Genetic transformation method of rehmannia glutinosa libosch

A genetic transformation method, Tianmu Dihuang technology, applied in biochemical equipment and methods, botanical equipment and methods, and the use of vectors to introduce foreign genetic materials, etc., can solve the problems of low genetic transformation efficiency and large genetic differences, and achieve healing Reduced browning rate, increased regeneration and differentiation rate, and convenient phenotypic identification

Inactive Publication Date: 2022-04-29
HENAN AGRICULTURAL UNIVERSITY +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the genetic transformation of its closely related species Rehmannia glutinosa has been established, the genetic transformation efficiency of Tianmu Rehmannia glutinosa is very low due to the large genetic differences between the two species.

Method used

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  • Genetic transformation method of rehmannia glutinosa libosch
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  • Genetic transformation method of rehmannia glutinosa libosch

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043]A method for genetic transformation of Rhizoma Rehmanniae, comprising:

[0044] Cloning of PDS Gene of Tianmu Dihuang

[0045] The young leaves of Tianmu Dihuang were selected to extract total RNA, and the extraction method was carried out with reference to the instructions of the RNA extraction kit (TaKaRa, Dalian);

[0046] Reverse transcription was performed using cDNA first-strand synthesis kit, and the method was carried out with reference to the reverse transcription kit type 6210A (TaKaRa, Dalian) kit instructions;

[0047] The transcript sequence encoding phytoene dehydrogenase (PDS) was screened in the Tianmu Dihuang transcriptome, and it was predicted to have a complete open reading frame (ORF), which was named RcPDS, and its nucleotide sequence is shown in SEQ ID NO. 1 mentioned;

[0048] According to the sequence of RcPDS, a pair of specific primers RcPDS_F and RcPDS_R were designed, and the cDNA synthesized by reverse transcription was used as a template, ...

Embodiment 2

[0072] A method for genetic transformation of Tianmu Rehmannia, comprising the following steps:

[0073] (1), Tianmu Dihuang PDS Gene Cloning

[0074] According to the transcriptome annotation results of Rehmannia glutinosa, a transcript with the highest homology to RgPDS1 gene of Rehmannia glutinosa was screened, specific PCR primers were designed, and the cDNA of Rehmannia glutinosa was used as a template to amplify the PDS gene of Rehmannia glutinosa with high-fidelity DNA polymerase The full-length coding sequence, the nucleotide sequence of its coding region is shown in SEQ ID NO.1;

[0075] (2), CRISPR / Cas9 gene editing vector construction and loaded into Agrobacterium tumefaciens

[0076] For the PDS gene obtained in the cloning step (1), select a target sequence that can be efficiently edited by the Cas9 gene at the end of the exon close to the ATG, and insert the sgRNA target sequence into the CRISPR / Cas9 gene editing vector by using a mixed restriction enzyme ligati...

Embodiment 3

[0096] A method for genetic transformation of Tianmu Rehmannia, comprising the following steps:

[0097] (1) Cloning of Tianmu Dihuang PDS Gene

[0098] According to the transcriptome annotation results of Rehmannia glutinosa, a transcript with the highest homology to RgPDS1 gene of Rehmannia glutinosa was screened, specific PCR primers were designed, and the cDNA of Rehmannia glutinosa was used as a template to amplify the PDS gene of Rehmannia glutinosa with high-fidelity DNA polymerase The full-length coding sequence, the nucleotide sequence of its coding region is shown in SEQ ID NO.1;

[0099] (2), CRISPR / Cas9 gene editing vector construction and loaded into Agrobacterium tumefaciens

[0100] For the PDS gene obtained in the cloning step (1), select a target sequence that can be efficiently edited by the Cas9 gene at the end of the exon close to the ATG, and insert the sgRNA target sequence into the CRISPR / Cas9 gene editing vector by using a mixed restriction enzyme liga...

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Abstract

The invention discloses a genetic transformation method of rehmannia glutinosa libosch. The genetic transformation method comprises the following steps: (1) cloning a PDS gene of the rehmannia glutinosa libosch; (2) constructing a CRISPR / Cas9 gene editing carrier, and loading agrobacterium tumefaciens; (3) carrying out genetic transformation on the rehmannia temmosissima; and (4) carrying out molecular detection on the transgenic plant. The method has the following beneficial effects: 1, the browning rate of the leaf callus of the rehmannia temmoku is obviously reduced, the differentiation rate of the regenerated buds is high, and the growth state of the regenerated buds is relatively good; 2, the identification method of the genetic transformation efficiency is improved; and 3, the concentration of the auxin NAA in the differential culture medium of the related species rehmannia glutinosa is 0.1-0.5 mg / L, when the same NAA concentration is used for genetic transformation of the rehmannia glutinosa, the regeneration bud differentiation rate is very low, and when the concentration of the improved NAA is 0.05 mg / L, the regeneration differentiation rate of the rehmannia glutinosa is remarkably improved.

Description

technical field [0001] The invention relates to the field of plant biotechnology, in particular to a genetic transformation method of rehmannia rehmanniae. Background technique [0002] Rehmannia chingii (Rehmannia chingii) is cold in nature, sweet and bitter in taste. Because it is mainly distributed in the Tianmu Mountain area of ​​Zhejiang Province, it is also called Zhejiang Rehmannia, and it is one of the important wild germplasm resources of Rehmannia genus. [0003] According to folk medicine records, Tianmu Dihuang is used as a medicine with the whole plant and root, which has the effects of clearing heat and cooling blood, nourishing yin and promoting body fluid, and nourishing liver and kidney. Tianmu Dihuang is rich in iridoid glycosides, phenylethanol glycosides, rehmannia glycosides, leonurin and polysaccharides. Studies have shown that the whole plant and roots of Tianmu Dihuang are rich in catalpol and verbascoside, which have important pharmacological activi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/53C12N15/84C12N15/82A01H5/00A01H6/00
CPCC12N9/001C12Y103/99029C12N15/8205C12N15/825
Inventor 王丰青左鑫魏荷孙红正张重义李烜桢郭林秀
Owner HENAN AGRICULTURAL UNIVERSITY
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