Sophora alopecuroides aquaporin and encoding gene and application thereof
A technology of aquaporin and bitter bean, which is applied in the field of genetic engineering, can solve problems such as affecting the growth and development of crops, making it difficult to exert genetic potential, and reducing crop yields.
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Embodiment 1
[0032] Example 1 Screening and Cloning of SaAQP Gene of Sophora Sophora
[0033] Select 10 g of full-grained Sophora sophora seeds, add 5 ml of concentrated sulfuric acid with a concentration of 98%, and soak for 20 minutes. Wash the seeds and sow them in potted soil. The culture conditions are: 16 hours of light, temperature of 26°C, humidity of 65%, light intensity of 30000 lux. Four weeks after germination, the seedlings were transferred to NaCl concentrations of 200 mmol, Na 2 CO 3 The concentration is 140mmol and PEG6000 concentration is 8% in hoagland nutrient solution, respectively, for 3h, 12h, 24h, 72h. The roots of Sophora sophora under different treatments were taken, and the total RNA of Sophora sophora roots under different treatment conditions was extracted respectively. The RNA extracted from the 4 treatments of Sophora japonica roots was taken, and the samples of each group were mixed with equal mass for cDNA library construction. The constructed cDNA libr...
Embodiment 2
[0042] Example 2 Tissue-specific expression of SaAQP gene in Sophora sophora
[0043] Sophora Sophora is carried out NaCl salt stress treatment, and the treatment method is the same as in Example 1. The processing time is 1h, 2h, 4h, 8h, 12h, 24h, 48h respectively. The roots, stems and leaves of Sophora sophorae cultivated in hoagland nutrient solution were taken at the same time. The total RNA of the processed material was extracted with reference to the column type plant total RNA extraction and purification kit of Sangon Company, and the integrity of the RNA was detected by 1% agarose electrophoresis. cDNA was synthesized according to Reverse Transcriptase M-MLV (RNase H - ) instructions. Real-time fluorescence quantitative PCR was used to detect the expression of SaAQP gene in different tissues and roots of Sophora sophorae in different salt treatment time. The experimental operation was carried out in a real-time fluorescent quantitative PCR instrument ABI 7500 accord...
Embodiment 3
[0051] The expression of embodiment 3SaAQP in soybean and the analysis to salt tolerance
[0052] The plant expression vector pCHF-1301-SaAQP was constructed. The plant expression vector pCHF-1301-SaAQP( Figure 5 ) to transform soybean Jilin 35, and detect the expression of GUS in the hairy roots of the transgenic soybeans, detect the expression level of the target gene in the positive plants at the same time, and analyze the salt tolerance of the hairy roots of the transgenic soybeans. The specific methods and results are as follows:
[0053] 3.1 Genetic transformation and screening of soybean hairy roots
[0054] 1) Processing of plant material
[0055] Select plump, disease-free soybean Jilin 35 seeds, place them flat in a petri dish, place the petri dish in a closed desiccator, and put a beaker with 96mlNaClO and 4ml concentrated HCl in the desiccator, ClO 2 Sterilize for 14-h16h.
[0056] 2) Germination of soybean seeds
[0057] Take the above-mentioned sterilized ...
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