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Ultra-binary vector as well as construction method and application thereof

A technology of binary vector and construction method, applied in the field of genetic engineering, can solve the problems of long cycle, difficult directional optimization, laborious and so on

Inactive Publication Date: 2013-08-21
INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The site-specific recombination system can accurately introduce foreign genes, but requires secondary transformation or hybridization, which is time-consuming and laborious
Although the selection marker gene can be completely deleted by using the transposon system, it needs to separate the target gene and the marker gene through sexual reproduction, which has a long cycle, low efficiency, and instability, making it difficult to optimize

Method used

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  • Ultra-binary vector as well as construction method and application thereof
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  • Ultra-binary vector as well as construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1, construction of super binary vector pDT1301

[0053] Step 1: Design and synthesize T-DNA right and left borders containing restriction sites

[0054] According to the published sequence of the pCMBIA1301 vector (GenBank: AF234297.1), design the "Xba I-RB-LB-Sal I including restriction sites Xba I and Sal I, and T-DNA right and left border sequences "The structure, the sequence is shown in SEQ ID NO1, synthesized by Beijing Aoke Company, the synthetic fragment is 98bp. The SEQ ID NO1 sequence is as follows:

[0055] GCTCTAGAGtaaacctaagagaaaagagcgtttattagaataacggatactgttggctggctggtggcaggatatattgtggtgtaaaca GTCGACGT.

[0056] Step 2: Construction of intermediate vector I containing two pairs of T-DNA borders

[0057] 45ng of the synthesized product of SEQ ID NO1 that has been sequence verified was digested by Xba I and Sal I at 37°C for 8h, then heat-treated at 65°C for 20min; at T 4 Under the action of ligase, with the vector pCMBIA1301-hpt which has also bee...

Embodiment 2

[0071] Embodiment 2, construction and genetic transformation of RNA interference vector resistant to rice stripe virus (RSV) and rice black-streaked dwarf virus (RBSDV)

[0072] Step 1: Construction of RNA interference (RNAi) vector

[0073] Using the intermediate vector pMCG161+ / -D containing the RSV-RBSDV-HCP hairpin structure as a template, and using the sequences SEQ ID NO9 and SEQ ID NO10 as primer pairs, high-fidelity LATaq enzyme was used for PCR amplification to obtain rice Intron and its flanks The sense and antisense strands of RSV-RBSDV-HCP and the hairpin structure of the restriction site Xho I, the amplified product is about 2309bp, and the negative control (pMCG161) only amplifies to a fragment of 1233bp, indicating that it has successfully obtained RSV-RBSDV -HCP hairpin structure.

[0074] The PCR product was digested with Xho I and ligated into the T-DNA1 region of the super binary vector pDT1301, which was also digested with Xho I, to obtain the RNA interfer...

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Abstract

The invention relates to an ultra-binary vector as well as a construction method and an application thereof. The ultra-binary vector is characterized in that a framework vector of the ultra-binary vector is pCMBIA1301-hpt<->, and Xba I-RB-LB-Sal I is inserted between restriction enzyme cutting sites Xba I and Sal I of the framework vector, thereby forming two T-DNA (transferred-deoxyribose Nucleic Acid) regions together with original left and right border sequences; and hpt II is connected into a T-DNA2 region through the restriction enzyme cutting sites, so as to acquire the ultra-binary vector pDT1301 containing the hpt II. The pDT1301 disclosed by the invention contains the hpt II and is convenient for screening callus-resistant and transgenic regenerated plants; and exogenous target genes of the hpt II are not in the same T-DNA region, so that the self-fertilization and separation of transgenic descendants are facilitated, the safe transgenic plants which do not contain selective marker genes are acquired, and obstacles for the safe release of the transgenic plants are cleared.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a super binary vector for cultivating non-selectable marker transgenic plants, a construction method and its application, and more specifically relates to a non-selectable marker gene transgenic and resistant to rice stripe virus and rice black-streaked dwarf Obtaining transgenic rice plants with shrink virus. Background technique [0002] Since the birth of transgenic plants in the 1980s, plant transgenic engineering, as one of the effective means to improve crop quality, yield, and stress resistance, has remained unabated. It has now entered the stage of large-scale commercial production and has become a One of the fastest growing biotechnology fields with the greatest application potential. According to the statistics of the International Agricultural Biotechnology Application Service (ISAAA), since the commercialization of GMOs in 1996, the planting o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/84C12N15/64C12N15/66A01H5/00
Inventor 李莉李羽王锡锋雷阳刘艳吴蓓蕾
Owner INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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