pAp) containing allene oxide synthase gene fragment was successfully constructed. The sequence analysis identified the ORF encoding 518 amino acid,which showed 100%(518/518) homologous to the allene oxide synthase in Arabidopsis thaliana from Genebank.3.2 Construction of the patchouli regeneration system3.2.1 Inducition of the callusCallus are successfully induced using patchouli in vitro leaf.The influences of different hormone concentrations were inspected on the callus.The suitable medium for the callus induction is found to be MS+KT(0.5mg/L)+2,4-D(0.05mg/L).3.2.2 Induction of the regeneration plants and differentiation of callusUsing the patchouli in vitro leaf and the successful induced callus as explant materials, respectively, we successully induced the regeneration plants of patchouli in medium MS+6-BA(1 mg/L).3.2.3 Confirmation of the antibiotic concentrationSupplementation of carbebucullin ranged from 0 mg/L to 2000 mg/L to the callus inducing medium MS+KT(0.5 mg/L)+2,4-D(0.05 mg/L) was tested for pressure tolerance sreening to the patchouli callus. The maximum concentration of carbenicillin to the patchouli is found to be 500 mg/L.3.3 AtAOS Gene transforming to patchouli3.3.1 The pre-training of the patchouli leafAccording to the growth conditions of the patchouli leaf under infection, we found that the time of the pre-training of patchouli leaf for a better transformation is 2 days.3.3.2 Infection and the selection of the antibioticUsing 1 to 20 times dilution of the agrobacterium suspension which carrying expression plasmids, we conducted different treatment times on the callus of the patchouli and the pre-cultured leaf. According to different materials'growth on the selection medium (MS+KT 0.5 mg/L+2,4-D 0.05 mg/L contains 500 mg/L carbenicillin and 75 mg/L Kan), the optimum condition is found to be 5 to 10 times' dilution of agrobacterium suspension with an infection time of 15 min. Because of the obvious inhibitory effect on the inducing callus of Kan, we chose MS+KT 0.5 mg/L+2,4-D 0.05 mg/L+500 mg/L carbenicillin as the selection medium.3.3.3 Detection of the transgenic regerenation plantsPCR screening of the AtAOS gene on 142 transgenic plants, resulted in 3 positive transformants PCR amplification using GUS specific primers and RT-PCR amplification of the AtAOS gene further confirmed, the target genes are successfully transformed into patchouli plants and the AtAOS had been expressed.Histochemical GUS assay showed that callus with green spot were stainable with X-gluc,indicating that meristematic zones are transformed.4 ConclusionIn the current work, AtAOS genes was successfully cloned and ligated into plant expression vector pBI121, followed by successfully constructing the expression plasmid pGEM-AtAOS-pBI (pAp).Three transgenic patchouli plants were obtained using the optimized transformation procedure.