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Construction of Expression Vectors for Skti and p-lec, and the Study on Their Transformation in Eustoma Grandiflorum

Author: ChenHongMei
Tutor: ChenLiMei
School: Kunming University of Science and Technology
Course: Biochemical Engineering
Keywords: Eustoma grandiflorum insect-resistance Genetic engineering SKTI P-Lec
CLC: S682.19
Type: Master's thesis
Year: 2009
Downloads: 19
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Abstract

At present, Eustoma grandiflorum has become one of the most popular cut flowers on the international markets. However, the fast production development, expanding cultivated area, and frequent import, export and domestic trade, has made Eustoma grandiflorum subject to many kinds of pest hazards in production, which seriously affected the quality of cut flowers and steady development of the plant industry. The birth of plant insect-resistant genetic engineering provides a new way for pest prevention and treatment. Since most researches focus on economic crops, insect-resistant genetic engineering of flowers although have developed, but also in infancy, and the insect-resistant genetic engineering on Eustoma grandiflorum has not been reported almost. Therefore, the insect-resistant genetic engineering in Eustoma grandiflorum not only can solve the pest problem in Eustoma grandiflorum production and lay the foundation for further development of Eustoma grandiflorum industry, but also broadens the germplasm resources of Eustoma grandiflorum.Soybean Kunitz-type trypsin inhibitor (SKTI) has a strong inhibitory effect on the protease activity of Lepidoptera pests. To over express SKTI in Eustoma grandiflorum against the Lepidoptera pests which hurt the root of the plant, we constructed a constitutive plant expression vector for skti in this study. First, two specific primers were designed for amplification of skti coding region. Then, the cDNA of skti was amplified from soybean total RNA via RT-PCR and subcloned into vector pMD18-T to yield pMD18-skti. Then, skti was excised from pMD18-skti and ligated into a modifed Gateway entry vector pENTR*-as (Zhou,Master thesis) between Sal I and BamHI to produce an entry vector pENTR*-skti. Finally, the skti fragment from the pENTR*-skti was recombinated into the Gateway plant expression vector pK2GW7 containing 35S promoter by gateway LR reaction to make the constitutive plant expresssion vector pK2-35S-skti.Pea lectin (P-Lec) is low toxicity to humans and animals, and the transgenic plants overexpressing P-Lec have obvious resistance to Homopteran and Coleoptera. To overexpress P-Lec against the Homopteran and Coleoptera pests which hurt the green young tissues in Eustoma grandiflorum, we constructed a light inducible plant expression vector for p-lec in this study. First, two specific primers were designed for amplification of p-lec coding region. Then, the p-lec coding region was amplified from soybean genome DNA via PCR and subcloned into vector pMD18-T to yield pMD18-p-lec. In order to express P-Lec in the green young tissues of the transgenic plants under a tissue-specific expression promoter (PrbcS), p-lec was excised from pMD18-p-lec and ligated into a modifed Gateway entry vector pENTR*-PrbcS-adh (Wang, Doctor thesis) which contains a light inducible PrbcS of tomato Rubico small subunit rbcs-3C between Sph I and EcoR I to produce an entry vector pENTR*-PrbcS-p-lec. Finally, the PrbcS-p-lec fragment from pENTR*-PrbcS-p-lec was recombinated into the Gateway plant expression vector pH2GW7 by gateway LR reaction to make the light inducible plant expresssion vector pH2-35S-PrbcS-p-lec.pK2-35S-skti and pH2-35S-PrbcS-p-lec was used to transform Eustoma grandiflorum (Prairie gentian and Polestar yellow) via Agrobacterium-mediated method. In order to optimize the transformation conditions for Eustoma grandiflorum, the regeneration system and antibiotics sensitivity were also investigated. The results showed that the best medium for Prairie gentian regeneration was MS+6-BA0.5 (mg/L)+IBA0.2 (mg/L). The highest concentration of Kanamycin and Hygromycin which Prairie gentian and Polestar yellow could tolerate was 30mg/L and 15mg/L, respectively. The highest concentration of Cef (Cefotaxime, Sodium Salt) suitable to inhibit the growth of Agrobacterium was 150mg/L. The best transformation conditions for Prairie gentian and Polestar yellow was pre-cultured for two days before transfection and transfected with 25mg/L acetosyringone (AS) for 20 min and then co-cultured for three days. The transgenic plants were selected on medium containing antibiotics and then verifyed by PCR. 2 transgenic lines were obtained for pK2-35S-skti and 12 transgenic lines were obtained for pH2-35S-PrbcS-p-lec, respectively. The examination of skti and p-lec expression in the transgenic plants and the larvae feeding will be performed in our future study.

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