RNAi (RNA interference)-based transgenic novel watermelon material construction method
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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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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
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[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):
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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,...
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