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Cloning of PLDα cDNA in Strawberry and Studies of Antisense Gene Genetic Transformation

Author: LuXiuLi
Tutor: LuoShuPing；YuanHaiYing
School: Xinjiang Agricultural University
Course: Biochemistry and Molecular Biology
Keywords: Strawberry PLDα gene Antisense plant expression vector Genetic Transformation Southern hybridization
CLC: S668.4
Type: Master's thesis
Year: 2009
Downloads: 82
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Abstract

Strawberry (Fragaria ananassa Duch.) Is a perennial herb, are distributed in many countries of the world. Its fruit is a typical non-climacteric fruit can easily rot after harvest, intolerance storage, and played a catalytic role in cell wall and cell membrane structure in the process of catabolism. Therefore, to maintain the integrity of the cell membrane structure and cell regionalized structure plays an important role in fruit ripening and senescence process. The activity helps the phospholipase da (PLDα) strawberry fruit mature, the increased activity is the first step in damage to the cell membrane aging, thereby effectively control PLDα strawberry fruit research PLDα gene storability. In this study, the Plant RNA Reagent Kit from 达赛莱克特 strawberry (Darselect) End cooked fruit extract high purity and integrity better total RNA by RT-PCR amplified gene sequences HKD2 get PLDα gene functional areas, the TA cloning to pMD19-T simple vector sequencing cloned vector by double digestion purpose reverse fragment inserted into a plant expression vector pBI121 build the strawberry PLDα antisense plant expression vector pBI121-PLD. To deficient plants obtained by freeze-thaw method plants carrying antisense cDNA expression vector plasmid introduced into Agrobacterium LBA44404, and genetic transformation of strawberry, gene expression, and then determine the regulation of strawberry fruit ripening and senescence effect. During the experiment while building strawberry PLDα gene Justice plant expression vector pBI121-PLD and its genetic transformation in the subsequent Storability experiment to compare the two. Agrobacterium-mediated genetic transformation system, the culture to the logarithmic growth phase of the Agrobacterium infection solution was diluted to OD600 value of 0.3 to 0.6, with liquid MS medium without hormones, infection has been pre-incubated 3d strawberry leaf disc 10min, co-cultured 3d, rinse the leaves with liquid MS medium containing Cef 300mg · L ^{-1 dry filter paper to the differentiation medium cultured for 7 to 10 days, and then go to the CEF 300 mg · L ^{-1 , km 20 mg · L ^{-1 selective medium selective culture. The selection pressure Km preliminary selection of six genetically modified strawberry plants. PCR amplification and Southern blotting hybridization analysis of the resistant plants were obtained, 4 strains were positive, indicating that the target gene was integrated into the genome of strawberry.}}}

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