Application of FveCHA1 gene and RNAi vector in promoting strawberry callus formation and in vitro bud regeneration
A technology of RNA interference and gene, applied in application, genetic engineering, plant genetic improvement, etc.
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Embodiment 1
[0020] Example 1: Cloning of FveCHA1 gene
[0021] Find the FveCHA1 gene corresponding to forest strawberry in GDR (https: / / www.rosaceae.org / ), mainly based on the full-length cDNA of Arabidopsis CHA1 gene (sequence As shown in SEQID NO.1), (1840bp, the sequence is shown in SEQ ID NO.2, the amino acid sequence encoded by the FveCHA1 gene is shown in SEQID NO.3), the total RNA of the wild-type forest strawberry variety Ruegen was extracted, reversed Recorded as cDNA, the CDS sequence of the forest strawberry is obtained from the GDR website (the download URL is ftp: / / ftp.bioinfo.wsu.edu / species / Fragaria_vesca / Fvesca-genome.v2.0.a2 / genes / ) with wild The cDNA of FveCHA1 gene was amplified by PCR as a template, and the forward fragment was inserted. The primers SEQ ID NO.4 and SEQ ID NO.5 were designed with Snap gene software to amplify the forward target fragment of the gene. The PCR reaction system is shown in Table 1.
[0022] Table 1:
[0023]
[0024] The primer sequenc...
Embodiment 2
[0035] Embodiment 2: the construction of GN2300-FveCHA1-RNAi carrier
[0036] (1) Connection of forward fragments
[0037] Using the One Step Cloning Kit (Vazyme) recombinant cloning kit, Sal I was selected as the restriction site, and on the GN2300-35S-FAQ-NOS vector, the cloning vector was linearized with restriction endonucleases and placed at 37 ℃ water bath, react for 4 hours. The reaction system is as shown in Table 2:
[0038] Table 2:
[0039]
[0040] After digestion, inactivate at 65°C for 20 minutes, and use for recombination reaction.
[0041] Prepare the reaction system in an ice-water bath, as shown in Table 3:
[0042] table 3:
[0043]
[0044]
[0045] After preparing the above mixed reaction solution, place it in a water bath at 37°C for 30 minutes. Cool the reaction solution on ice for 5 min;
[0046] (2) Connection of reverse fragments
[0047] Select XbaI as the enzyme cutting site, connect the reverse fragment to the GN2300-35S-FAQ-NOS ca...
Embodiment 3
[0057] Example 3: Transformation of strawberries for functional verification
[0058] (1) Preparation of explants
[0059] For the explants, young leaves of forest strawberry ecotype Ruegen were selected.
[0060] Use tweezers to peel off the seeds of Ruegen's fruit, put them on filter paper, and after air-drying, pick about 200 plump seeds and put them in a 2ml centrifuge tube.
[0061] Perform the following operations on the ultra-clean bench: add 1ml of 75% alcohol to the centrifuge tube, shake it upside down for about 5 minutes, and suck out all the alcohol. Then add 1ml of 10% sodium hypochlorite (sodium hypochlorite needs to be stored away from light), shake for 8 minutes and suck out the sodium hypochlorite. Add sterile water to the centrifuge tube and wash it 7-8 times to remove sodium hypochlorite and avoid residues. After cleaning, pour the seeds on sterile filter paper, place 4-5 seeds in each tissue culture bottle containing MS solid medium with sterile cooled t...
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