Fruit germplasm culture method capable of spontaneously eliminating agriculture remanet
A breeding method and fruit technology, applied in the field of plant germplasm cultivation, can solve problems such as excessive pesticide residues, and achieve the effect of enhancing food safety
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[0034] Example 1: Tomato Germplasm Cultivation for Spontaneous Elimination of Pesticide Residues
[0035] (1) Clone the fruit-specific promoter according to the conventional method:
[0036] Genomic DNA was extracted from tomato leaves, and the 1.1KbE8 promoter was cloned by PCR (see figure 1 ), PCR primers were designed according to the E8 promoter sequence provided in Genbank ID: AF515784.1, synthesized by Shanghai Jierui Bioengineering Company, the sequence is:
[0037] P1: 5'-CCCAAGCTTAGGAATTTCACGAAATCG-3';
[0038] P2: 5'-CGCGGATCCTCTTTTGCACTGTGAATG-3';
[0039] The upstream primer introduces a HindIII restriction site, and the downstream primer introduces a BamHI restriction site. According to the instructions of the plasmid pGEM-T easy, connect the cloned product to the plasmid pGEM-T easy to transform E.coli TG1, obtain positive clones through blue-white screening, and send them to Beijing Sanbo Yuanzhi Biological Company for sequencing to obtain the correct E8 prom...
Example Embodiment
[0058] Example 2: Cucumber Germplasm Cultivation for Spontaneous Elimination of Pesticide Residues
[0059] (1) Clone the fruit-specific promoter according to the conventional method:
[0060] With embodiment 1;
[0061] (2) Synthetic gene of organophosphate hydrolase OPH:
[0062] With embodiment 1;
[0063] (3) Construct the fruit-specific OPH plant expression vector:
[0064] With embodiment 1;
[0065] (4) transformation of soil Agrobacterium and cucumber explants
[0066] The transformation soil agrobacterium is the same as embodiment 1;
[0067] Cucumber seeds Jinyan No. 4 were purchased from Tianjin Academy of Agricultural Sciences. Soak ripe cucumber seeds in warm water for 1 hour, soak them in 75% alcohol for 45 seconds after peeling, and then wash them with 0.1% HgCl 2Soak for 5 minutes, wash 5 times with sterile water, dry the water on sterilized paper and connect it to MS medium. When the seed germinates to a height of 5 cm and the cotyledon is fully stretch...
Example Embodiment
[0075] Example 3: Capsicum Germplasm Cultivation for Spontaneous Elimination of Pesticide Residues
[0076] (1) Clone the fruit-specific promoter according to the conventional method:
[0077] With embodiment 1;
[0078] (2) Synthetic gene of organophosphate hydrolase OPH:
[0079] With embodiment 1;
[0080] (3) Construct the fruit-specific OPH plant expression vector:
[0081] With embodiment 1;
[0082] (4) transformation of soil Agrobacterium and capsicum explants:
[0083] The transformation soil agrobacterium is the same as embodiment 1;
[0084] Pepper seeds Tianjiao No. 1 were purchased from Beijing Jintianli Company. Soak the surface of the seeds in 95% alcohol for 45 seconds, then soak them in 50% bleach for 10 minutes, wash them with sterile water for 5 times, dry them with sterilized paper, put them on MS medium, and culture them under light at 25°C. When stretching, the hypocotyls were excised and cultured on the pre-medium for 2 days. The Agrobacterium LB...
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