Low-temperature resistant plant protein TCF1 (transcription factor1) and encoding genes and application thereof
A technology of LC-35S-GFP-TCF1 and gene, which is applied in the field of plant low temperature resistance protein TCF1 and its coding gene and related transgenic applications, can solve the problems of long period, lagging research on plant cold resistance mechanism, low efficiency, etc., and achieve improvement The effect of cold resistance
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Embodiment 1
[0041] Embodiment 1, the expression characteristic of TCF1 gene and the cloning of TCF1 gene
[0042] 1. Analysis of the expression characteristics of TCF1 gene under stress
[0043] Arabidopsis Col-0 seedlings grown at room temperature for about 12 days were subjected to the following stress treatments:
[0044] (1) Low temperature treatment: put Arabidopsis thaliana Col-0 seedlings in a 4°C incubator, light culture for 0 hours, 1 hour, 3 hours, 6 hours, 12 hours, 24 hours, take them out and freeze them with liquid nitrogen, -80°C Save for later.
[0045] (2) Abscisic acid treatment: Arabidopsis thaliana Col-0 seedlings were placed in 100 μM abscisic acid (ABA) solution, cultured in light for 3 hours, taken out and quick-frozen in liquid nitrogen, and stored at -80°C for later use.
[0046] (3) Salt treatment: Arabidopsis Col-0 seedlings were placed in 150 mM NaCl solution, cultured under light for 5 hours, the materials were taken out, quick-frozen in liquid nitrogen, and ...
Embodiment 2
[0057] Example 2, TCF1 gene deletion mutant tcf1-1 and overexpression TCF1 transgenic plant TCF1OX all improve cold resistance
[0058] 1. Obtained tcf1-1 and TCF1OX transgenic plants
[0059] SAIL Lines seeds from ABRC (Arabidopsis Biological Resource Center) were homozygously identified to obtain a homozygous mutant tcf1-1 with TCF1 gene insertion. Semi-quantitative RT-PCR results showed that tcf1-1 was a nonsense mutant of the TCF1 gene ( image 3 ); the pCAMBIA1300-35S-TCF1 vector was used to transform Arabidopsis thaliana into Col-0 to obtain pure transgenic plants, and the RT-PCR results showed that the TCF1 gene was overexpressed in TCF1OX ( Figure 4 ).
[0060] 2. Both tcf1-1 and TCF1OX transgenic plants showed cold resistance
[0061] In the case of cold treatment without cold acclimation, the survival rates of tcf1-1 after freezing treatment at -6°C, -8°C and -10°C for 2 hours were 91.3%, 52.2% and 17.4%, respectively, while Col-0 (WT ) at the corresponding trea...
Embodiment 3
[0063] Example 3, Analysis of biochemical characteristics of TCF1 protein
[0064] 1. Analysis of subcellular localization of TCF1 protein
[0065] Cut off the leaves of seedlings (cold-acclimated or non-cold-acclimated transgenic homozygous plants) grown in a petri dish for 12 days, put them in a disposable syringe (without a needle), mix the leaves with water, and then block them by hand Needle, pulled several times to make the leaves translucent, put the air hole side up on the glass slide, add a drop of water, cover the cover glass, and put it upside down on the Confocal microscope stage, Mobilize the bright field, and then scan the laser, the excitation light is 488nm; the microscope inspection is the same as above. The results showed that the TCF1 protein was nuclear localized ( Figure 8 ).
[0066] 2. Analysis of GEF Activities
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