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Construction of Plant Expression Vectors for as and Chr Genes, and the Study on Their Transformations in Eustoma Grandiflorum

Author: ZhouYuLong
Tutor: ChenLiMei
School: Kunming University of Science and Technology
Course: Biochemistry and Molecular Biology
Keywords: Lisianthus Chalcone reductase Aureusidin synthase Transgenic plants
CLC: S682.19
Type: Master's thesis
Year: 2009
Downloads: 35
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Abstract

Lisianthus (Eustoma grandiflorum) as the Gentianaceae (Gentianacea) prairie gentian genus. Lisianthus plants light stagnation sprinkle, elegant colors and crisp, chic and cute flower-shaped. After 30 years of research, the Lisianthus most an international market is very popular potted plant and cut flower species, has a dark blue, pink, red roses, yellow, white, and blue color. Small but complex color flower types and colors for the rich and raise the Lisianthus complex color flower species, this study attempts through genetic manipulation in a the Lisianthus variety (Eustoma grandiflorum, Prairie gentian 315P) overexpression as and chr expect to change this Lisianthus suit to produce purple lace yellow petals at the bottom of molecular breeding flowers provide a theoretical basis. In this study, mainly to obtain the following results: 1. Extracts of yellow snapdragon petals RNA was reverse transcribed to cDNA. As gene amplified by PCR using the TA cloning technique to get pMD19-as subcloned into pMD19-T vector using the restriction enzyme cutting the pMD19-as plasmid containing the light-inducible promoter Gateway entry vector pENTR PrbcS *-PrbcS-* T-gfp (Marlee, master's thesis) purified plasmid, carrier recovery and the target gene fragment of the target gene fragment and the vector fragment with ligase connect the entry vector pENTR containing PrbcS promoter * -PrbcS-as. through the gateway LR reaction PrbcS-as fragment was subcloned into the plant in expression vector pK2WG7, produce inducible plant expression vector pMD19 pK2-35S-PrbcS-as2. with a restriction endonuclease cutting a purified -as plasmids and Gateway entry vector pENTR *-2B purified plasmid recovery carrier and target gene fragment, connect with ligase target gene fragment and the vector fragment, as the entry vector pENTR *-as through the gateway technology LR reaction as fragment was subcloned into the plant in expression vector pK2WG7, to generate constitutive plant expression vector PK2-35S-as.3 at. extraction yellow alfalfa petal RNA was reverse transcribed to synthesize cDNA. Chr gene amplification by PCR out using the TA cloning technique subcloned into pMD19-T vector pMD19-chr. restriction endonuclease cutting the the pMD19-chr plasmid containing the light-inducible promoter pENTR PrbcS Gateway entry vector *-PrbcS-adh (Song Zhongbang LR through gateway technology, master's thesis) purified plasmid recovery carrier and target gene fragment with ligase target gene fragment and the vector fragment connection entry vector pENTR containing PrbcS promoter *-PrbcS-chr. reaction PrbcS-chr fragment was subcloned into the plant in expression vector pH2WG7, to produce the light-inducible pH2-35S-PrbcS-chr plant expression vector. 4 with a restriction endonuclease cutting purified of pMD19-chr and plasmid and Gateway entry vector pENTR *-2B plasmid was purified and recovered by the carrier and gene The fragment to be connected in the target gene fragment and the vector fragment by ligase, containing the entry vector pENTR * chr-chr. Via the gateway LR reaction chr fragment was subcloned into the plant in expression vector pK2WG7, forming the composition type pK2-35S-chr plant expression vector. 5 of Eustoma (Prairie gentian 315P), tissue culture and transformation methods are not mature enough, so this study this Lisianthus leaves adventitious bud induction medium MS 6-BA 1.0mg / L IBA 0.2mg / L determined by experiments. Eustoma grandiflorum antibiotic sensitivity experiments, Lisianthus transformed adventitious buds screening antibiotics (kanamycin, Km) of the optimal concentration of 15 mg / L, effective to inhibit Agrobacterium cefotaxime sodium (Cef) concentration of 300mg / L. 6. Electric conversion of building a good plant vector into Agrobacterium C58C1 (pPMP90), then the use of Agrobacterium-mediated transformation of Eustoma. Three kinds of transgenic Lisianthus lines, turn pK2-35S-as four transgenic lines, turn of pK2-35S-PrbcS-as gene lines 3 and turn pK2-35S-chr strains 2. The genomic PCR detection experimental results show that as and chr genes have been transferred to Lisianthus genome. as chr detection of gene expression levels and petal color changes observed in the research plan.

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CLC: > Agricultural Sciences > Gardening > Ornamental Horticulture ( flowers and ornamental trees) > Perennial Flowers > Perennial Flowers Class > Other
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