A lily susceptibility fungal gene lrwrky-s1 and its application

A technology of lrwrky-s1 and fungus, applied in the fields of application, genetic engineering, plant gene improvement, etc., to achieve the effect of improving lily disease resistance and good application prospects

Inactive Publication Date: 2021-08-03
YANGTZE NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are few reports on the susceptibility genes of fungi in Lily of Minjiang River

Method used

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  • A lily susceptibility fungal gene lrwrky-s1 and its application
  • A lily susceptibility fungal gene lrwrky-s1 and its application
  • A lily susceptibility fungal gene lrwrky-s1 and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1 Cloning and Sequence Analysis of Laijiang Lily LRWRKY-S1 Gene

[0025] With the material of Lijiang lily blade, use Trizol TM Plus Rna Purification Kit (12183555, Invitrogen TM The total RNA is extracted according to the instruction step, and the residual micro DNA is removed by DNase I (18047019, InvitroGENTM), and the concentration of RNA is measured, and the concentration of RNA is measured.

[0026] Take the total RNA of the Lijiang Lancang Lijiang Lijiang Lani, synthesized the first chain cDNA according to the Primescript II First-Strand CDNASYNTHESIS KIT (6210A, TAKARA).

[0027] PCR amplification system: 2xFast PFU Master Max 10UL, forward primer (lrwrky-S1-1-F, 10 μm) 1 μL, reverse primer (lrwrky-S1-R, 10 μm) 1 μL, Template (cDNA) 1 μL, sterile DDH 2 O Adhesive to 20 μL.

[0028] The forward primers and reverse primers are:

[0029] LRWRKY-S1-F: 5'-gtccgaatatcatgggagag-3 '

[0030] LRWRKY-S1-R: 5'-ctacaacatttaaacgaagaaggcag-3 '

[0031] The PCR reaction pr...

Embodiment 2

[0035] Example 2GFP fusion expression vector construction and subcellular positioning analysis

[0036] (1) Construction of PTF101-P35S :: GFP-LRWRKY-S1 expression vector

[0037] According to the PTF101-GFP carrier sequence and the full length sequence (SEQ ID NO.1), the forward primer (lrwrky-S1-INF-F1) and the reverse primer (LRWRKY-S1-INF-R1) are designed. The positive cloned massage of the TA connected in Example 1 was a template, and the PCR amplification of the LRWRKY-S1 gene fragment was performed.

[0038] The primer sequence is as follows:

[0039] LRWRKY-S1-INF-F1:

[0040] 5'- ATGGACGGAGGCTGGGGAGAG-3 '

[0041] LRWRKY-S1-INF-R1:

[0042] 5 ' TTAAACGAGAGGCAGAGGCATCG-3 '

[0043] The underscore is a sequence of in-fusion cloned carrier joints, and the bold sequence is the enzyme sink.

[0044] PCR reaction system: High-fidelity expansion enzyme PrimeStarHS (R010 A, TAKARA) 0.5μL, 5XPrimesTarbuffer (Mg 2+ Plus) 10 μL, forward primer (10 μm) 1 μL, reverse primer (10 μm) ...

Embodiment 3

[0054] Example 3 LrWrky-S1 gene overduced vector constructs and genetic transformation of Arabidopsis

[0055] (1) Construction of PBI121-P35S :: lrwrky-S1 overexpression carrier

[0056] Depending on the PBI121 vector sequence and the Lrwrky-S1 gene sequence (SEQ ID NO.1), the primer lrwrky-S1-INF2 and LRWRKY-S1-INF-R2 are designed to introduce seamless cloned (in-fusion) carrier joint sequences. And the enzyme sequence sequence. The positive cloned massage of TA connection in Example 1 was a template, and the specific amplification of the LRWRKY-S1 gene fragment was performed.

[0057] The primer sequence is as follows:

[0058] LRWRKY-S1-INF-F2:

[0059] 5'- GGACTCTAGAGGATCC Gtccgaatatcatgggacgag-3 '

[0060]LRWRKY-S1-INF-R2:

[0061] 5'- GatcggGGAAATTCGAGCTC CTacaactttaaacgaagaaggcAg-3 '

[0062] Among them, the underscore is a sequence of the in-fusion cloned carrier joint.

[0063] The PCR reaction system is: high-fidelity expansion enzyme primestar HS (R010A, TAKARA) 0.5 μL...

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Abstract

The invention discloses a lily susceptibility fungal gene LrWRKY‑S1 and its application, the LrWRKY‑S1 The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the amino acid sequence encoded by it is shown in SEQ ID NO.2; the present invention studies the biological function of the protein through antibacterial experiments, and the experimental results show that the protein makes plants more Susceptible to pathogenic fungi such as Botrytis cinerea, Fusarium oxysporum and anthracnose. Also found that LrWRKY‑S1 The expression level of the gene was low in Minjiang lily with excellent resistance, while the expression level of its homologous gene was high in Lanzhou lily, Longya lily and Lilium lily with important economic value. The present invention provides a high-efficiency disease-susceptible gene for genetic engineering transformation, and lays the foundation for further cultivating lily or other plant new materials or new varieties with fungal resistance / sensitivity, and can provide a basis for using the gene to carry out plant genetic improvement in the future. Theoretical and technical support, but also broaden the thinking of plant disease control, has a very broad application value.

Description

Technical field [0001] The present invention belongs to the field of plant gene engineering, and in particular, the susceptible fungal gene LRWRKY-S1 and its application thereof are involved. Background technique [0002] In agricultural production, plant diseases, especially fungal diseases, has always been an important factor in limiting crop yield. The role of fungal pathogens and plant hosts can be divided into "resistance" or "susceptibility" based on fungal infection, which involves different host cell genes and regulating the mechanism of host cell gene expression. In resistance, the organism controls fungal invasion by producing an antifungal protein (AFP), antifungal peptide or compound. Antifungal proteins are evolutionary conservative components in congenital immune responses, and is the main effect molecule. Antifungal protein is involved in the compositional resistance or inducing resistance mechanism of organisms, enabling organisms to effectively resist the infring...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/29C07K14/415C12N15/82A01H5/00A01H6/82A01H6/20
CPCC07K14/415C12N15/8218C12N15/8282
Inventor 符勇耀杨利平吴涵徐文姬
Owner YANGTZE NORMAL UNIVERSITY
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