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High efficiency stable expression system of transgene tomato

A tomato and gene sequence technology, applied in genetic engineering, plant genetic improvement, introduction of foreign genetic material using vectors, etc., can solve the problems of low expression level, non-expression of foreign genes, differences in independent transformants, etc.

Inactive Publication Date: 2006-08-23
周晓红 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

A large number of experimental results show that in the process of plant gene transformation, the expression level of exogenous genes is generally low or even not expressed, and there are significant differences among independent transformants

Method used

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  • High efficiency stable expression system of transgene tomato
  • High efficiency stable expression system of transgene tomato
  • High efficiency stable expression system of transgene tomato

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0236] Embodiment 1 Construction of E81.1-pGEM-T and E82.2-pGEM-T

[0237] 1 Design of primers

[0238] P1 and P3 are primers at the 5' end, and P2 is a primer at the 3' end. P1 and P2 were used to amplify the E81.1 sequence of Zhongshu No.5 tomato; P3 and P2 were used to amplify E81.1 and its upstream 1.1kb sequence, which is the full-length 2.2kb fragment E82.2 of the fruit-specific E8 promoter. P1: 5'CCG AAG CTT TCT AGA AG G AAT TTC ACGAAA TCG 3'; introduce HindIII and XbaI enzyme cutting sites. P2: 5'GCC TCT AGA GGA TCC TCTTTT GCA CTG TGA ATG3'; introduce BamHI and XbaI enzyme cutting sites. P3: 5'GGC AAG CTT CCCTAA TGA TAT TGT TCA C3' introduces HindIII restriction site.

[0239] 2 PGR cycle conditions

[0240] 95°C, 5min; 95°C denaturation for 1min, 56°C annealing for 1min, 72°C extension for 90s, 30 cycles; 72°C, 8min.

[0241] 3 Identification and purification of PCR products

[0242] After identification by 1% agarose gel electrophoresis, use the freeze-...

Embodiment 2

[0245] Example 2 Construction of plant expression operon unit and its intermediate vector and expression vector

[0246] (1) The construction of the intermediate vector pB35MG and the plant expression vector pC35MG composed of E35S+MAG+NOS (see construction procedure Figure 5 )

[0247] The MAG in the pCRII-Topo-MAG plasmid was subcloned into the pBAC55 vector by BamHI single enzyme digestion and ligation, and the intermediate vector pB35MG was constructed, which uses E35S to drive MAG, and the 3' end is the terminator NOS3' sequence, forming E35S / MAG / NOS3 'Structural units. Cloning identification by enzyme digestion and sequencing: use BamHI enzyme digestion to identify the insert, XhoI to identify the forward and reverse of the insert, HindIII to identify the E35S / MAG / NOS3' structural unit; Shanghai Jikang Biological Co., Ltd. completed the E35S / MAG in the pB35MG vector Sequencing identification of the / NOS 3' structural unit. Then, the identified E35S / MAG / NOS3' structur...

Embodiment 3

[0264] Example 3 Establishment of Tomato High Frequency Regeneration System

[0265] (1) Selection of tomato explants

[0266] For the tomato seedlings germinated for 8-12 days, the cotyledons and hypocotyls (without terminal buds) are taken, the lower surfaces of the cotyledons and leaf discs are facing downward, and the wounds of the cuts are closely attached to the regeneration medium.

[0267] (2) Choice of hormone

[0268] Cytokinin selection: ZT, 6-BA; auxin selection: IAA.

[0269] The basal medium is MS1, and the selection of hormones is different. The following three hormone formulas can obtain high-frequency regeneration:

[0270] Regeneration medium 1: ZT0.2-0.5mg / L, IAA 0.1mg / L

[0271] Regeneration medium 2: ZT0.2-0.5mg / L

[0272] Regeneration medium 3: ZT0.2-0.5mg / L, 6-BA 0.5mg / L, IAA 0.1mg / L

[0273] (3) Culture conditions

[0274] Put it in an artificial climate box, the light conditions are the same as the cultivation of sterile seedlings, the temperatur...

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Abstract

Through cloning to obtain the specific fruit promoters E82.2 and E81.1 gene of excellent pure tomato breed Zhongshu No.5, driving the vaccine molecule with the promoter E82.2 and the compound double promoter E35S-E81.1 and constituting plant expression operon unit with NOS terminator, and introducing to Agrobacterium tumefaciens LBA4404, engineering bacterium is obtained. By means of optimizing the complete technological system, recombinant vaccine molecule protein with efficient and stable expression and its tomato transforming strain are obtained. The present invention constitutes technological platform of efficient and stable tomato expression for exogenous vaccine molecule and medicinal protein production and provides excellent technological platform for developing oral tomato vaccine and its medicinal protein.

Description

technical field [0001] The invention relates to a transgenic tomato system for efficiently expressing medicinal proteins, vaccine molecules and the like. The system is constructed with a dual promoter composed of tomato fruit-specific E8 promoter, enhanced cauliflower mosaic virus 35S promoter and E8 promoter to drive vaccine molecules and other target proteins, and a gene expression operon composed of NOS terminator. Intermediate vectors, plant expression vectors, tomato transformation, screening identification, and stable and high-efficiency expression of exogenous target gene transgenic plant strain cultivation and molecular identification system established after transformation of Agrobacterium LBA4404, including all involved vectors, strains, technical processes, Reagent formula, the vaccine or medicinal protein with highly efficient and stable expression obtained at last, its transgenic tomato strain and its application. Background technique ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C12N15/63C12N15/82C12N5/04A01H4/00A61K39/002
Inventor 周晓红陈晓光
Owner 周晓红
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