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Chinese cabbage B-type response regulatory factor gene BrRR10 and application thereof

A regulator, cabbage technology, applied in the fields of application, genetic engineering, plant gene improvement, etc., can solve the problem of limited function of B-type response regulator

Active Publication Date: 2021-07-02
ZHEJIANG UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, TCS-related genes similar to Arabidopsis have been found in many plants, but the function of B-type response regulators in Chinese cabbage is very limited

Method used

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  • Chinese cabbage B-type response regulatory factor gene BrRR10 and application thereof
  • Chinese cabbage B-type response regulatory factor gene BrRR10 and application thereof
  • Chinese cabbage B-type response regulatory factor gene BrRR10 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1: Construction of cabbage brrr10 subcellular positioning carrier

[0022] 1, plant inflorescence total RNA extraction

[0023] The total RNA was extracted from the Ombet of the Omega Plant RNA Kit Kit from the Chinese Cabbage 'CHIIFU-401-42'. The specific steps were as follows: liquid nitrogen polishing samples were about 100 mg, transferred into 1.5 ml of centrifuge tubes, and immediately added 500 μL RBBUFFER (added β-mercaptoethanol), severe vortex; 14000 rpm centrifugation 5min, taken into GDNAFILTERCOLUMN, 14000 rpm Centrifugal 2min; 0.5 times the volume of anhydrous ethanol, reverse mix; mixed solution into Hibind RNA mini column 1 min for 1 min, abandyed with 400 μlrwf Wash buffer, 1 min, 10000 rpm; 300 μl Rnawash Buffer II, 10000 rpm is centrifuged for 1min, discard the filtrate, repeat 2min, 300 rpm Centrifuge 2 min, discard the filtrate, dry column; In a clean 1.5 ml centrifuge tube, 30 μl DEPC was added to 3 min, 10000 rpm centrifuge for 1min, and column...

Embodiment 2

[0038] Example 2: Construction of cabbage brr10 heterologous expression vector

[0039] The cabbage cDNA is a template amplification gene fragment and adopts a gel recovery fragment (which is shown in Table 2), using homologous recombination method to convert the KPN I and BAM HI double-digested PAC007-3 * Flag vectors to transform Escherichia coli Feeling state DH5α, via a bacterial PCR verifying and sequencing the gene fragment and the connection correctly, the extraction carrier plasmid is preserved - 20 ° C. Specific steps are described in Example 1.

[0040] Table 2 Exhibits for the construction of heterologous expression vector

[0041] Primer name Primer sequence (5'-3 ') Brr10-f Gggcgcccggggtaccatgacattggaacaagatt (SEQ ID NO.4) Brr10-r Atagtccatgatcctatgcatgttctgatgatgcta (seq ID no.5)

[0042] Verify that the alignment of the successful heterologous expression vector plasmid is used to transform the Agrobacterium Agrobacterium in Agrobacterium in A...

Embodiment 3

[0045] Example 3: Screening of Xionguan Method Transforming Arabidopsis and Positive Transformation

[0046] 1, dip gossip in combination

[0047] Take 100 μL of activated heterologous expression vector plasmid ( figure 2 A) and PAC007-3 * FLAG Empty Gasal Agrobacterium liquids were added to 30 mL of RIF, STR, and CMR liquid Lb medium, and shake the bed in 28 ° C. When the bacterial fluid was cultured to OD600 was about 1.0, 8000 rpm was centrifuged for 10min, discarded, resuspended with an equal volume of resuspension (5 wt% sucrose, 200 μl / L Silwet L-77), stirring for 2 min. The wild-type Arabidopsis was removed and the open flowers were immersed in the fungus liquid in the bacterial liquid, and the water absorbed paper was removed with a pulsation of the multifunga liquid, moisturizing Dark culture for 24 hours, and then placed in normal culture. After a week, repeatedly wake up more transgenic seeds at a time.

[0048] 2, formulate hygromycin screening medium

[0049] Take 2...

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Abstract

The invention provides a Chinese cabbage B-type response regulatory factor gene BrRR10 and application thereof, and belongs to the technical field of plant genetic engineering. The DNA sequence of the Chinese cabbage B-type response regulatory factor gene BrRR10 is as shown in SEQ ID No. 1. A BrRR10 heterologous expression arabidopsis thaliana strain is obtained by transforming the gene into Columbia type arabidopsis thaliana through an agrobacterium flower immersion transformation method, and the result shows that the heterologous expression of the Chinese cabbage B-type response regulatory factor gene BrRR10 can lead to the increase of the number of leaves of the arabidopsis thaliana, the decrease of the leaves, the obvious increase of the number of branches and the obvious shortening of the root length of a primary root of the arabidopsis thaliana. The Chinese cabbage B-type response regulatory factor gene BrRR10 plays an important regulation role in leaf development, branch number regulation and primary root development, can be applied to breeding of Chinese cabbage vegetables and other horticultural plants, and has a good application prospect.

Description

Technical field [0001] The present invention belongs to the field of plant gene engineering, specifically, cabbage B-type response regulatory factor gene BRR10, its encoded protein and its application during plant breeding. Background technique [0002] Cabbage (Brassica Rapa L.Syn.b.campeStris L.) belongs to the crops, nutrients, and strong resistance to low temperatures, and has a very high economic value in production. Among them, the cabbage and cabbage are the largest cultivation area and the largest vegetable crops in my country. And it has a relatively close proximal relationship with the model plants of the Cross, and there is also an important research significance in plant basic science. Different crop product organs are different. According to the consumption site, you can separateize the vegetables, stems, leaves, flowers and vegetables, cabbage, and vegetables, which are important roast vegetables. Plants's leaves are more photosynthetic plants, and it is also an imp...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C12N15/82A01H5/00A01H5/06A01H5/12A01H6/20
CPCC07K14/415C12N15/8261
Inventor 余小林周芳园孔李俊赵彤宋建伟
Owner ZHEJIANG UNIV
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