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RNAi (RNA interference)-based transgenic novel watermelon material construction method

A watermelon and gene technology, applied in the field of molecular biology, can solve problems such as loss of the ability to mediate virus infection

Inactive Publication Date: 2017-10-20
NORTHWEST A & F UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In recent years, RNAi technology has been widely used in the study of plant virus gene function and pathogenic mechanism and plant disease resistance mechanism. The ability to mediate virus infection, so that plants can acquire virus resistance, especially in the research of watermelon anti-virus disease, has not been reported yet

Method used

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  • RNAi (RNA interference)-based transgenic novel watermelon material construction method
  • RNAi (RNA interference)-based transgenic novel watermelon material construction method
  • RNAi (RNA interference)-based transgenic novel watermelon material construction method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0071] Embodiment 1, PCR amplification obtains watermelon plant eIF4E gene:

[0072] According to the eIF4E gene sequence, the present invention first designs the primers of the watermelon eIF4E gene, as follows:

[0073] 4E-F: 5′-ATGGTAGTTGAAGAKWCGATSAAAGC-3′

[0074] 4E-R: 5′-TCACACYRWATATTTTRTTCTTYGCAT-3′

[0075] Using the total DNA of watermelon plant leaves as a template, the full-length eIF4E gene (708bp) was amplified by PCR ( figure 1 ).

Embodiment 2

[0076] Embodiment 2, PCR amplification obtains watermelon plant eIF (iso) 4E gene:

[0077] According to eIF (iso) 4E gene sequence, the present invention at first designs the primer of watermelon eIF (iso) 4E gene, as follows:

[0078] 4E-iso-F: 5′-ATGGCCGGTGAGGTAGCGGTGG-3′

[0079] 4E-iso-R: 5′-TCAAACACTRTATCGAGCTTTTGC-3′

[0080] Using the total DNA of watermelon plant leaves as a template, the full-length eIF(iso)4E gene (612bp) was amplified by PCR ( figure 1 ).

Embodiment 3

[0081] Example 3, PCR amplification of eIF4E and eIF (iso) 4E gene sense fragments, connected to form 4E-iso sense fragments

[0082] According to the gene sequences of eIF4E and eIF(iso)4E, the present invention firstly designs primers for respectively amplifying the gene fragments of eIF4E and eIF(iso)4E. The primers contain enzyme cutting sites and can be connected to the target vector. Use primer 4E-iso-1 and primer 4E-iso-2 to amplify the sense fragment of eIF4E gene; use primer 4E-iso-3 and primer 4E-iso-4 to amplify the sense fragment of eIF(iso)4E gene; use primer 4E- iso-1, 4E-iso-2, 4E-iso-3 and 4E-iso-4, overlap PCR amplification to get 4E-iso sense fragment.

[0083] The specific primers are as follows (the underlined part is the restriction site):

[0084] 4E-iso-1: 5′-TTGA CCATGG CCTGGGGTGCGTCTATCCG-3′

[0085] 4E-iso-2: 5′-TCTGCATTAGCCGGCGCCAGCCATTATCAG-3′

[0086] 4E-iso-3: 5′-CTGATAATGGCTGGCGCCGGCTAATGCAGA-3′

[0087] 4E-iso-4:5′-GAGCT GGTCACC CGCACTGGC...

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Abstract

The invention relates to an RNAi (RNA interference) vector construction method and an application thereof in watermelon genetic transformation, relates to the technical field of plant virus control and belongs to the application technology in biological and modern agriculture technique fields. The mediated virus infection function is destroyed by acting on host factors and thus watermelons obtain antiviral activity, which has not been reported yet at present. An RNAi vector is constructed and genetically transformed to watermelons, the constructionmethod is characterized in that watermelon eIF4E and a homological isomer eIF(iso)4E gene thereof are cloned and connected, 4E-iso sense and antisense fragments are obtained and connected to two sides of an intron sequence, and a 4E-iso hairpin structure fragment is obtained; the 4E-iso sense fragment, the 4E-iso antisense fragment and the hairpin structure fragment are inserted into a pCAMBIA1301 vector through T4 connection and homologous recombination, and the RNAi vector is obtained; target gene fragments are transformed into watermelon cotyledon explants through agrobacterium tumefaciens-medicated transformation, and transgenic watermelons with the target gene fragments are obtained through screening cultivation. The method is mainly used for providing a novel material and a novel technology for watermelon virus and disease control.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to the construction of an RNA interference vector suitable for genetic transformation of dicotyledons and highly resistant to watermelon mosaic virus disease and its application in genetic transformation of watermelon. It further relates to the expression vector of the watermelon eIF4E gene and its homologous isomer eIF(iso)4E gene and the genetic transformation of the watermelon to obtain transgenic plants. technical background [0002] The cultivation area of ​​watermelon (Citrullus lanatus) in my country ranks first in the world, with an average annual cultivation area of ​​1.2 million ha 2 . Watermelon virus diseases represented by watermelon mosaic virus (WMV) are the main limiting factors for the production of cucurbit crops such as watermelon, and the annual incidence of watermelon virus diseases reaches 100 thousand hm 2 , because of its serious harm,...

Claims

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Application Information

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IPC IPC(8): C12N15/84C12N15/66A01H5/00
CPCC12N15/66C12N15/8218C12N15/8283
Inventor 郝兴安赵磊吴云锋
Owner NORTHWEST A & F UNIV
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