Application of plant stress tolerance-related protein gmstop1 and its encoding gene
A technology of plant stress tolerance and related proteins, applied in the field of application of plant stress tolerance related protein GmSTOP1 and its encoding gene
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Embodiment 1
[0036] Cloning of Example 1 Soybean GmSTOP1 Gene
[0037] The total RNA of the leaves and roots of the soybean variety "Kefeng 1" was extracted according to the instructions of the spin-column plant total RNA extraction kit, and the total RNA was extracted using RT Master Mix Perfect Real Time kit was used for reverse transcription to synthesize cDNA. Primers GmSTOP1-F and GmSTOP1-R were designed according to the cDNA sequence of the Arabidopsis thaliana AtSTOP1 homolog gene Glyma16g27280 in soybean, and the cDNA after reverse transcription of the RNA treated with aluminum stress for 6 h was used as a template to perform gene amplification with high-fidelity enzymes. The target band was recovered by gel cutting, connected to the pMD19-T vector, transformed into Escherichia coli DH5α competent cells, 10 single colonies were randomly picked for expansion culture, and the plasmid was extracted according to the instructions of the plasmid DNA mini kit for PCR identification, and ...
Embodiment 2
[0044] Example 2 Subcellular localization of GmSTOP1 encoded protein
[0045] Use GmSTOP1-GFP-F / GmSTOP1-GFP-R as primers to amplify the GmSTOP1 gene, cut the gel to recover the amplified product, digest the amplified product and pJIT166-GFP plasmid with Sal I and BamH I; The PCR product and the plasmid restriction fragment were connected to construct the vector pJIT166-GmSTOP1-GFP, which was transformed into Escherichia coli DH5α, and then the positive clones were screened by bacterial liquid PCR and enzyme digestion, and the positive clones were sequenced and verified. Plasmids were prepared using an endotoxin-free plasmid extraction kit, and Arabidopsis protoplasts were transformed with reference to the method of Yoo et al. (2007). The cultured protoplasts were detected with a confocal laser microscope for GFP signals. At the same time, a gene gun was prepared to bombard the onion epidermis to observe the transient expression system of GFP.
[0046] where GmSTOP1-GFP-F: 5'-...
Embodiment 3
[0050] Example 3 Fluorescence Quantitative Analysis of Soybean GmSTOP1 Gene Expression
[0051] 3.1 Fluorescent quantitative analysis of GmSTOP1 gene expression in different soybean tissues
[0052] Extract the root tip of the soybean variety "Kefeng 1" 3 days after sand germination, the shoot tip meristem of the 14-day seedling age, the stem and the first compound leaf of the 18-day seedling age, the flower at the full flowering stage, and the green pod at the full-blooming stage and mature seed RNA, reverse transcribed into cDNA, and diluted 10 times as a template, using a two-step method with reference to the operation manual of SYBR Premix Ex Taq II (Perfect Real-time), using soybean ACT11 gene as an internal reference gene, using fluorescence quantification Quantitative analysis of GmSTOP1 gene expression in different tissues by PCR.
[0053] The PCR reaction system is:
[0054]
[0055] Wherein qRT-GmSTOP1-F: 5'-CCTTGCTCCTCATACCCATTTCTG-3'(SEQ ID NO.5)
[0056] qRT...
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