Method of improving plant type of dendrocalamus latiflorus with GRG1 gene and application of method

A kind of bamboo and gene technology, which is applied in the field of plant type improvement of bamboo with GRG1 gene, can solve the problems of restricting the process of plant genetic improvement, low feasibility of hybrid breeding, and plants with irregular flowering, and achieves convenient and precise orientation for functional research Editing effect, effect of promoting job development

Active Publication Date: 2020-01-24
FUJIAN AGRI & FORESTRY UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

For Ma bamboo, the feasibility of hybrid breeding is not great, mainly due to the special biological characteristics of bamboo plants - irregular flowering and plant death after flo...

Method used

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  • Method of improving plant type of dendrocalamus latiflorus with GRG1 gene and application of method
  • Method of improving plant type of dendrocalamus latiflorus with GRG1 gene and application of method
  • Method of improving plant type of dendrocalamus latiflorus with GRG1 gene and application of method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Embodiment 1 GRG1 gene cloning and target site design in Mazhu

[0028] Use the Plant Genomic DNA Extraction Kit (#DP305) from Tiangen Company to extract the genomic DNA from the leaves of Majuba bazaar; use the Plant RNA Extraction Kit from OMEGA Company (#R6827) to extract the total RNA from the leaves of Majuba japonica, and use the RNA reaction kit from TAKARA Company The transfer kit (#6110) reverses the cDNA of Mazhu from the extracted RNA. All the experimental operations involved in the above are described in the kit.

[0029] Previous studies have shown (Zhang, H., H. Wang, Q. Zhu, Y. Gao, H. Wang, L. Zhao, Y. Wang, F. Xi, W. Wang, Y. Yang, C. Lin and L . Gu. 2018. Transcriptome characterization of moso bamboo (Phyllostachys edulis) seedlings in response to exogenous gibberellin applications. BMC Plant Biol . 18:125.) GRG1 gene (GA-responsive gene 1, PH01004823G0070 ) was strongly induced by gibberellin, suggesting that this gene may be involved in the grow...

Embodiment 2

[0040] Example 2 Construction of Gene Editing Vectors

[0041] The intermediate vector pYLsgRNA-OsU6c used in the construction of pYLCRISPR / Cas9Pubi-H (hereinafter referred to as PUBI-H) vector and sgRNA expression cassette was introduced by the research group of Professor Liu Yaoguang of South China Agricultural University ( figure 1 ). The vector construction process follows the literature MaX., Zhang Q., Zhu Q., et al. (2015). A Robust CRISPR / Cas9 System forConvenient, High-Efficiency Multiplex Genome Editing in Monocot and DicotPlants. Mol Plant. 2015 Aug; 8( 8): The procedure mentioned in 74-84. was carried out (Ma X et al., 2015).

[0042] The specific process is:

[0043] (1) Preparation of target linkers

[0044] For different promoter-driven sgRNA expression cassettes, different primers containing special base adapters were designed, and the primers are as follows

[0045] Table 1 Primers containing special base linkers

[0046]

[0047] Target adapters are pr...

Embodiment 3

[0069] Embodiment 3 Ma bamboo genetic transformation and mutant plant acquisition

[0070] (1) Conversion material

[0071] The materials used in the genetic transformation system of Bamboo bamboo were calluses of Bamboo bamboo (Ye, S., C. Cai, H. Ren, W. Wang, M. Xiang, X. Tang, C. Zhu, T. Yin, L. Zhang and Q. Zhu. 2017. An Efficient Plant Regeneration and Transformation System of Ma Bamboo (Dendrocalamus latiflorus Munro) Started from Young Shoot as Explant. Front Plant Sci. 8:1298.);

[0072] (2) Agrobacterium transformation

[0073] Take about 100 grains of Mazhu callus in good growth state, soak in OD 600 =0.6 In the EHA105 Agrobacterium suspension carrying the gene editing vector for 10-20min, blot the excess bacterial solution with sterile filter paper, place it in the co-cultivation medium, and culture it in the dark at 26°C for 3 days.

[0074] (3) Sterilization and resistance screening

[0075] The calli after co-cultivation were concentrated in a sterile Erlenme...

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Abstract

The invention discloses a method of improving a plant type of dendrocalamus latiflorus with a GRG1 gene and application of the method and belongs to the technical field of plant gene editing and dendrocalamus latiflorus breeding. In the method disclosed by the invention, a CRISPR/Cas-9 gene editing technology is utilized to perform directional editing on a gene conserved functional domain of GRG1in the dendrocalamus latiflorus, and directional knockout is performed on a genome by Cas9 proteins under the guidance of the sgRNA of a GRG1 targeted gene; by taking calluses of the dendrocalamus latiflorus as a receptor material for genetic transformation, a gene editing tool box is introduced into callus cells by an agrobacterium-mediated method for screening and identifying positive calluses;and for a transgenic dendrocalamus latiflorus plant obtained through the callus differentiation process, a positive plant subjected to genetic transformation is identified, the targeting gene editingcondition is determined, and finally, a dendrocalamus latiflorus plant line with GRG1 gene function deletion and accelerated growth is obtained.

Description

technical field [0001] The invention belongs to the technical fields of plant gene editing and bamboo breeding, and in particular relates to a method and application of a GRG1 gene for improving the plant type of bamboo. Background technique [0002] Mazhu is a large sympodial bamboo, a hexaploid species with three subgenomes (2n=72, AABBCC), widely distributed in the southern provinces of my country. It produces nutrient-rich bamboo shoots up to 20 meters high and 25-30 cm in diameter. Its products are widely used in construction, handicrafts, papermaking, water and soil conservation and other fields. Mazhu is an excellent dual-purpose bamboo for bamboo shoots. Its shoots are fresh and sweet, and the bamboo shoots last for a long time. Its bamboo poles are tall and strong. It can be used as building materials such as bamboo rafts and water pipes. It also has high ornamental value and economic value. As an important plant material, hemp bamboo has been difficult to meet t...

Claims

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Application Information

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IPC IPC(8): C12N15/29C12N15/113C12N15/82A01H4/00A01H5/00A01H6/46
CPCA01H4/00C07K14/415C12N15/113C12N15/8218C12N15/8261C12N2310/20
Inventor 朱强陈刚叶善汶
Owner FUJIAN AGRI & FORESTRY UNIV
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