Soybean leaf specific promoter SRS4 and application thereof
A specific, promoter technology, applied in the direction of application, angiosperm/flowering plants, DNA/RNA fragments, etc., can solve the problems of breaking the metabolic balance of plants, hindering the normal growth of plants, and being toxic
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Embodiment 1
[0019] Example 1 Extraction of soybean "Jinong 13" genomic DNA
[0020] Seeds of soybean "Jinong 13" were sown in a nutrient bowl filled with sand, and placed in an RXZ intelligent artificial climate incubator for cultivation. The seeds were cultured in dark before germination, and cultured in Hoagland nutrient solution after germination, at 25°C during the day, 16°C at night, 16h / d of light, light intensity of 3000lx, and relative humidity of 75%. The young leaves of soybean seedlings grown for 2 weeks were taken according to the AxyPrep genome DNA mini-extraction kit to extract soybean "Jinong 13" genomic DNA;
[0021] Measure the absorbance of DNA at 260nm and 280nm with an ultraviolet spectrophotometer, calculate the DNA concentration and purity, and obtain the DNA concentration as OD 260 / OD 280 =1.865, the purity is 850ng / μl;
Embodiment 2
[0022] Example 2 Cloning of soybean rbcS gene SRS4 promoter 5' flanking sequence
[0023] The 5' flanking sequence of soybean rbcS promoter was cloned by TAIL-PCR, and the operation procedure was referred to the method of Liu Yaoguang et al.
[0024] Source of primers: Three specific primers SP1 and SP2 with higher annealing temperature (60°C-65°C) were designed according to the upstream DNA sequence of the coding region of soybean RuBP carboxylase small subunit gene (SRS4) registered in NCBI (GeneBank accession: M16889) , SP3; the uniquely designed amalgamation primer AD2 with a lower annealing temperature (40°C-45°C) is kept in our laboratory (Table 1).
[0025] Table 1: Primers and base composition
[0026]
[0027] Using soybean "Jinong 13" genomic DNA as a template, three rounds of PCR amplification were carried out with specific nested primers SP1, SP2 and SP3 and annexation primer AD2 respectively; the first-round PCR product was diluted 50 times as the second-round...
Embodiment 3
[0030] Example 3 Obtaining of soybean rbcS gene SRS4 promoter SRS4 (1538bp)
[0031] Design specific primers according to the sequences obtained by TAIL-PCR and known sequences:
[0032] SS: 5`-GTGGATGACTCAAGTGCTGG-3`
[0033] SA: 5`-TCATTGAGGAAGCCATTTGC-3`
[0034] Using soybean "Jinong, 13" genomic DNA as a template, using SS and SA as primers to amplify the target gene, and through 1% agarose gel electrophoresis detection, the results show that there is a single specific band at about 2000bp ( figure 2 ), which is consistent with the expected theoretical value.
[0035] The PCR product was recovered with a DNA gel recovery and recovery kit; the purified product was connected to the pMD18-T vector, transformed into E.coliJM109 competent cells, screened for Amp and blue and white spots, and the recombinant plasmid was selected for PCR and enzyme digestion identification ( image 3 ) positive clones were sent to Shanghai Sangon for sequencing; the analysis of the sequencin...
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