Method for obtaining rape variety with low seed glucosinolate content

A rape and glucosinolate technology, which is applied in the directions of chemical instruments and methods, botanical equipment and methods, biochemical equipment and methods, etc., can solve the problems of reduced disease resistance and aggravation of diseases and insect pests in rapeseed, and achieves easy operation and guaranteed disease resistance. high-efficiency editing effect

Inactive Publication Date: 2019-11-12
WUHAN ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

When the content of glucosinolates in rapeseed is reduced, the content of glucosinolates in its vegetative organs will also be reduced, which leads to the decrease of rapeseed's disease resistance and the aggravation of pests and diseases

Method used

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  • Method for obtaining rape variety with low seed glucosinolate content
  • Method for obtaining rape variety with low seed glucosinolate content
  • Method for obtaining rape variety with low seed glucosinolate content

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] The acquisition of the rape plant of embodiment 1 seed low glucosinolate content

[0033] Brassica napus 13CK-1 (119 μmol / g glucosinolate content) and 13CK-3 (129 μmol / g glucosinolate content) were selected as the recipient lines for genetic transformation, both of which belong to rape materials with high glucosinolate content in seeds. According to the sequence information of Brassica napus reference genome website (http: / / www.genoscope.cns.fr / brassicanapus / ), BnaA02g33530D was isolated from the materials of 13CK-1 and 13CK-3 and named as BnGTR2A2. The gene sequence is shown as SEQ ID NO .1 shown.

[0034] The sequence of the BnGTR2A2 gene was submitted to the CRISPR-P (http: / / cbi.hzau.edu.cn / crispr / ) website, and the gene editing target sites with a low off-target rate were screened and named GTR2SgRNA1 and GTR2SgRNA2. The sequence of the GTR2SgRNA1 is as follows: Shown in SEQ ID NO.2, specifically: CTTGGGCCTGTACACGTGA GGG , GGG It is a PAM sequence; the sequence...

Embodiment 2

[0041] Example 2 Analysis of Editing Sites in Transgenic Positive Plants

[0042] In order to further confirm the editing efficiency of positive individual plants, primers were designed to analyze the sequences of the target sites GTR2A2SgRNA1 and GTR2A2SgRNA2 of the BnGTR2A2 gene in the gene-edited 13CK-1 material (K1m for short). In order to obtain the target gene sequence from the K1m material, the target fragment was amplified by checkGTR2A2-F and checkGTR2A2-R, and the mutation type was analyzed by sequencing. At the same time, T1-N2gtr2a2-F and T1-N2gtr2a2-R, T2-N2gtr2a2-F and T2-N2gtr2a2-R primers were respectively designed to amplify the two target sites of GTR2A2SgRNA1 and GTR2A2SgRNA2 and analyzed by polyacrylamide gel electrophoresis. The sequences are shown in Table 3, and the detection results are as follows Figure 4 with Figure 5 shown.

[0043] Table 3 Primers for detecting gene editing sites

[0044]

[0045] Sequencing analysis of the GTR2A2SgRNA1 tar...

Embodiment 3

[0048] Example 3 Detection and Analysis of Seed Glucosinolate Content of Gene Edited Plants

[0049] Using liquid chromatography detection method to analyze the non-transgenic material and the target gene mutated T 0 generation plants and gene-edited T 1 Contents of glucosinolates in mature seeds harvested from generations of families,.

[0050] The determination of glucosinolate content adopts the international standard method of ISO9167-1:1992 (E) high performance liquid chromatography, as follows: ① glucosinolate extraction: Accurately weigh 0.2000g of the prepared sample into a centrifuge tube, and keep it warm in a 75°C water bath Inactivate for 1 min, add 2ml 70% methanol extract and 200μl internal standard, centrifuge, transfer the supernatant to a test tube, then add 2ml 70% methanol to extract and centrifuge, repeat 3 times, mix the supernatant, take 2ml solution on Acetic acid type DEAE-Sephadex A-25 anion exchange column, add 500 μl of sulfatase, take out after 20...

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Abstract

The invention provides a method for obtaining a rape variety with the low seed glucosinolate content. A rape glucosinolate transporter (GTR) is edited through CRISPR / Cas9, specifically, GTR editing target sites are determined through screening, a plurality of primers are synthesized according to information of a CRISPR / Cas9 carrier and target sites, and the primers are constructed into the CRISPR / Cas9 carrier, so that the rape GTR is mutated, and glucosinolates are inhibited from being transferred to rape seeds. According to the method, through the mutated rape GTR, the new rape variety with the low glucosinolate content in the seeds is obtained, the content of glucosinolates in rape leaves and stalks is not affected, disease resistance of rape is ensured, and good application prospects are realized.

Description

technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to a method for obtaining rapeseed varieties with low glucosinolate content in seeds by using CRISPR / Cas9 to edit the rapeseed glucosinolate transport gene GTR. Background technique [0002] Rapeseed is the largest oil crop in my country, and domestic rapeseed oil accounts for more than 55% of the oil production of domestic oil crops. The remaining cake after rapeseed oil extraction is rich in protein and reasonable in amino acid composition, which is a high-quality feed protein source. However, the oil-extracted cakes of traditional rapeseed varieties contain a large amount of glucosinolates (glucosinolates for short), which will cause goiter and even poisoning when eaten by livestock and poultry, which greatly limits the rational utilization of rapeseed cakes. However, glucosinolates and their degradation products can participate in plant defense mechanisms, which are clos...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12N15/29
CPCC07K14/415C12N15/8218C12N15/8243
Inventor 万丽丽杨光圣王转茸洪登峰孙玉宏高红霞杨皓琼谭庆
Owner WUHAN ACADEMY OF AGRI SCI
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