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Method for cultivating high-TYLCV (tomato yellow leaf curl virus)-resistant tomatoes

A tomato, high resistance technology, applied in the biological field, to achieve the effect of shortening the breeding cycle

Active Publication Date: 2015-01-28
SHANXI AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In 2005, Bai Qingrong et al. transferred PVX-CP and PVY-CP chimeric genes into tobacco and obtained 6 transgenic strains immune to the two viruses; in 2008, Zhu Changxiang et al. Tobacco that is resistant to all three viruses; in 2011, Niu Yanbing and others obtained genetically modified tobacco that is immune to two viruses (TMV and CMV) at the same time; so far, there is no relevant research on the use of gene technology to aggregate different genes of the same source of resistance at home and abroad Therefore, it is of great significance to carry out multi-chimeric gene silencing technology and related research on new tomato lines with high resistance to TYLCV

Method used

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  • Method for cultivating high-TYLCV (tomato yellow leaf curl virus)-resistant tomatoes
  • Method for cultivating high-TYLCV (tomato yellow leaf curl virus)-resistant tomatoes
  • Method for cultivating high-TYLCV (tomato yellow leaf curl virus)-resistant tomatoes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1 RNAi vector construction of AV1, AC1 and AC3 gene fragments of TYLCV

[0041] The construction process of the RNAi vector of the three-gene fragment chimeric gene is as follows: figure 1 As shown, the main steps are as follows:

[0042] 1.1 Genomic DNA extraction of susceptible tomato

[0043] In this example, a modified CTAB method was used to extract the genomic DNA of tomato leaves. The specific experimental steps are as follows: Weigh 2 g of susceptible tomato leaves into a mortar pre-cooled at -20°C, add an appropriate amount of liquid nitrogen, and then quickly grind the leaves into powder. After grinding, put the powder into a 1.5 mL EP tube; add 600 μL of 2% CTAB solution (preheated at 60 °C) to the EP tube, shake and mix well, and take a water bath at 60 °C for 15 min; add 300 μL of chloroform, shake slightly, put in 4 Centrifuge at 13,000 rpm for 5 min. Transfer the supernatant to another new 1.5mL EP tube; add an equal volume of 25:24:1 phenol / c...

Embodiment 2

[0067] Example 2 Agrobacterium-mediated tomato transiently

[0068]2.1 Preparation of Agrobacterium EHA105 competent cells

[0069] Agrobacterium EHA105 was picked and streaked on a YEP solid plate containing 25 μg / mL rifampicin, and cultured at 28 °C for 36-48 h; a single colony was picked and inoculated in 2 mL YEP medium, and cultured overnight at 28 °C and 200 rpm; 2 mL of bacterial solution was inoculated into Continue to culture in 50 mL YEP medium for 6-8 h until OD600 = 0.5, ice bath for 10 min; centrifuge at 7,000 rpm for 5 min at 4 °C, discard the supernatant, and resuspend the bacterial weight in 5 mL of 20 mM CaCl2 solution; centrifuge at 7,000 rpm for 1 min at 4 °C, Discard the supernatant, gently suspend the cells with 1 mL of 20 mM pre-cooled CaCl2, aliquot 100 μL per tube, snap-frozen in liquid nitrogen, and store at -80 °C for later use.

[0070] Note: All steps in contact with air in the experiment are operated in a clean bench. After the preparation of NaCl...

Embodiment 3

[0080] Example 3 Identification of TYLCV resistance of transgenic tomato

[0081] 3.1 Agrobacterium injection inoculation

[0082] 3 weeks after inoculation with RNAi vector, Agrobacterium containing TYLCV infection plasmid was inoculated on the new leaves of transgenic tomato plants, and the tomato plants were infected. The plant transformed with empty load was used as a negative control, and the method was the same as that in 2.3. 5 weeks after inoculation, the symptoms of tomato leaves were observed, and the results were as follows image 3 , 4 , 5 shown. Depend on image 3 It can be seen that A is an untransformed but virus-infecting tomato plant. From the leaf symptoms, it can be found that the tomato has been severely infected with TYLCV; Figure 4 It can be seen that B is a transformed empty tomato plant, and its leaf symptoms are similar to A, indicating that the tomato is also infected with TYLCV; Figure 5 It can be seen that C is a tomato plant transformed wit...

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Abstract

The invention discloses a method for cultivating a high-TYLCV (tomato yellow leaf curl virus)-resistant tomatoes. The method comprises the following steps: selecting fragments of AV1 gene, AC1 gene and AC3 gene in a TYLCV genome for gomphosis to obtain polygenes chimera AV1-AC1-AV3, and constructing an interference vector RNAi of a palindromic sequence; carrying out transient expression of dsRNA on tomato cotyledon by virtue of agrobacterium-mediated transformation to obtain a transgenic tomato for transforming the polygenes chimera AV1-AC1-AV3; and if the transgenic tomato shows TYLCV resistance after system infection of the TYLC, is not observed with obvious symptoms and has high-TYLCV resistance, screening out the high-TYLCV-resistant tomato. By utilizing the method, the high-TYLCV-resistant tomato strains can be quickly screened out and cultivated in 2 months, the cultivation period is greatly shortened, and a technical support is provided for preventing and curing TYLCV.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for cultivating tomato with high resistance to TYLCV. Background technique [0002] Tomato yellow leaf curl virus (TYLCV) belongs to the genus Begomovirus of the Geminiviridae family, and is the only type of single-stranded circular DNA with twinning particle morphology among plant viruses. (single stranded DNA, ssDNA) virus. The TYLCV genome encodes six ORFs: AV1 and AV2 on the viral strand, and AC1, AC2, AC3, and AC4 on the complementary strand. AV1 encodes the coat protein (CP) of the virus, which is related to the encapsidation, mediator transmission and interaction with the host of the virus; AV2 encodes a protein related to the move protein (MP), which mainly assists The function of mobile proteins; AC1 encodes viral replication-associated protein (Rep), which is involved in the initiation of replication of viral genomic DNA; AC2 encodes a transcriptional...

Claims

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Application Information

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IPC IPC(8): C12N15/84A01H5/00
Inventor 孙胜邢国明邢晓娟亢秀萍陈志峰李兴桃许小勇
Owner SHANXI AGRI UNIV
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