Stress inducible promoter, stress inducible promoter plant expression vector and expression method of induced target gene

[0024] Example 1

[0025] This example is about Brachypodium BdDREB-47 Cloning of gene promoters.

[0026] 1.1 Preparation of genomic DNA of Brachypodium

[0027] The leaves of normally growing Brachypodium bismuth were extracted by CTAB method, the concentration and quality of the extracted DNA were detected by ultra-trace nucleic acid protein analyzer Q5000, and the DNA concentration was diluted to 100ng/μl for use.

[0028] 1.2 Cloning of promoters

[0029] Using the above-extracted Brachypodium edodes DNA as a template, using the primers of SEQ ID NO: 2 and SEQ ID NO: 3, PCR amplification was performed to obtain the sequence of SEQ ID NO: 1.

[0030] The PCR reaction system is as follows:

[0031] reagent

[0032] PCR reaction program: pre-denaturation at 98°C for 5 min; denaturation at 98°C for 30s, annealing at 55°C for 20s (annealing temperature increased by 0.5°C for each cycle), extension at 72°C for 90s, and amplification for 20 cycles; denaturation at 98°C for 30s, annealing at 65°C for 20s , 72 ℃ extension for 90s, amplification for 15 cycles; 72 ℃ extension for 10min; 4 ℃ incubation.

[0033] Pipette 10 μl of the above PCR products for 1% agarose gel electrophoresis detection. For the specific results, see figure 1 , where the Marker is DL2000, and the target band size is 1388bp.

[0034] Connect the above-mentioned remaining PCR products to T carrier, the present invention uses Takara Prime Star Taq high-fidelity enzyme, the amplification product is blunt end, and the T carrier is pEASY-Blunt Cloning Vector (purchased from Beijing Quanshijin Biotechnology Co., Ltd.), according to The kit was operated according to the instructions of the kit, and the ligation system was 5 μl (4 μl PCR product + 1 μl T vector).

[0035]The above ligation product was transferred into Escherichia coli competent cells Trans-T1 (purchased from Beijing Quanshijin Biotechnology Co., Ltd.) by the heat shock method, and the specific operation method can be found in the kit instruction manual. Single clones were picked for colony PCR detection, and positive clones were selected for sequencing. The sequencing results showed that the amplified sequence was completely consistent with SEQ ID NO:1.

[0036] The clones with correct sequencing were amplified, and the plasmids were extracted with a plasmid mini-extraction kit (purchased from Tiangen Biochemical Technology Co., Ltd.), and the concentration of the extracted plasmids was detected by an ultra-micro nucleic acid protein analyzer Q5000.

[0037] Embodiment two

[0038] This example is about the construction of a stress-inducible promoter (pBdDREB-47) plant expression vector.

[0039] use Eco RI and Hin dⅢ (purchased from NEB Company) double-digested the recombinant plasmid pBdDREB-47-T and the vector pCAMBIA1381-GUS, see figure 2 , respectively recovered the target fragment (1388bp) and the pCAMBIA1381-GUS vector fragment, connected them with T4 DNA ligase (purchased from NEB Company), transferred them into E. coli competent cells Trans-T1 by heat shock method, and picked a single clone for positive See results after testing image 3 , amplify and extract the plasmid, and further digest and identify the positive plasmid ( EcoR I and Hin dⅢ double enzyme digestion) see the results Figure 4 , and the sequencing results are correct, indicating that the plant recombinant expression vector pC1381-pBdDREB-47-GUS was successfully constructed, and the pC1381-pBdDREB-47-GUS recombinant plasmid can be further transformed into Agrobacterium competent cells EHA105 (purchased in Shanghai Shifeng Biotechnology Co., Ltd.), positive test results such as Figure 5 As shown, it can be used for subsequent plant transgenesis after being correct.

[0040] The schematic diagram of constructing the plant expression vector containing pBdDREB-47 promoter is as follows Figure 7 As shown, 35S Pro and pBdDREB-47 are promoters, 35S Ter and Nos Ter are terminators, the selection marker is hygromycin resistance, and the GUS gene is a reporter gene. The expression of GUS gene can be used to judge pBdDREB-47 Promoter activity.

[0041] Embodiment Three

[0042] This example is about transforming tobacco with pC1381-pBdDREB-47-GUS recombinant plasmid through Agrobacterium-mediated method.

[0043] 3.1 Preparation of transgenic recipient material

[0044] Seeds of wild-type tobacco were sown in flower pots and placed in a 25-degree cultivation room (12h light/12h dark), covered with plastic wrap to keep moisture, and the plastic wrap was gradually removed after the seedlings emerged. Two weeks later, the plants were transplanted, and when the tobacco was 2 months old, the leaves could be used for transgenic experiments.

[0045] 3.2 Disinfection and pre-cultivation of tobacco leaves

[0046] Tobacco leaves with good growth conditions were selected, and the tender leaves were cut off with scissors, and the tobacco leaves were soaked and sterilized with 75% ethanol for 50 seconds on the ultra-clean workbench. Then soak and sterilize with 2.5% sodium hypochlorite solution for 10 minutes, shake continuously during this period to fully sterilize, and finally wash the tobacco leaves 5 times with sterile water.

[0047] Put the sterilized tobacco leaves on sterile filter paper to fully absorb the surface moisture, and cut the tobacco leaves into about 0.5×0.5 cm with a sterile blade 2 The small pieces (note avoiding the leaf veins), the cut leaves were spread on the tobacco co-cultivation medium (MS+6-BA (2 mg/L)+NAA (0.2 mg/L)+Agar (8 g/L) + sucrose (30g/L) pH 5.8), pre-cultured for 3 days at 25°C in the dark.

[0048] 3.3 Preparation of Agrobacterium liquid

[0049] Streak the Agrobacterium liquid containing the pC1381-pBdDREB-47-GUS recombinant plasmid on an LB solid plate containing 50mg/L kan, culture in the dark at 28°C for 1 day, resuspend in 50ml MS liquid medium, and store at 28°C Shake at 200r/min and shake for 30min, and proceed to the next experiment immediately.

[0050] 3.4 Agrobacterium infection

[0051] Tobacco leaves pre-cultivated for 3 days were placed in fully shaken Agrobacterium bacteria solution, soaked for 10 minutes, and shaken constantly during the period. After the infection is complete, put the leaves on sterile filter paper to fully absorb the residual bacterial solution on the leaves, and then disperse the infected leaves on the co-culture medium covered with sterile filter paper, and cultivate in the dark at 25°C for 3 days .

[0052] 3.5 Differentiation and rooting of tobacco leaves

[0053] Tobacco leaves after dark culture for 3 days were transferred to differentiation medium (MS+ 6-BA (2 mg/L) + NAA (0.2 mg/L) + Cb (500 mg/L) + Kan (100 mg/L) + Agar (8 g/L) + sucrose (30 g/L) pH 5.8), cultured at 25°C under 12h light/12h dark conditions until differentiation and budding.

[0054] When the buds grow to 2~3 cm, cut off the buds and insert them into the rooting medium (1/2MS+ Kan (100 mg/L) + Cef (250 mg/L) + Agar (8 g/L) + Sucrose (30 g/L, pH 5.8), cultured at 25°C under 12h light/12h dark conditions. Generally, the explants can take root after two weeks, and when the root system grows more developed, they are transplanted into flower pots and continued to be cultivated in the greenhouse. Extract the genomic DNA of the transgenic T0 tobacco, and use the GUS gene primers for positive detection. For the detection results of some plants, see Image 6 , SEQ ID NO:4 and SEQ ID NO:5 are GUS gene PCR detection primers.