Method for specifically knocking out soybean SOC1 gene by CRISPR/Cas9 and application of method

A SOC1, specific technology, applied in the field of plant genetic engineering and bioengineering, can solve problems such as unclear specific functions, achieve the effect of increasing the number of main stem nodes, increasing the yield of a single plant, and increasing the yield

Active Publication Date: 2021-08-17
GUANGZHOU UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the functions and molecular regulatory pathways of SOC1-like genes regulating flowering and plant development in Arabidopsis and other species have been preliminarily studied, their specific functions in soybean are still unclear

Method used

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  • Method for specifically knocking out soybean SOC1 gene by CRISPR/Cas9 and application of method
  • Method for specifically knocking out soybean SOC1 gene by CRISPR/Cas9 and application of method
  • Method for specifically knocking out soybean SOC1 gene by CRISPR/Cas9 and application of method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 Design and construction of CRISPR / Cas9 vector

[0032] In this example, based on the CRISPR / Cas9 gene editing system, the soybean homologous genes SOC1a and SOC1b were edited, and a CRISPR / Cas9 knockout vector for simultaneously targeting and editing the coding regions of SOC1a and SOC1b was created. The specific creation method is as follows:

[0033] 1. Target primer design

[0034] According to SOC1a provided by NCBI (National center for biotechnology, https: / / www.ncbi.nlm.nih.gov / ) and Phytozome (https: / / phytozome.jgi.doe.gov / pz / portal.html) two databases (Glyma.18G224500) and SOC1b (Glyma.09G266200) gene sequences, using the CRISPR direct website (http: / / crispr.dbcls.jp / ) to design target site adapter primers. Click Specificity check-Soybean(Glycine max)genome,v2.0(Nov, 2015)-design, and select the first 20 bp of the appropriate PAM sequence (NGG) as the target primer, including 1 / 3 / 4 targets located in SOC1b and Located on 2 / 4 targets of SOC1a.

[003...

Embodiment 2

[0051] Example 2 Identification of CRISPR / Cas9 Knockout Vector Genetically Transformed Soybean and Transgenic Soybean

[0052] 1. Stable genetic transformation of soybean with CRISPR / Cas9 knockout vector

[0053] 1) Hair root transformation and target editing effect detection

[0054] The CRISPR / Cas9 knockout vector constructed in Example 1 containing four tandem target sites was transformed into Agrobacterium K599, cultured at 28°C for 36 hours, and positive clones were selected to infect the cotyledon of Willimas82 soybean to induce hairy roots. The hairy roots grown for 15 days under light culture at 25°C were taken for DNA extraction (CWBIO DNA Kit). Cas9-specific primers (SEQ ID.NO.9 and SEQID.NO.10) were used to detect positive hairy roots, and target editing detection primers (SEQ ID.NO.11~SEQ ID NO.20) were used to perform PCR amplification and analysis of positive hairy roots. Sequencing, to count the editing efficiency of the target. The sequencing results showed ...

Embodiment 3

[0067] Phenotype identification of embodiment 3 soc1a1b double mutant plants

[0068] Homozygous soc1a1b double mutant plants and control Willimas82 (W82) plants were planted in the potted field of the Northeast Institute of Geography and Agroecology, Chinese Academy of Sciences, under natural long-day conditions (16 hours of light / 8 hours of darkness) and artificially set short-days (light 12 hours / dark 12 hours). Observe their flowering and maturity stages, and count the number of nodes and yield per plant when they are mature. The flowering period, maturity period, number of nodes and yield per plant of mutant plants and W82 plants are as follows: Figure 4 shown.

[0069] in, Figure 4 A is the phenotype of flowering stage, maturity stage, number of nodes and yield per plant of W82 and soc1a1b double mutant plants under short-day conditions in 2019; Figure 4 B is the phenotype of flowering stage, maturity stage, number of nodes and yield per plant of W82 and soc1a1b d...

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Abstract

The invention belongs to the field of plant genetic engineering, and particularly relates to a method for specifically knocking out a soybean SOC1 gene through CRISPR/Cas9 and application of the method. The CRISPR/Cas9 knockout vector capable of simultaneously performing targeted editing on coding regions of homologous genes SOC1a and SOC1b in the soybean is obtained through construction, and the knockout vector is subjected to stable genetic transformation by utilizing an agrobacterium-mediated method to obtain a homozygous soc1a1b double-mutant soybean plant. Compared with a control soybean plant, the soc1a1b double mutant plant shows obvious phenotypes of delayed flowering period and mature period and increased node number, and the soybean yield and the regional adaptability of varieties are remarkably improved.

Description

technical field [0001] The invention belongs to the field of bioengineering, relates to the field of plant genetic engineering, in particular to a method for specifically knocking out soybean SOC1 gene by CRISPR / Cas9 and its application. Background technique [0002] Soybean [Glycine max(L.)Merr.] provides human beings with vegetable protein and vegetable oil, as well as industrial raw materials, and is closely related to human life. Cultivated soybeans were domesticated in the Huanghuaihai region of China about 5,000 years ago, and are known as the "king of beans". Soybean is a typical short-day dicotyledonous plant, which is extremely sensitive to photoperiod. The flowering time determines the regional adaptability, sowing time and harvesting time of soybean. SOC1 is an important flowering-inducing gene in Arabidopsis, which is ubiquitous in a variety of angiosperms. Although the functions and molecular regulatory pathways of SOC1-like genes regulating flowering and pla...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12N15/55C12N15/29A01H5/02A01H5/04A01H5/00A01H6/54C12Q1/6895C12N15/11
CPCC12N15/8218C12N15/827C12N15/8261C12N15/8205C12N9/22C07K14/415C12Q1/6895C12Q2600/156C12Q2600/13
Inventor 孔凡江杨慧寇坤
Owner GUANGZHOU UNIVERSITY
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