Developmental related gene BrCRF6 of brassica rapa pistils and application of developmental related gene BrCRF6
A cabbage, gene technology, applied in the application, genetic engineering, plant genetic improvement and other directions, can solve the problem of CRF rarely reported
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Embodiment 1
[0031] Example 1 Overexpression of Chinese cabbage BrCRF6 and construction of suppression expression vector
[0032] 1. Construction of overexpression vector
[0033] (1) Take the pistil tissue sample of Chinese cabbage 'Chiifu-401-42' and use TRIzol reagent to extract total RNA. The synthesis of cDNA is completed with TAKARA reverse transcription kit. The specific method is as follows: 5 × gDNA Eraser Buffer 2 μL, gDNA Eraser 1 μL, RNA 1 μg, RNase Free H 2 Make up to 10 μL with O, remove genomic DNA at 42°C for 2 minutes, add 4 μL of 5×Primer Script Buffer, 1 μL of RT Primer Mix, 1 μL of Primer Script RT EnzymeMix, RNase Free H to the reaction solution in the previous step 2 O 4 μL, after pipetting and mixing, 37°C for 20 minutes, and 85°C for 5 seconds to complete cDNA synthesis, and cDNA was stored in a -20°C refrigerator.
[0034] (2) Using the pistil cDNA of cabbage as a template, use the primers in Table 1 to amplify the target fragment of the BrCRF6 gene through high-...
Embodiment 2
[0040] Embodiment 2 cabbage genetic transformation
[0041] 1. Transfer the vectors pBI121-BrCRF6, pBI121-amiRBrCRF6 and pBI121 empty vectors obtained in Example 1 into Agrobacterium GV3101, the specific method is as follows: take the thawed Agrobacterium competent and mix with 5 μL of plasmid, and ice bath for 10 minutes, React in liquid nitrogen for 5 minutes, 5 minutes in a water bath at 28°C; add 1 mL of liquid LB medium without any antibiotics on a clean bench, and incubate on a shaker at 28°C at 200 rpm for 4 to 5 hours; centrifuge at 10,000 rpm for 1 minute, and discard most of the supernatant , and the remaining about 100 μL of bacteria was resuspended and spread on a solid LB plate containing Rif and Kan; placed in a positive direction at 28°C for 30 minutes, then inverted for 1 to 2 days. After the positive colonies were detected correctly by PCR, the strains were resuspended in 25% glycerol LB and stored at -75°C for later use.
[0042] 2. Prepare culture medium
...
Embodiment 3
[0069] Example 3 Phenotype Observation of Transgenic Chinese Cabbage Plants
[0070]1. In the flowering stage of the transgenic Chinese cabbage plants, it was found that the transgenic Chinese cabbage plants with BrCRF6 overexpression and suppressed expression had nested flower phenotypes, while the control group transgenic Chinese cabbage plants did not appear nested flowers ( Figure 5 , 7 ). Further observations revealed that nested flowers were produced by regenerated inflorescences in pistils ( Figure 6 , 8 ).
[0071] 2. Statistics on the proportion of nested flowers of transgenic plants and the size of floral organs
[0072] After the transgenic plants bolted and bloomed, 10 plants were taken from each line, and the number of nested flowers and normal flowers was counted, and the proportion of nested flowers was calculated ( Figure 9 ). The results showed that the disrupted expression of BrCRF6 could make the transgenic plants produce a certain proportion of nes...
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