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Gene silence indicating gene and virus induced silencing vector and construction method and infection method

A technology of gene silencing and carrier, applied in the field of molecular biology, can solve the problems that the CHll gene has not been confirmed and reported, and achieve good indication effect, simple construction, and long-lasting effects of traits

Active Publication Date: 2017-07-11
CITRUS RES INST OF CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The CH11 gene of citrus has not yet been confirmed and reported

Method used

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  • Gene silence indicating gene and virus induced silencing vector and construction method and infection method
  • Gene silence indicating gene and virus induced silencing vector and construction method and infection method
  • Gene silence indicating gene and virus induced silencing vector and construction method and infection method

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] The construction of embodiment 1 citrus CH11 gene virus-induced silencing vector

[0039] 1. Extract cDNA from citrus leaves: use cDNA extraction kit PrimeScript TM 1st Strand cDNASynthesis Kit extracts cDNA from citrus leaves.

[0040] 2. Amplification of the citrus CH11 gene

[0041] The amino acid sequence of CH11 was searched from the Arabidopsis genome database, and the homologous gene sequence was obtained after Blast comparison with the genome database of citrus (http: / / phytozome.jgi.doe.gov / pz / portal.html), according to This homologous gene design has the PCR amplification primers CiCHll-V2-F and CiCHll-V2-R of the citrus CHll gene of restriction site, and primer sequence is:

[0042] CiCH11-V2-F: 5'-TCTAGAGGTCTGTGGGACGATTGACAT-3' (SEQ ID No: 2),

[0043] CiCH11-V2-R: 5'-GAGCTCCGAGCTCTCTCCTCCACAATC-3' (SEQ ID No: 3).

[0044] Using citrus leaf cDNA as a template, using primers CiCHll-V2-F and CiCHll-V2-R, using Ex (Refer to the instructions for the PCR re...

Embodiment 2

[0049] Embodiment 2 prepares the multiplication bacteria liquid

[0050] Take 0.5 μL of TRV1, TRV2-GFP, and TRV2-CiCH11 plasmids, respectively, and transform them into 20 μL of EHA105 Agrobacterium competent cells by electric shock method, and spread them on LB solid medium (containing 50 mg / L kanamycin, 50 mg / L L gentamicin and 50mg / L rifampicin), cultured in the dark at 28°C for 2-3 days. Pick the single clone that contains TRV1, TRV2-GFP, TRV2-CiCH11 Agrobacterium to LB liquid culture medium (50mg / L contains kanamycin, 50mg / L gentamycin and 50mg / L rifampicin), place Cultivate overnight at 200r / min on a shaker at 28°C, collect Agrobacterium cells, and use MMA (containing 10mM MgCl 2 , 10mM 2-morpholineethanesulfonic acid (MES), 100μM acetosyringone (acetosyringone) liquid resuspended cells, adjusted OD600 to above 0.5. Add an equal volume of TRV1 resuspension to the TRV2-GFP and TRV2-CiCH11 resuspensions respectively, then add Silwet L-77 surfactant with a volume of 0.05-0...

Embodiment 3

[0051] Example 3 Dip Dyeing

[0052] Use the control group prepared in Example 2 and the resuspension of the treatment group to dip different citrus seedlings respectively, then detect whether the infection is successful, and operate according to the following steps:

[0053] (1) Immerse the citrus seedlings in the resuspension solution, and carry out dipping under vacuum conditions for 10s-30mins. Or inject the resuspension into the leaves with a sterile syringe.

[0054] (2) After rinsing the dipped part with clear water, move the seedlings to a light incubator for cultivation under the conditions of 19-28° C., 16 hours of light during the day, 8 hours of darkness at night, and 50-95% humidity for cultivation.

[0055] (3) Detection of infection results

[0056] A, the citrus seedlings infected with the control group and the treatment group can detect green fluorescence, and the fluorescence observation of the treatment group seedlings is as follows: figure 1 shown.

[0...

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Abstract

The invention discloses an indicating gene used in a TRV-mediated gene silencing system, the nucleic acid sequence of the indicating gene is as shown in SEQIDNo:1, and the invention discloses a virus induced silencing vector comprising the sequence and a construction method thereof and an infection method of citrus seedlings by use of the virus induced silencing vector. The effective indicating gene is provided for the TRV-mediated virus induced gene silencing system, the TRV-mediated gene silencing system is firstly successfully established in citrus fruits, and the indicating gene in the citrus fruits is successfully silenced. The silencing system constructed by the silencing vector has the advantages of simple operation and high conversion efficiency, can rapidly carry out functional verification of the gene in the citrus fruits, and can effectively inhibit the expression of endogenous genes in the citrus fruits, and the phenotype is remarkable. The silencing vector provides a reliable technique for rapid and effective performing of gene function verification in the citrus fruits.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to a gene silencing indicator gene, a virus-induced silencing vector, and a construction and infection method thereof. Background technique [0002] Citrus is a perennial woody plant. The gene function has been verified through conventional genetic transformation technology, but it has been troubled by low genetic transformation rate, long regeneration cycle and long childhood. Virus-induced gene silencing (VIGS) is a powerful tool to inhibit the expression of endogenous genes. It has been used in potato (Solanum tuberosum) , Nicotianabenthamiana, Arabidopsis thaliana, tomato, rice (Oryza sativa) and corn (Zeamays) and other gene function research. Currently, multiple VIGS vectors are used to resolve metabolic pathways, plant development, and biotic and abiotic stresses. In recent years, a variety of VIGS vectors have been developed and widely used in various...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C12N15/11C12N15/83C12N15/66A01H5/00C12Q1/68
CPCC07K14/415C12N15/66C12N15/8269C12Q1/6895
Inventor 王福生赵晓春申晚霞刘小丰朱世平
Owner CITRUS RES INST OF CHINESE ACAD OF AGRI SCI
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