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Plant transgenic visual tracking expression system and application thereof

A plant expression vector and gene technology, applied in the direction of using vectors to introduce foreign genetic material, recombinant DNA technology, etc., can solve the problem of transgenic plants purple and other problems, achieve the improvement of accuracy and reliability, strong purpose, and reduce the complexity of work. Effect

Inactive Publication Date: 2013-05-01
BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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Problems solved by technology

Han et al. (Han Y J, Kim Y M, Lee J Y, et al. Production of purple-colored creeping bentgrass using maize transcription factor genes Pl and Lc through Agrobacterium-mediated transformation. Plant Cell Rep.2009, 28(3): 397-406 ) have also obtained similar results in using the transcription factor regulation genes P1 and Lc of the anthocyanin synthesis pathway of maize or jointly transforming creeping bentgrass. The degree shows purple, the regulatory effect of Lc gene is stronger than that of Pl gene, but under the condition of joint action, the whole transgenic plant will appear purple

Method used

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  • Plant transgenic visual tracking expression system and application thereof
  • Plant transgenic visual tracking expression system and application thereof
  • Plant transgenic visual tracking expression system and application thereof

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Experimental program
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Effect test

Embodiment 1

[0035] Embodiment 1, the synthesis of maize anthocyanin regulation gene and the construction of plant expression vector

[0036] Considering the safety of antibiotics in biolistic transformation, in order to facilitate the deletion of antibiotic genes in subsequent experiments, the pGreen vector was transformed, and two loxP sites in the same direction were inserted on both sides of the Kan gene expression frame, which were identified by sequencing. The vector was named pBAC814. Insert the EPSP gene (EPSP gene expression cassette) driven by the Intron1 of the CaMV 35S promoter and the maize Adh1 gene at the NdeI restriction site before the pBAC814 vector multiple cloning site afterwards, and the EPSP gene is used as a selection marker gene for screening transgenic plants . The correct vector identified by sequencing was named pBAC823.

[0037] Digest the above pBAC823 vector with DraIII, add AgeI / PacI linker, then insert the expression cassettes (sequence 4) of the two trans...

Embodiment 2

[0039] Embodiment 2, transient expression of anthocyanin gene

[0040] Take wheat leaves and wheat Baofeng 104 15-17 days after flowering (Wang Junwei, Yang Fengping, Zhang Xiaodong, etc., Induced expression of DREB transcriptional factor and study on its physiological effects of drought tolerance in transgenic wheat.Acta Genetica Sinica, 2006, 33( 5): 468~476) immature embryos, after removing the top part of the growth, inoculate the scutellum upside down on MS solid medium, and use the purified concentration of 1 μg μL -1 The pBAC9008 vector, according to Zhang Xiaodong et al. (Zhang Xiaodong, Liang Rongqi, Chen Xvqing, Yang Fengping, Zhang Liquan, Transgene inheritance and quality improvement by expressing novel HMW glutenin subunit (HMW-GS) genes in winter wheat. 48 (8): 771-776.) The method described in the preparation of gene gun microprojectiles, PDS-1000 / He type gene gun bombards scutellum and blade, after 48h, observe anthocyanin gene in different tissues under a Leic...

Embodiment 3

[0042] Embodiment 3, corn biolistic transformation

[0043] Induction medium: N6 basic medium + 10mg L -1 AgNO 3 +100mgL -1 Inositol+2mg L -1 2,4-D+30g L -1 Sucrose+7g L -1 agar.

[0044] Differentiation medium: N6 basic medium + 0.5mg L -1 AgNO 3 +100mgL -1 Inositol+1.5mg L -1 KT+30g L -1 Sucrose+7g L -1 Agar+0.5mgL -1 glyphosate.

[0045]The ears of corn elite inbred line 501 about 12 days after pollination were taken, and the immature embryos were picked and inoculated on the induction medium after surface disinfection with 75% ethanol, and embryogenic callus was induced after dark culture for 3-5 days. Adjust the concentration of the purified pBAC9008 vector to 1 μg μL -1 , according to Zhang Xiaodong et al (Zhang Xiaodong, Liang Rongqi, Chen Xvqing, Yang Fengping, Zhang Liquan, Transgene inheritance and quality improvement by expressing novel HMW glutenin subunit (HMW-GS) genes in winter wheat. ): 771-776.) The method carries out the preparation of gene...

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Abstract

The invention discloses a plant transgenic visual tracking expression system and application thereof. The plant transgenic visual tracking expression system is a plant expression vector which contains a visual marker gene expression kit, the visual marker gene expression kit consists of an expression kit of a transcription factor bi gene in the anthocyanin biosynthetic pathway and an expression kit of a transcription factor cl gene in the anthocyanin biosynthetic pathway. The color regulator gene is applied on plant genetic transformation operation, the screening work is effectively relieved after the transformation, according to the gene performance on the plant, the valuable transformation materials can be selectively kept, accordingly, the screening time and the research cost are greatly saved, the accuracy and the reliability of the experimental result are greatly improved, so good foundation is laid for carrying out analysis work after the transformation. In addition, the transient expression of the color gene is utilized, the transformation effect can be visually and more directly analyzed, and accordingly, various transformation influencing factors are prevented from being generated.

Description

technical field [0001] The invention relates to a plant transgene visual tracking expression system and application thereof, in particular to a plant expression vector using two transcription factors bi and cl genes in an anthocyanin synthesis pathway as a visual marker gene during the plant transgene process. Background technique [0002] Among the numerous plant genetic transformation reporter genes, the anthocyanin metabolism regulation gene is the most convenient one. After it is introduced into plant cells, anthocyanins will accumulate in the vacuoles after expression, turning the cells into red or purple, which can be observed with a general dissecting microscope or even the naked eye, without special treatment or selection of special detection parts of plants. Instruments for observation, not to mention killing plant tissue for histochemical testing. Therefore, applying this type of gene to the operation of plant genetic transformation can effectively reduce the comp...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/82
Inventor 杨凤萍张晓东陈绪清薛静张立全李向龙宫硖
Owner BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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