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Chinese cabbage pistil development related gene BrCRF11a and application thereof

A cabbage and genetic technology, applied in the fields of application, genetic engineering, plant genetic improvement, etc., can solve the problems of CRF that are rarely reported

Active Publication Date: 2019-03-29
ZHEJIANG UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the participation of CRF in the regulation of many cytokinins in plants has been found, but the role of CRF in flower development is rarely reported

Method used

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  • Chinese cabbage pistil development related gene BrCRF11a and application thereof
  • Chinese cabbage pistil development related gene BrCRF11a and application thereof
  • Chinese cabbage pistil development related gene BrCRF11a and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 Overexpression of Chinese cabbage BrCRF11a 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 obtain the target fragment of the BrCRF11a gene through hi...

Embodiment 2

[0040] Embodiment 2 cabbage genetic transformation

[0041] 1. Transfer the vectors pBI121-BrCRF11a, pBI121-amiRBrCRF11a and pBI121 empty vector obtained in Example 1 into Agrobacterium GV3101, the specific method is as follows: take the thawed Agrobacterium competent and mix it with 5 μL of plasmid, and put it in an 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. Prepar...

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 overexpression and suppressed expression of BrCRF11a all had the phenotype of nested flowers, while the transgenic Chinese cabbage plants of the control group 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 BrCRF11a could make the transgenic plants produce a c...

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Abstract

The present invention provides a Chinese cabbage pistil development related gene BrCRF11a and an application thereof, and belongs to the technical field of plant genetic engineering. A DNA sequence ofthe Chinese cabbage BrCRF11a is shown as SEQ ID No. 1 and a gene artificial miRNA sequence is shown as SEQ ID No. 2. The gene and the artificial miRNA designed for the gene are respectively transferred into brassicae parachinensis bailey, var. Youqing-Siqiu by an agrobacterium-mediated method to obtain transgenic brassicae parachinensis strains with overexpression and inhibition expression of BrCRF11a, expression changes of Chinese cabbage BrCRF11a enable the brassicae parachinensis to show a phenotype of nested flowers and the phenotype is caused by continuous maintenance of stem cell activity by floral meristem. The Chinese cabbage BrCRF11a is closely related with pistil development. The gene is applied in Chinese cabbages and other horticultural plant breeding and has good applicationprospects.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering, and specifically relates to Chinese cabbage BrCRF11a, its encoded protein, its artificial miRNA and its application in flower development. Background technique [0002] Chinese cabbage (Brassica rapa L.syn.B. campestris L.) belongs to Brassicaceae Brassica Brassica crops, mainly including heading cabbage, non-heading cabbage, turnip, cabbage sprouts, laver sprouts, sprouts, There are 8 varieties such as Wutaicai and Japanese Mizuna. They are not only important vegetables and oil crops with high economic value in production, but also have a close relationship with the Brassicaceae model plant Arabidopsis thaliana, and have important research significance in basic plant science. The normal development of flowers is the basis of sexual reproduction of angiosperms, and is closely related to the quality and quantity of seeds of plants. The research on the mechanism of flower develo...

Claims

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Application Information

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IPC IPC(8): C12N15/29C07K14/415C12N15/113C12N15/82A01H5/02A01H6/20
CPCC07K14/415C12N15/113C12N15/8218C12N15/827C12N2310/141
Inventor 余小林孔李俊缪黎明赵坤邓航
Owner ZHEJIANG UNIV
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