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RNAi anti-ASPV (Apple Stem Pitting Virus) expression vector and application thereof in apple breeding for disease resistance

A technology of apple stempox virus and expression vector, which is applied in the field of biological breeding, can solve the problems of large application barriers, non-implementation of apples, and lagging development level of disease-resistant breeding technology.

Active Publication Date: 2016-04-06
ZHENGZHOU FRUIT RES INST CHINESE ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although the virus coat protein (CP) gene silenced by RNA interference technology has achieved good antiviral effects in some crops (such as soybeans, tobacco, etc.), since apples are perennial woody plants, compared with herbaceous plants, the use of The development level of genetic engineering for disease-resistant breeding technology lags behind obviously, so there are relatively large obstacles to its application in apples, and it has not been implemented for a long time

Method used

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  • RNAi anti-ASPV (Apple Stem Pitting Virus) expression vector and application thereof in apple breeding for disease resistance
  • RNAi anti-ASPV (Apple Stem Pitting Virus) expression vector and application thereof in apple breeding for disease resistance
  • RNAi anti-ASPV (Apple Stem Pitting Virus) expression vector and application thereof in apple breeding for disease resistance

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] RT-PCR amplification obtains the target gene fragment (ASPV conservative segment), which specifically includes the following steps:

[0070] (1.1) According to the full-length sequence of the ASPV viral coat protein gene published by Genebank (accession number HM12156.1), primers were designed and synthesized in the conserved segment:

[0071] ASPV-F (5'- CACC AGTTGCATTGATTGGGAT-3', CACC Sequence adapters necessary for the added TOPO clones) and ASPV-R (5'-TCCAATTTTTCCCCCAGTGACT-3'),

[0072](1.2) Total plant RNA was extracted from susceptible apple leaves by the Trizol method. In this example, the plant material used for RNA extraction was the 7-year-old apple variety “Royal Gala”. Take 2 μg of purified RNA, under the action of M-MLV reverse transcriptase, use random primer OligodT to carry out reverse transcription to synthesize the first strand of cDNA, and then use this as a template to carry out PCR amplification with primers ASPV-F, R and pfu enzyme The PCR r...

Embodiment 2

[0076] RNAi antiviral expression vector construction specifically comprises the following steps:

[0077] (2.1) Use TOPO-Cloning technology to construct an entry-level cloning vector:

[0078] Take 1 μL of the PCR recovery product containing the target gene fragment, and construct a 6 μL reaction system (the specific reaction system used in this example is: recover the target gene fragment ASPV 1 μL, salt solution 1 μL, pENTR? / SD / D-TOPO? vector (produced by Invitrogen, USA) 1 μL , sterilized ultrapure water 3 μL) at 25°C for 30 min, transform Escherichia coli TOP10 competent cells by heat shock method, spread the transformed cells evenly on LB solid medium containing 50 mg / L kanamycin (Kan), 37 After culturing at ℃ for 16 hours, pick a single clone into LB liquid medium containing the same concentration of kanamycin, culture with shaking at 37 ℃ for 14 hours, and culture with ASPV-F, ASPV-R and M13-F (5'-GTAAAACGACGGCCAGT-3' ) and ASPV-R as primers for bacterial liquid PCR am...

Embodiment 3

[0083] The RNAi antiviral expression vector is transformed into Agrobacterium tumefaciens, specifically comprising the following steps:

[0084] (3.1) Transformation of Agrobacterium strains by alternate freeze-thaw method: Transform the plasmid pH-ASPV into Agrobacterium EHA105 competent cells by alternate freeze-thaw method, and spread evenly on the medium containing 100mg / LSpc and 50mg / L rifampicin (Rif). On the YEB plate medium, culture it upside down at 28°C for 2 days, pick a single clone into the YEB liquid medium containing the same concentration of Spc and Rif, and culture it with shaking at 28°C for 48 hours;

[0085] (3.2) Screening and identification of positive clones after transformation: After transforming the pH-ASPV plasmid into Agrobacterium strain EHA105, use ASPV-F and ASPV-R, P35S-F (5'-GACGCACAATCCCACTATCC-3') and ASPV-R and NPTⅡ-F (5'-ACAATCGGCTGCTCTGATG-3') and NPTⅡ-R (5'-TCAGAAGAACTCGTCAAGAAG-3') were used as primers for bacterial liquid PCR amplificat...

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Abstract

The invention belongs to the field of biological breeding and particularly relates to an RNAi anti-ASPV (Apple Stem Pitting Virus) expression vector and an application thereof in apple breeding for disease resistance. The expression vector is prepared by the following steps: (1) RT-PCR amplification to obtain the target gene segment; and (2) establishment of the expression vector. An application method of the expression vector in apple breeding for disease resistance comprises the following steps: (1) transferring the RNAi anti-ASPV expression vector into agrobacterium tumefaciens; (2) performing aseptic culture of an apple leaf disc acceptor material; (3) transforming the apples with an agrobacterium-mediated method to obtain a transgenic material; (4) detecting the transgenic plant; and (5) performing in-vitro propagation of the obtained transgenic positive plant and transplanting. In the invention, the RNAi anti-ASPV expression vector containing the conservative segment of ASPV coast protein (CP) gene is adopted for genetic transformation of apples, the obtained transgenic plant can acquire the anti-virus character, and the RNAi anti-ASPV expression vector has a good application prospect in fruit tree production.

Description

technical field [0001] The invention belongs to the field of biological breeding, and in particular relates to an RNAi anti-apple stem pox virus expression vector and its application in apple disease-resistant breeding. Background technique [0002] Apple is one of the main tree species of fruit trees in my country, and its cultivated area and output both rank first in the world. However, in recent years, with the rapid development of the fruit tree industry and the increasing frequency of seedling transportation, the spread of viral diseases has become increasingly serious. Apple Stem Pox Virus (ASPV) is one of the main latent viruses in production. After the plants are infected by it, the tree vigor is weakened, resulting in a decline in yield and fruit quality. Some plants show leaf curling, xylem stem pox spots, Fruit sag, etc., cause serious economic losses to fruit tree production, and there is no effective chemical control method at present. [0003] In recent years...

Claims

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Application Information

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IPC IPC(8): C12N15/84A01H5/00
CPCC12N15/8218C12N15/8283
Inventor 田莉莉牛良
Owner ZHENGZHOU FRUIT RES INST CHINESE ACADEMY OF AGRI SCI
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