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Study on the Construction of a Plant Expression Vector with ACO1 Antisense Gene Regulated by Fruit Specificity Promoter and Its Transformation to Cucumis Melon ’Yu Jingxiang’

Author: ZhaoZuo
Tutor: ChenBaiHong
School: Gansu Agricultural University
Course: Pomology
Keywords: Melon ACC oxidase CM - ACO1 promoter Tissue-specific expression Transformation
CLC: S652
Type: Master's thesis
Year: 2010
Downloads: 32
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Abstract

Melon ( Cucumis melon L. ) during storage, transportation , easily soften and rot. By transgenic technology can effectively extend the melon ripening process and prevent storage rot, the antisense technology melon storage and transportation has been successful . But ships constructed vector are through 35S promoter regulation ACC antisense gene , although the target gene can be stably expressed , will cause a waste of energy but non-specific expression of exogenous gene expression affected . Therefore, this study for the melon turn ACC oxidase antisense gene 35S constitutive promoter , as well as clear melon ACC oxidase gene promoter with specific regulatory function on the basis of building -specific promoter regulation antisense gene expression vector , and transformed melon . The main results are as follows : 1 antisense expression vector PCB - aACO1 to remove 35S promoter with Hind III and the XBAR I digested large fragment , pCAM-ACO 1-promoter with Hind III and BamHI digested 886bp promoter The restriction sites do not match, the 3 'ends blunted with DNA polymerase Klenow fragment and blunt end ligation . Were transformed into E. coli DH5a Colonies were screened for identification of resistance and amplified by PCR to verify specific promoter and ACC antisense gene in order to build the plant with specific promoter expression carrier PCB - aACO1 - P . 2 with the freeze-thaw method the PCB - aACO1 - P directly into the Agrobacterium tumefaciens strain EHA105 , antibiotic resistance and PCR confirmed the PCB - aACO1 - P has been introduced into Agrobacterium . Agrobacterium tumefaciens -mediated transformation of tobacco leaf disc transformation method , antibiotic resistance screening and PCR detection of transgenic tobacco plants containing the carrier the PCB - aACO1 - P . 3 melon cultivars ' Yujinxiang '5 D the seedling age cotyledons MS 2.0 mg / L 6-BA adventitious buds induced differentiation medium , adventitious bud regeneration rate of up to 85.39% ; induce adventitious shoot elongation medium MS 0.1mg / L6 - BA for the best, good elongation of adventitious buds in 1/4MS rooting explants rooting rate was 92.15% , can be used as the the melon genetic transformation of the receptor . 4 leaf disc transformation using Agrobacterium tumefaciens -mediated transformation Melon, after antibiotic resistance and PCR confirmed the PCB - aACO1 - P imported melon . Get 12 Kana resistance , PCR -positive transgenic lines .

Study on the Construction of a Plant Expression Vector with ACO1 Antisense Gene Regulated by Fruit Specificity Promoter and Its Transformation to Cucumis Melon ’Yu Jingxiang’

Abstract

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