Phytophthora resistance application utilizing soybean agglutinin gene lec-s
A technology of soybean lectin and lec-s, which is applied in the field of genetic engineering, can solve problems such as the impact of chemicals on the environment and the safety of agricultural products
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Embodiment 1
[0033] Embodiment 1 pBI121:: the construction of lec-s plasmid 1.1 The cloning of lec-s
[0034] The leaves of soybean variety Hefeng No. 29 were taken, and total soybean RNA was extracted using the Plant RNA Kit plant RNA extraction kit from Omega Company, and treated with RNase-free DNase I for purification. Using PrimeScript TM Reverse Transcriptase (Takara) performs RNA reverse transcription to synthesize cDNA. Use 1 μg of total RNA to obtain the first strand of cDNA by reverse transcriptase as a template, lec-s-F / lec-s-R as primers, and amplify lec-s containing Xba I / Sma I restriction sites at both ends through PCR reaction Gene fragment. The PCR electrophoresis bands were recovered using the AxyPrep DNA Gel Recovery Kit from Axygen. The recovered lec-s fragment was connected to the sequencing vector pMD18-T Simple Vector to obtain the plasmid pMD-18T Simple vector::lec-s. Transform DH5α competent cells with the plasmid pMD-18T Simple vector::lec-s, use the AxyPrep P...
Embodiment 2
[0045] Example 2 Agrobacterium-mediated genetic transformation of tobacco
[0046] Tobacco was transformed using the Agrobacterium-mediated leaf disc method. Tobacco leaf discs (0.5 cm in diameter) were taken as transformation recipients, pre-cultured in the pre-medium for 1-2 days, then immersed in the prepared Agrobacterium solution for infection for 20 minutes, and the tobacco leaf discs were co-cultured with Agrobacterium in the dark for 2 days, and then transformed into Into the screening medium containing carbenicycin (500mg / L) and kanamycin (100mg / L). Culture at 25°C, L:D=16h:8h, subculture once a week. About 1 week later, the leaf discs began to differentiate into resistant callus, and then gradually differentiated into resistant buds. Bud differentiation begins in 3 to 6 weeks. After the green shoots grow to a height of 2 to 3 cm, cut off the regenerated shoots and place them in rooting medium for cultivation. After the transformed seedlings have a plant height of...
Embodiment 3
[0048] Example 3 Transgenic Tobacco T 0 generation of PCR detection
[0049] A total of 45 transgenic tobacco-resistant seedlings were obtained in the previous step, and the total plant DNA was extracted using the CTAB method. transgenic tobacco T 0 The total DNA of generation leaves was used as a template, the total DNA of empty vector-transformed plants and non-transformed plants was used as a negative control, and the plasmid pBI121::lec-s was used as a positive control for PCR amplification. Primers lec-s-F / lec-s-R (SEQ ID NO.1 / SEQ ID NO.2) were designed according to the target gene sequence to amplify the target gene (amplification product 849bp), and 31 strains were detected positive. The test results showed that the transgenic tobacco plants, like the positive control plasmid, could amplify the specific fragment 849bp of the corresponding target gene, while the non-transformed plants and the transformed plants had no amplified bands ( Image 6 A). Therefore, the res...
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