A kind of transgenic method of paphiopedilin particle room injection method

A transgenic and injection technology, applied in the field of plant biology, to achieve the effect of high result rate, simple operation and high transformation rate

Active Publication Date: 2021-02-26
SOUTH CHINA BOTANICAL GARDEN CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, orchids generally take dozens of days from pollination to fertilization, and in the short period before fertilization, because the germ cells or fertilized eggs have no cell walls or the cell walls are relatively weak, they are in a more sensitive competent state and are easier to be transformed. Appropriate injection time is the key to the success of ovary injection transgene

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] 1. Preparation of plasmid injection

[0020] Construction of the expression vector containing the target gene: the Jianlan flowering integrated gene (CeFT) (GenBank accession number: HM803115) was used as the target gene, and the primers were designed: CeFT-F: 5'- TCTAGA ATGAATAGAGAGAGAGACTC-3' (the underlined part is the Xba I restriction site), CeFT-R: 5'- GGATCC TCAATCCTGCATCCTTCTTC-3' (the underlined part is the BamH I restriction site), with the cDNA of Jianlan flower buds as a template, CeFT-F and CeFT-R as primers to amplify the Jianlan CeFT gene with restriction sites XbaI and BamH I, Connected to Takara's PMD18-T vector, and then transformed and identified according to J. Sambrook et al. "Molecular Cloning Experiment Guide". The identified positive plasmid utilizes restriction sites Xba I and BamH I to excise the CeFT gene from the T vector, and uses the same Xba I and BamH I enzymes as the plant expression vector pCAMBIA1301 (GenBank accession number: AF2342...

Embodiment 2

[0031] 1. Preparation of plasmid injection

[0032] Construction of the expression vector containing the target gene: the Jianlan flowering integrated gene (CeFT) (GenBank accession number: HM803115) was used as the target gene, and the primers were designed: CeFT-F: 5'- TCTAGA ATGAATAGAGAGAGAGACTC-3' (the underlined part is the Xba I restriction site), CeFT-R: 5'- GGATCC TCAATCCTGCATCCTTCTTC-3' (the underlined part is the BamH I restriction site), with the cDNA of Jianlan flower buds as a template, CeFT-F and CeFT-R as primers to amplify the Jianlan CeFT gene with restriction sites XbaI and BamH I, Connected to Takara's PMD18-T vector, and then transformed and identified according to J. Sambrook et al. "Molecular Cloning Experiment Guide". The identified positive plasmid utilizes restriction sites Xba I and BamH I to excise the CeFT gene from the T vector, and uses the same Xba I and BamH I enzymes as the plant expression vector pCAMBIA1301 (GenBank accession number: AF2342...

Embodiment 3

[0043] 1. Preparation of plasmid injection

[0044] Construction of the expression vector containing the target gene: the Jianlan flowering integrated gene (CeFT) (GenBank accession number: HM803115) was used as the target gene, and the primers were designed: CeFT-F: 5'- TCTAGA ATGAATAGAGAGAGAGACTC-3' (the underlined part is the Xba I restriction site), CeFT-R: 5'- GGATCC TCAATCCTGCATCCTTCTTC-3' (the underlined part is the BamH I restriction site) uses the cDNA of Jianlan flower buds as a template, uses CeFT-F and CeFT-R as primers to amplify the Jianlan CeFT gene with restriction sites Xba I and BamH I, Connected to Takara's PMD18-T vector, and then transformed and identified according to J. Sambrook et al. "Molecular Cloning Experiment Guide". The identified positive plasmid utilizes restriction sites Xba I and BamH I to excise the CeFT gene from the T vector, and uses the same Xba I and BamH I enzymes as the plant expression vector pCAMBIA1301 (GenBank accession number: A...

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Abstract

The invention discloses a transgenosis method for paphiopedilum by a plasmid ovary injection method. The transgenosis method comprises the steps that a plasmid injection solution of a plant expression vector containing a carrying target gene is injected into an ovary after pollination and before double fertilization of the paphiopedilum; a developed seed as an explant is subjected to aseptic seeding; and protocorm induction, resistant protocorm screening and molecular detection are performed to obtain a transgenic paphiopedilum plant. The transgenosis method for the paphiopedilum by the plasmid ovary injection method has the advantages of simplicity in operation, high fruitbearing rate and high conversion rate, facilitates molecular breeding of the paphiopedilum, and accelerates a breeding progress of the paphiopedilum.

Description

Technical field: [0001] The invention belongs to the field of plant biotechnology, and in particular relates to a method for genetically transforming genes through the injection method of a paphiopediphyllin particle chamber. Background technique: [0002] Paphiopedilum (Paphiopedilum) has a very high ornamental value because of its unique flower shape, gorgeous flower color, and long-lasting ornamental flowering period. It is a very popular high-end flower in the international flower market. However, due to over-harvesting and habitat destruction, Papuopsis has become one of the most endangered plant species in the world, and all wild species are included in the appendix of the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) I was prohibited from trading. Through various breeding methods, new orchid varieties that can be freely traded in the international market and have high ornamental value can be bred to meet market needs. The tr...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/82A01H5/00A01H6/62
CPCC12N15/8207
Inventor 曾宋君罗白雪张建霞张莉邓莎吴坤林郑枫张新华马国华段俊
Owner SOUTH CHINA BOTANICAL GARDEN CHINESE ACADEMY OF SCI
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