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Construction of Transgenic Pogostemon Cablin (Blanco) Benth. Plant with Allene Oxide Synthase Gene (AOS) from Arabidopsis Thaliana

Author: MaLiMin
Tutor: HeGuoZhen
School: Guangzhou University of Traditional Chinese Medicine
Course: Of Pharmacy
Keywords: AtAOS gene Clone Expression Patchouli
CLC: S567.239
Type: Master's thesis
Year: 2010
Downloads: 54
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Abstract

1 Purpose Jasmonates (jasmonates, JAs) are a class of plant hormones and the important injury signaling molecules, play a vital role in the plant defense response. JAs can induce the plant to produce secondary metabolites, improve stress resistance in plants, enhance the defense capability of the pest. This project aims JAs biological synthesis of specific enzymes - allene oxygenase (AOS) gene was cloned using Agrobacterium-mediated transformation of patchouli (Pogostemon cablin (Blanco) Benth), get turn AtAOS transgenic plants, in order to get a broad-spectrum disease and insect resistance and high efficacy ingredients transgenic patchouli strains. 2 Methods 2.1 Purpose Cloning 2.1.1 Objective Cloning published under Genebank AtAOS (AY128755) sequences, the design of the specific RT-PCR primers, to the Columbia ecotype of Arabidopsis (Arabidopsis thaliana) total RNA as template for RT -PCR, obtain AtAOS gene fragment. 2.1.2 Cloning plasmid The RT-PCR product was recovered, purified the connection T vector (pDriver Cloning vector pGEM-T easy, etc.), and transformed into E.coli DH5α competent cells and ampicillin resistance by blue-white screening, screening and identification of positive recombinants and by digested sequencing methods. Establishment of Regeneration System 2.2 patchouli 2.2.1 callus induction MS basal medium, add 2,4 - dichlorophenoxyacetic acid (2,4-dichiorophenoxyacetic; 2,4-D) and cell mitogen kinetin (kinetin; KT), screening patchouli best callus induction conditions. 2.2.2 induction and plant regeneration of callus differentiation MS basal medium, add the cytokinin benzyladenine (6-benzyladenine; 6-BA), screening the patchouli shoots renewable induced conditions and callus differentiation conditions. 2.2.3 antibiotic concentration conditions determine patchouli callus induction medium based add carbenicillin penicillin (carbenicillin; Carb,), set different concentration of patchouli selection pressure screening. To get the the patchouli plant regeneration maximum tolerated level carbenicillin concentration. 2.3 allene the lipoxygenase (AtAOS) transformed patchouli 2.3.1 plant expression vector 2.3.1.1 expression plasmid digestion processing of to carry AtAOS gene fragment cloning plasmid pGEM-AtAOS (pA), electrophoretic separation recovery and purification of the objective DNA fragment obtained sticky ends, connected to the same digested processing and after CIAP dephosphorylated processing plant expression vector pBI121, and the recombinant was transformed into E. coli competent cells E. coli DH5a and Carb resistance screening positive clones were screened by restriction enzyme digestion, sequencing and identification of positive recombinants. 2.3.1.2 expression plasmid electroporation Agrobacterium EHA1 05 recombinant expression plasmid and Agrobacterium Agrobacterium tumefaciens EHA105 competent cells mixed and added to the shock of electric shock Miriam cup, choose the the Agr program of electric shock. Transformed by electroporation broth coated in resistance YEB tablet positive recombinants were screened by chloramphenicol and kanamycin resistance and by PCR assay to determine the positive recombinants. Save with the positive recombinant bacterium. 2.3.2 patchouli leaves the preculture in regenerated plants differentiation medium patchouli leaves, set different preculture days, in order to get the best conversion patchouli leaves into the state. 2.3.3 the conversion of the target gene will carry the expression plasmid of the Agrobacterium culture to log phase, diluted infestation patchouli callus and pre-cultured blade. After a lump of cultured, transfer to the the the antibiotic selection meristematic medium light culture. Transgenic regenerated plants blade Transgenic Plant Regeneration 2.3.4 detection of extracting DNA and RNA, respectively, for each plant target gene (AtAOS), GUS gene PCR amplification, RT-PCR amplification of the AtAOS gene; filtered prognosis injured tissue and transformed plants the histochemical staining finalized transgenic regenerated plants. 3 Results of 3.1 AtAOS genes cloned the 3.1.1 AtAOS RT-PCR amplification according to agarose gel electrophoresis results, RT-PCR product was in line with expectations, can initially determine the successful amplification AtAOS gene fragment length of 1.6 kb. 3.1.2 cloning plasmid and identification of positive recombinants were screened by blue-white screening and colony PCR method identified by agarose gel electrophoresis and sequencing methods. The results show that: to successfully build cloning plasmid pGEM-AtAOS (pA) carry AtAOS gene. Sequence alignment recombinant cloning plasmid pGEM-AtAOS (pA), AtAOS gene nucleotide sequence and Genebank registered Arabidopsis thaliana allene oxide synthase gene sequence (serial number AY1 28755) 100% (1645 bp consistency / 1645 bp), containing the entire coding sequence of the target gene. 3.2 patchouli regeneration system build 3.2.1 callus induction Zhaoqing patchouli plantlets leaves explant material, successfully induced the callus, and examine the concentration of combinations of different plant hormones callus Induction of differences, the best callus induction MS KT (0.5 mg / L) 2,4-D (0.05 mg / L). 3.2.2 induction and plant regeneration callus differentiation Zhaoqing patchouli plantlets leaves and callus induced material successfully induced in differentiation medium patchouli plant regeneration, differentiation culture The group identified as MS 6-BA (1 mg / L). 3.3 allene lipoxygenase transformation the patchouli system build 3.3.1 expression plasmid PCR, single colonies cloning, agarose gel electrophoresis and sequencing results show that successfully constructed expression plasmid pGEM-AtAOS carry AtAOS gene- pBI (pAp), ORF correct, encoding 518 amino acids, the amino acid sequence (accession number AAM91155.1) homology to 100% (518 aa/518, AA) and from Genebank AtAOS. 3.3.2 determine antibiotic concentrations add different concentrations Carb to callus induction medium MS KT (0.5 mg / L) 2,4-D (0.05 mg / L), Zhaoqing patchouli leaves plant selection pressure of tolerance screening to determine the the explant Carb maximum tolerated concentration of 500 mg / L. Preculture 3.3.3 patchouli leaves the patchouli leaves infected by the growth conditions, derived patchouli leaves the pre-incubation time of 2 days. 3.3.4 infection, antibiotic screening will carry the recombinant expression plasmid of Agrobacterium culture to log phase, was diluted 20-fold after the preculture patchouli leaves and callus different time colonization process, according to the organizations of the material in the filter medium (MS KT 0.5 mg / L 2,4-D 0.05 mg / L Carb 500 mg / L Kan 75 mg / L), the growth of state determine infection: Agrobacterium were diluted 5-10 times the infection time of 15 min best. The Kan inhibitory effect on callus induction screening methods are improved, that is followed by a double-antibody screening and relieve the inhibition of Kan continue to single antibiotics carbenicillin selection medium (MS KT 0.5 mg / L 2,4-D0 .05 mg / L Carb 500 mg / L) were cultured and screened. 3.3.5 Transgenic regenerated plants for PCR amplification, the detection of 142 transgenic plants AtAOS from which three are obtained of the product is consistent with the expected fragment size; on GUS PCR amplification, AtAOS the RT-PCR expansion growth, has also been a consistent product with the size of the expected fragment. Filtered callus apparent after staining method staining blue the transformed plants obvious staining results, the test results showed that the target gene has been successfully transformed into patchouli plants, and expressed. 4 Conclusion clone AtAOS gene connected to the plant expression vector pBI121; successfully constructed expression plasmid pGEM-AtAOS-pBI (pAp); and has received three transgenic patchouli plants.

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