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Tobacco ferredoxin capable of improving disease resistance of rice and coding gene of tobacco ferredoxin

A technology for encoding genes and disease resistance, applied in the field of tobacco ferredoxin Ferredoxin and its encoding gene NbFd, to achieve the effect of improving bacterial blight resistance

Inactive Publication Date: 2019-04-02
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the targets of most Xoo type III effector proteins in plants remain unreported

Method used

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  • Tobacco ferredoxin capable of improving disease resistance of rice and coding gene of tobacco ferredoxin
  • Tobacco ferredoxin capable of improving disease resistance of rice and coding gene of tobacco ferredoxin
  • Tobacco ferredoxin capable of improving disease resistance of rice and coding gene of tobacco ferredoxin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Cloning and expression of embodiment 1.xopL gene

[0036] (1) Cloning of xopL gene

[0037] Take PXO99 AThe genome is used as a template, and the xopL gene is amplified with primers XopL-F (see SEQ ID NO.4) and XopL-R (see SEQ ID NO.5), cloned into the binary vector pHB using the XbaI site, and the obtained plasmid pHBxopL Transformed into Agrobacterium EHA105 for expression of xopL.

[0038] (2) Agrobacterium-mediated transient expression of xopL

[0039] After the Agrobacterium containing the plasmid pHBxopL was cultured overnight, induction buffer (10 mM MgCl 2 , 0.2mMacetosyringone, 200mM MES, pH 5.6) After washing once, adjust the concentration of bacteria solution OD 600 to 1.0. After standing at room temperature for 1 hour, inject tobacco leaves; inject Agrobacterium containing empty vector pHB as negative control. After 7 days of expression, XopL induced HR responses and cell death in tobacco leaves (see figure 1 A), this process is accompanied by the acc...

Embodiment 2

[0042] Example 2. Interaction between Ferredoxin protein and XopL obtained by yeast two-hybrid

[0043] In this example, the full-length tobacco ferredoxin gene was cloned, and its interaction with XopL was verified by yeast two-hybrid technology. Specifically: XopL was used as a bait to screen interaction factors from a tobacco cDNA library, and it was confirmed by sequencing that the positive clone contained the gene encoding Ferredoxin protein. The Ferredoxin gene was amplified in tobacco cDNA with primers Fd-F (SEQ ID NO. 6) and Fd-R (SEQ ID NO. 7). The PCR product was digested with NdeI and EcoRI and ligated into the pGBKT7 vector to obtain pGBKT7-Fd. After co-transforming yeast AH109 with the pGADT7-xopL plasmid expressing xopL, double hybridization was performed to confirm the interaction between Ferredoxin protein and XopL (see figure 2 A).

Embodiment 3

[0044] Example 3. Verification of the interaction domain between XopL and Ferredoxin

[0045] In this example, mutants of XopL and Ferrdoxin proteins were constructed by truncation, and the interaction domains of the two proteins were further verified. Specifically, the N-terminal (1-452aa) domain XopL-N of XopL is amplified with primers XopL-F (SEQ ID NO.4) and XopL-N-R (SEQ ID NO.8); primer XopL-C-F (SEQ ID NO.9) and XopL-R (SEQ ID NO.5) amplify the C-terminus (453-656aa) of XopL, namely XopL-C. The PCR products of each mutant of XopL were cloned into pGADT7 vector respectively. The C-terminal (51-144aa) of Ferredoxin was amplified with primers Fd-C-F (SEQ ID NO.10) and Fd-R (SEQ ID NO.7), and cloned into pGBKT7 vector. Yeast two-hybrid results proved that both the N-terminal and C-terminal of XopL interact with Ferredoxin protein, and the C-terminal of Ferredoxin protein still interacts with XopL (see figure 2 B).

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Abstract

The invention relates to tobacco ferredoxin capable of improving disease resistance of rice and a coding gene of the tobacco ferredoxin, and belongs to the field of disease-resistant genetic engineering of the rice. Tobacco ferredoxin Ferredoxin (NbFd) in effector protein XopL mediated plant cells generates ubiquitination, then the tobacco ferredoxin is degraded by proteasomes, and the number of plant cell chloroplasts entering the NbFd is reduced, so that transmission of electrons in photosynthesis is restrained, accumulation of active oxygen is caused, and plants are induced to produce an allergic reaction (HR) type disease-resistant reaction. Ferredoxin protein in the rice does not interact with XopL, but after Ferredoxin protein coding genes from tobacco are transferred into the rice,the resistance of the rice to bacterial leaf spot can be improved. The tobacco Ferredoxin protein coding gene NbFd obtained in the way of protein interaction can be used as a breeding gene resource ofthe rice for resisting bacterial leaf spots, and the resistance of obtained NbFd transgenic rice to the bacterial leaf spots can be notably improved.

Description

technical field [0001] The invention belongs to the field of rice disease-resistant genetic engineering, and in particular relates to tobacco ferredoxin Ferredoxin and its coding gene NbFd, which interact with Xanthomonas oryzae effector protein XopL and can significantly improve rice disease resistance. Background technique [0002] Rice bacterial blight, caused by Xanthomonas oryzae pv. oryzae (Xoo), is an important rice disease. The disease occurs in various rice areas in my country, and the disease is severe in the southern rice areas, which can lead to a 20%-50% reduction in yield, and can result in a complete failure of the harvest in severe cases. Planting resistant varieties is the most cost-effective means of controlling bacterial blight. [0003] Xoo's type III secretion system (type III secretion system, T3SS) is a key pathogenic factor for bacterial infection and establishment of a parasitic relationship with plants. It is a hrp gene cluster formed by nearly 30 ...

Claims

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Application Information

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IPC IPC(8): C12N9/00C07K14/415C12N15/29C12N15/82A01H5/00A01H6/46
CPCC07K14/415C12N9/93C12N15/8281C12Y603/02019
Inventor 陈功友马文秀邹丽芳徐夏萌朱章飞
Owner SHANGHAI JIAO TONG UNIV
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