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An expression vector and vector technology, applied in the field of plant expression vector, can solve the problem of inability to remove bacterial screening markers, and achieve the effects of ensuring biological safety, simplifying operation steps, and improving work efficiency
Inactive Publication Date: 2011-02-02
INST OF CROP SCI CHINESE ACAD OF AGRI SCI
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Biolistic-mediated co-transformation removes plant selectable markers but not bacterial selectable markers
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Embodiment 1
[0039] Embodiment 1, preparation and identification of pWMB014 universal expression vector
[0040] Expression vectors pRTL2, pZP101, pZP201, pCAMBIA3301 and pBluescript II-KS(-) were purchased from Biovector Science Lab ( http: / / biovector.blog.163.com / ; Tel: 18901268599 ).
[0041] Cloning vector pEASY TM -T1 Simple was purchased from Beijing Quanshijin Biotechnology Co., Ltd., the product catalog number is CT111.
[0042]Expression vectors pAHC25 and pTCK303 have been disclosed in literature (Christensen et al., Plant Molecular Biology, 1992, 18: 675-689) and (Wang et al., Plant Molecular Biology Reporter, 2004, 22: 409-417), and the public can obtain them from China Agriculture Acquired from the Institute of Crop Science, Academy of Sciences.
[0043] SEQ ID NO: 1: a T-DNA region containing ubi promoter, before replacement; corresponding Figure 14 ;
[0044] SEQ ID NO: 2: multiple cloning site from pBluescript II-KS(-), corresponding to Figure 15 ;
[0045] SEQ ID...
Embodiment 2
[0065] Embodiment 2. The application of vector-the insertion and identification of GUS gene in pWMB014
[0066] C58C 1 Agrobacterium tumefaciens were purchased from Biovector Science Lab ( http: / / biovector.blog.163.com / ; Tel: 18901268599). The wheat variety Xinchun 9 is preserved by the National Crop Germplasm Bank and provided to users free of charge (Institute of Crop Science, Chinese Academy of Agricultural Sciences, Tel: 010-62189650).
[0067] 1. Insertion of GUS gene into pWMB014
[0068] Design a pair of PCR primers GUS014XbaI2F: 5′-AT TCTAGA ATGGTAGATCTGAGGGTAAATTTCT-3' and GUS014SacI2R:5'-AT GAGCTC TCACACGTGGTGGTGGTGGT-3′, amplify the 2050bp full-length sequence of the GUS gene from the pCAMBIA3301 vector, add XbaI and SacI restriction sites at both ends, and first connect the full-length sequence of the GUS gene to pEASY TM -T1 Simple cloning vector. Then, use XbaI and SacI respectively to pWMB014 vector and pEASY TM -T1 Simple recombinant cloning vector wa...
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Abstract
The invention discloses a plant expression vector and application thereof. The vector is obtained by the following steps of: inserting a DNA segment shown as SEQ ID No. 4 between enzyme cutting sites of HindIII and EcoRI of a starting expression vector pZP201 to obtain an intermediate vector I; and inserting the DNA segments including the DNA segments expressed by LB, RB and SEQ ID No. 3 between the enzyme cutting sites of ScaI and ScaI in the intermediate vector I to obtain a target expression vector. A target gene can be conveniently and rapidly inserted into the vector of the invention, and a foreign gene can be directly inserted into the expression vector of the invention by a one-step cloning vector, so that the vector has the advantages of greatly simplifying the operating steps, saving time and labor, reducing the cost, improving the working efficiency, along with no need of construction of series vectors, and is simple and convenient to operate. The invention of the vector can provide technical support for the breeding of the plant gene engineering and lay a foundation for the safety evaluation and the commercial planting of transgenic plants.
Description
technical field [0001] The invention relates to a plant expression vector. Background technique [0002] Since the 20th century, genetic engineering breeding has been considered as an effective way to improve plant yield, quality and stress resistance. In fact, some new varieties of transgenic plants, such as transgenic soybeans, cotton, corn and rapeseed, have been bred by the use of genetic engineering breeding technology, and have been widely used in production practice. Especially in the past five years, the research on plant transgenics has developed very rapidly, and the number of plant varieties bred by transgenic methods has increased year by year. At the same time, because many national governments and consumers pay attention to the safety of genetically modified plants, as well as the inherent safety obstacles of genetically modified plants, it is difficult for most genetically modified plant varieties to be approved for commercial production. Among them, the exi...
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