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Silence vector for silence of Gprotein beta subunit coding gene CsGbeta2 of Ciboria shiraiana as well as application and method thereof

A technology that encodes genes and β subunits, applied in the field of genetic engineering, can solve the problems of long breeding cycle, limited improvement of plant resistance, and large differences in tolerance of pathogenic bacteria, achieving good silencing effect, continuous antibacterial effect, and stable genetic effect

Pending Publication Date: 2021-11-02
SOUTHWEST UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] Due to the shortcomings of traditional breeding methods such as long cycle, large difference in tolerance to pathogenic bacteria, and limited improvement of plant resistance, the present invention uses host-induced gene silencing technology to significantly improve the resistance of transgenic tobacco to the CsGβ2 sequence of C. The resistance of C. mulberry, and provides an effective target for the control of the bacterium

Method used

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  • Silence vector for silence of Gprotein beta subunit coding gene CsGbeta2 of Ciboria shiraiana as well as application and method thereof
  • Silence vector for silence of Gprotein beta subunit coding gene CsGbeta2 of Ciboria shiraiana as well as application and method thereof
  • Silence vector for silence of Gprotein beta subunit coding gene CsGbeta2 of Ciboria shiraiana as well as application and method thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] The present invention passes the nucleotide sequence of the mulberry cup csgβ2 by the local saunamer genome data comparison by NCBI retrieval, gray, and brewing yeast. According to the sequence of the genome, the primers CSGβ2-F / R, the specific primers are as follows:

[0028] CSCSGβ2-F: 5'-AtgggggaacaCTTATCCT-3 '(SEQ ID No.3);

[0029] CSCSGβ2-R: 5'-TtacgctctCgacataAcC-3 '(SEQ ID No.4);

[0030] PCR amplification was carried out by saunasian cup cDNA as a template, and the product was connected to the PMD19-T vector after recovery, and the CSGβ2 nucleotide sequence was obtained after segmentation, such as SEQ ID NO.1.

Embodiment 2

[0032] Selecting a specific sequence of about 300 bp on CSGβ2 to construct a silencing vector targeting the sullen-solid cup CSGβ2, the selected silencing fragment SICSGβ2, such as SEQ ID NO.2, a silencing fragment of the host induced gene silencing carrier constructed SACSGβ2 Enzymatic plasmid KPNI and XHOI enzyme digestion, the intermediate carrier PHANNIBAL-SICSGβ2-F was obtained, and then SiCSGβ2 was subjected to a BamHI and Hindш-Sichase digestion of the PHANNIBAL-SCSG [beta] 2-F plasmid to give the carrier PHANNIBAL-SICSGβ2-FR.

[0033]The primitive to silencing fragment amplification primers is as follows:

[0034] SICSGβ2-F: 5'-ccgctcgagacatcaaccggtcaccatc-3 '(SEQ ID No.5);

[0035] SICSGβ2-R: 5'-cggggtacctgttatctgtgtaacctGCA-3 '(SEQ ID No.6);

[0036] The reverse silent fragment amplification primer is as follows:

[0037] SICSGβ2-F1: 5'-cgcgatccacatcaaccggtcaccatc-3 '(SEQ ID No.7);

[0038] SICSGβ2-R1: 5'-cccaagcttgttatctgtgtaacctGCA-3 '(SEQ ID No.8).

[0039] Subsequent...

Embodiment 3

[0048] The silent expression vector PCAMBIA1300-SicSGβ2 was used to obtain a corresponding interference strain using the protoplast transformation method. Wild-type tobacco was cultured at 25 ° C, 16H light / 22 ° C for 8h dark incubator for 40 days, followed by the same leaf, and the size of the blade was placed in a petri dish with wet sterile filter; Flat fungus (wild type, no-load, three CSGβ2 interference strains), using a yellow gun head punched in the edge of mycelium, inoculation of the same size in a 25 ° C incubator on tobacco, after inoculation 48 hours a photo, the result is like figure 1 Indicated.

[0049] The final silencing expression vector Pbin19-SiCSGβ2 was transferred into root cancer LBA4404 by chemical transformation, and positive transformants were identified by the bacterial liquid PCR. The detection primer is as follows:

[0050] KAN-F: 5'-ggtgccctgaatgaactgca-3 '(SEQ ID NO.13);

[0051] KAN-R: 5'-GGTAGCCAACGCTATGTCCT-3 '(SEQ ID NO.14).

[0052] Such as fi...

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Abstract

The invention discloses a silence vector for silence of a Gprotein beta subunit coding gene CsGbeta2 of Ciboria shiraiana as well as application and a method thereof. The nucleotide sequence of the Gprotein beta subunit coding gene CsGbeta2 of Ciboria shiraiana is as shown in SEQ ID NO. 1; a vector containing a silent fragment of a Gprotein beta subunit coding gene CsGbeta 2 of the Ciboria shiraiana is expressed in a plant by utilizing a host-induced gene silencing technology, and the resistance of the transgenic plant to the Ciboria shiraiana can be remarkably improved by identifying the pathogenicity of the Ciboria shiraiana, therefore, the method can be used for improving the resistance of the plant to the Ciboria shiraiana and preventing and treating the infection of the Ciboria shiraiana.

Description

Technical field [0001] The present invention relates to the field of genetic engineering, and more particularly to the silencing carrier of the silent mulberry mulberry bump G protein β subunit encoding gene CSGβ2, and also relates to the application and method of the carrier. Background technique [0002] Mulberry (Morus Alba L.) is an important economic forest in China. It not only produces mulberry leaves, but also obtain silkworm woven silk silk, but also produces mulberry. Mulberry fruit meat, taste sweet, rich in amino acids, vitamins, flavonoids, and anthocyanins rich in human body, have been listed by the Ministry of Health as one of the agricultural products of "medicine and food", with high food supplement value. In the fruit smell planting, mulberry is extremely easy to explosive disaster disease - "mulberry nucleus". Among them, mulberry hypertrophic psychosis is a mulberry pathogen that is the largest and wide range of mulberry. At present, the prevention and control...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/84C12N15/113C12N15/31A01H5/00A01H6/82A01H6/00
CPCC12N15/8218C12N15/8282C12N15/113C07K14/37C12N2310/14
Inventor 赵爱春朱攀攀张帅李若兰刘长英范伟夏中强马舒煜向仲怀
Owner SOUTHWEST UNIVERSITY
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