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Construction of Actinidia Latifoia Merr.Galdh Plant Expression Vector and Its Transformation into Tomato

Author: WangAiLian
Tutor: MaFengWang
School: Northwest University of Science and Technology
Course: Pomology
Keywords: Latifolia Ascorbic acid L-galactose dehydrogenase Construction of expression vector
CLC: S663.4
Type: Master's thesis
Year: 2011
Downloads: 57
Quote: 1
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Abstract


Ascorbic acid (ascorbic acid, AsA) in organisms with important metabolic functions and antioxidant effects. Studies have shown that AsA is not only necessary to ensure that human health is one of the nutrients, but also for the plant's own antioxidant protection and regulating photosynthetic growth and so has a very important role. L-galactose dehydrogenase (L-galactose dehydrogenase, GalDH) plants ascorbic acid L-galactose key enzyme in the biosynthetic pathway, it may be L-galactose regulation of biosynthesis locus. The test is obtained latifolia (Actinidia latifolia Merr.) GalDH on the basis of full-length cDNA gene construct GalDH excessive plant expression pCSB / GalDH and RNA expression vector pHGRV / GalDH, and overexpression vector pCSB / GalDH Transfer Agrobacterium tumefaciens EHA105, and then through Agrobacterium-mediated method pCSB / GalDH gene into the tomato (Solanum lycopersicum L) Micro-Tom, resistant plants were screened for gene function GalDH verify and higher plants synthesize ascorbic laid material foundation. The main conclusions obtained are as follows: 1. Establish a Micro-Tom tomato Regeneration System test the effect of different hormone concentration ratio, dark incubation time, cefotaxime concentration conditions on regeneration, and to explore the IAA on the rooting of the results showed the best shoot regeneration medium for: MS ZT 2.0mg / L IAA 0.2mg / L; in MS 0.3mg/LIAA can be regenerated on medium high-quality root transplanting survival rate of 80%. Preculture 2d, cefotaxime 200mg / L can regenerate a high frequency callus and adventitious buds. 2 gene was constructed GalDH excessive plant expression vector pCSB / GalDH and RNAi expression vector pHGRV / GalDH with restriction endonucleases BamHI and KpnI the cloned genes from A. latifolia GalDH pMD-19T cloning vector was cut, directional insertion into the plant expression vector pCSB, successfully constructed latifolia GalDH transgenic plants overexpressing vector pCSB / GalDH. GalDH length gene obtained after two rounds of PCR reactions with the attB site of the target gene, the BP Clonase under the action of the carrier pHGRV attP loci on homologous recombination, gene fragment into vector pHGRV, the end inverted repeat is formed carrying the target gene of the plant expression vector, named pHGRV / GalDH. 3 Micro-Tom tomato has been genetically modified plants in the establishment of Micro-Tom tomato cotyledons frequency plant regeneration system and GalDH gene overexpression vector pCSB / GalDH basis, Micro-Tom tomato cotyledons as explants using Agrobacterium tumefaciens gene transfer mediated method will GalDH Micro-Tom tomato. Discusses the hygromycin resistance screening as the best system. The results showed that pre-cultured cotyledons through 2d, at OD600 value of 0.3 to 0.4 of bacteria in infected 10min, 3d co-culture, hygromycin pressure of 2.5mg / L, the concentration of inhibitory factors cefotaxime 200mg / L of conditions, the highest conversion rate, after detection by PCR detection of GUS initially identified three resistant plants.

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