Peach dormancy related PpTCP20 gene and application thereof
A gene and gene coding technology, applied in the field of genetic engineering, can solve the problems of long cycle of varieties
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Embodiment 1
[0026] Example 1 Cloning of peach dormancy-related PpTCP20 gene
[0027] 1. RNA extraction and reverse transcription of peach flower buds
[0028] 1. Use the RNAprep Pure Polysaccharide Polyphenol Plant Total RNA Extraction Kit (Tiangen, Beijing) to extract plant RNA, and follow the instructions:
[0029] (1) Take 100 mg of 'Zhongyou No. 4' peach blossom buds, grind them into powder with liquid nitrogen, add 500 μL of Lysis Solution SL (add 25 μL of β-mercaptoethanol to a final volume concentration of 5%), and vortex to mix.
[0030] (2) Centrifuge at 12,000 rpm for 10 minutes.
[0031] (3) Add the supernatant A to the filter column CS, centrifuge at 12,000 rpm for 5 minutes, pour the supernatant B in the collection tube into a new centrifuge tube to avoid absorbing cell debris and precipitation.
[0032] (4) Slowly add absolute ethanol 0.4 times the volume of the supernatant B into the centrifuge tube, mix well and pour into the adsorption column CR3 together, centrifuge at...
Embodiment 2
[0059] Example 2 The expression of peach dormancy-related PpTCP20 gene during the dormancy period of peach flower buds
[0060] The dormant state of peach buds was determined by investigating the germination rate of buds on peach branches after 25 days of cultivation. It is in the natural dormancy stage from October 15 to December 15; it is in the release stage of natural dormancy from November 15 to December 15. From December 30 to January 30 of the next year, the flower bud germination rate exceeds 50%. This stage is the ecological dormancy period.
[0061] The expression of peach dormancy-related PpTCP20 gene in peach flower bud dormancy period is as follows: figure 2 shown.
[0062] Depend on figure 2 The results showed that the expression of peach dormancy-related PpTCP20 gene was up-regulated during the release period of natural dormancy (11th and 15th to December 15th), indicating that peach dormancy-related PpTCP20 gene can regulate the release of natural dormancy...
Embodiment 3
[0063] The acquisition of embodiment 3 transgenic tobacco
[0064] In order to verify the function of peach dormancy-related PpTCP20 gene, the full-length ORF of peach dormancy-related PpTCP20 gene was amplified, connected with PRI-GFP expression vector, and transformed into Agrobacterium GV3101. Adopt Agrobacterium-mediated leaf disk method to transform tobacco, the steps are as follows:
[0065] (1) Tobacco seeds of Nicotiana benthamiana were sterilized with 75% alcohol for 5 minutes, washed once with sterile water, then sterilized with 5% sodium hypochlorite solution for 10 minutes, and washed 5 times with sterile water.
[0066] (2) plant the sterilized tobacco seeds in a sterilized culture bottle, and wait until completely healthy green leaves grow out.
[0067] (3) Select the above-mentioned leaves, remove the petioles and leaf tips, cut into squares, cut a few times continuously in the middle, do not cut off, in the pre-differentiation medium (MS+6-BA 3mg L -1 +NAA 0....
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