Specific promoter for growing point of cotton, cloning thereof and application thereof
A promoter and growth point technology, applied in the fields of application, botany equipment and methods, angiosperms/flowering plants, etc., can solve problems such as energy waste, normal plant growth and development burden, etc.
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Embodiment 1
[0022]The implementation steps of the present invention are to clone the promoter sequence of the gene according to the DNA sequence of the cotton GhMPK7 gene by reverse PCR method → promoter cis-acting element analysis and function prediction → design with enzyme cleavage sites HindIII and BamH I Amplify the target promoter sequence from the DNA with the primers → Digest pBI121 with the same enzymes HindIII and BamH I → Ligate the target fragment and digested pBI121 → Transform Arabidopsis → GUS histochemical staining to detect GUS activity and analyze the promoter Features. All other technologies in this embodiment adopt existing technologies. Cloning of embodiment 1 cotton GhMPK7 gene promoter
[0023] 1.1 Genomic DNA extraction
[0024] 1. Add 40μl β-mercaptoethanol to 5ml extraction buffer (100mM Tris, 500mM NaCl, 50mM EDTA, used at 4°C after autoclaving), and ice-bath;
[0025] 2. Weigh 1g of young leaves, grind in liquid nitrogen, quickly transfer to the extraction...
Embodiment 2
[0052] After the reaction, 1% agarose gel electrophoresis detection, the target fragment size is correct, recover the target fragment, such as figure 1 . Construction and Transformation of Embodiment 2 Plant Expression Vector
[0053] 2.1 Fusion of cotton GhMPK7 gene promoter sequence and GUS reporter gene
[0054] Select pBI121 as the plant expression vector, digest the target fragment obtained by PCR amplification, electrophoresis and gel recovery. At the same time, the expression vector was digested with restriction endonucleases HindIII and BamHI, and the vector fragment was recovered. The gene fragment and the vector fragment were connected, and the pBI121-GhMPK7 promoter was screened by colony PCR and plasmid digestion. The specific process can refer to the attached image 3
[0055] 1. Using primers Sz1 and Szx1 with restriction sites to amplify the promoter sequence of the cotton GhMPK7 gene, and gel electrophoresis to separate and recover the target fragment.
...
Embodiment 3
[0080] Example 3 GUS activity histochemical staining of transgenic plants at different stages
[0081] 3.1 GUS active histochemical staining method:
[0082] 1. Place the sample in an ice-water mixture for two minutes;
[0083] 2. Dry the sample with filter paper, put it into a staining bottle, add 90% acetone and soak the sample for 10 minutes;
[0084] 3. Use the buffer to wash it quickly and discard it;
[0085] 4. Add GUS staining solution to rinse twice, each time for 5 minutes, and shake gently;
[0086] 5. Pump air twice for 10 minutes;
[0087] 6. Store overnight at a constant temperature of 37°C. Observe with a microscope.
[0088] 3.2 GUS staining buffer formula
[0089] 100ml buffer:
[0090]
[0091] wxya 2 O was adjusted to 100ml.
[0092] 3.3GUS staining solution formula
[0093] Dissolve 100mg of X-GluC with 800μl DMSO, add GUS staining buffer to make the volume to 100ml.
[0094] 3.4 Histochemical staining of GUS activity on transgenic plants at d...
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