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In-vivo expression method of woody plant

A technology of live expression and woody plants, which is applied in the field of live expression of woody plants, can solve the problems of long growth cycle of chestnut and achieve intuitive results, short time and simple operation

Inactive Publication Date: 2015-05-13
BEIJING UNIV OF AGRI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the growth cycle of Chinese chestnut is long, and the transgenic regeneration system of Chinese chestnut has not yet been established, so an effective method is urgently needed to verify the function of KAO gene

Method used

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  • In-vivo expression method of woody plant
  • In-vivo expression method of woody plant
  • In-vivo expression method of woody plant

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] A method for in vivo expression of woody plants, comprising the steps of:

[0060] (1) Extraction and purification of total RNA from male inflorescences

[0061] Chestnut genetic transformation experiments were carried out on chestnut trees grafted in 2000 in the chestnut producing area of ​​Miyun County, Beijing. After sampling, they were quickly frozen in liquid nitrogen and stored in a -80°C refrigerator. Male inflorescence RNA was extracted by EASYspin Plant RNA Rapid Extraction Kit. And add RNase-FREE DNase I to digest the DNA in the total RNA. The integrity of the total RNA was detected by 1% agarose gel electrophoresis, the concentration of the total RNA was measured by a nucleic acid analyzer, and whether the ratio of OD260 / 280 and OD260 / 230 met the requirements. The remaining samples were temporarily stored in an ice box for subsequent cDNA synthesis; or stored in a -80°C refrigerator for a long time after quick freezing in liquid nitrogen.

[0062] (2) Syn...

Embodiment 2

[0076] Adopt the chestnut male inflorescence used in embodiment 1, what differ from embodiment 1 is that what step (5) constructs is the CmKAO-RNAi interference carrier, and the construction of described interference carrier is as attached figure 2 shown. The RNAi vector pFGC5941 was used to replace pCAMBIA1304 to construct the CmKAO-RNAi interference vector; in step (6), Agrobacterium tumefaciens EHA105 was used to replace LGV3101 Agrobacterium for CmKAO-RNAi plasmid transformation and subsequent in vivo infection of chestnut male inflorescences.

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Abstract

The invention discloses an in-vivo expression method of a woody plant. The in-vivo expression method comprises the following steps: preparing a plant sample, establishing an over-expression vector, performing infectious transformation on agrobacterium, and performing genetic transformation on the plant over-expression vector. Through establishment of the over-expression vector pCAMBIA1304-CmKAO and an interference vector CmKAO-RNAi, in a formation stage of a male inflorescent primordium of a wild bud-mutated short male inflorescence, the agrobacterium is used for mediating KAO gene-carrying vector bacterial liquid, the male inflorescence is injected and smeared respectively, and an instantaneous expression and interference system of an chestnut inflorescence is established; through establishment of an in-vivo-instantaneous transformation system of the chestnut male inflorescence, a function of verifying a gene on a parent of a plant with a long juvenescent phase and an imperfect regeneration system is realized, the transformation period is greatly shortened, and a valuable reference is provided for transformation of the in-vivo-instantaneous system of other plants, especially the woody plant.

Description

technical field [0001] The invention relates to a living body expression method of the plant, in particular to a living body rapid expression method of Chinese chestnut. Background technique [0002] With the completion of the sequencing of a large number of plant genomes, more and more homologous genes and unknown gene information have been discovered, and the functions of these genes are waiting to be verified. Mutant-based reverse genetics technology is the most effective means of verifying gene function. This technology first needs to obtain the source of mutants. Artificial mutants can be obtained through T-DNA insertion and transposon insertion. For plants that are difficult to obtain mutants through artificial modification, natural mutation is to obtain mutants. important source of . For plants with mutants but no complete regeneration system, it can be verified with the help of homologous gene mutants of model plants such as Arabidopsis, tobacco, etc., but it is di...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/84C12N15/53A01H5/00
Inventor 秦岭曹庆芹邢宇张卿杨柳房克凤
Owner BEIJING UNIV OF AGRI