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Method for rapidly identifying gene for regulating and controlling oil content of rape based on genomics approach and application thereof

A genomics and oil content technology, applied in chemical instruments and methods, botanical equipment and methods, biochemical equipment and methods, etc. Good application prospects, the effect of shortened time

Pending Publication Date: 2021-11-19
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, it takes a long time and a lot of work to mine the genes that regulate the oil content of rapeseed through the traditional QTL mapping method.

Method used

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  • Method for rapidly identifying gene for regulating and controlling oil content of rape based on genomics approach and application thereof
  • Method for rapidly identifying gene for regulating and controlling oil content of rape based on genomics approach and application thereof
  • Method for rapidly identifying gene for regulating and controlling oil content of rape based on genomics approach and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 Rapid Identification of Genes Regulating Rapeseed Oil Content Based on Genomics Approach

[0032] 1. RNA sample preparation of high oil content inbred line 1L99 and low oil content inbred line 1L363

[0033] Brassica napus high oil content inbred line 1L99 and low oil content inbred line 1L363 were planted in the experimental field of Huazhong Agricultural University during the sowing season. The siliques at different developmental stages were collected at 11 days, 16 days, 23 days, 30 days, 37 days and 44 days after flowering, and three biological replicates were taken for samples at each stage. According to the instructions of the RNA extraction kit (TransZol Plant, BioTeke, China), the sample RNA was extracted, and the RNA was treated with RNase-free DNase-I (Thermo Scientific, USA) to remove gDNA contamination, and a nucleic acid / protein analyzer was used 730 (Beckman ) for quantification.

[0034] 2. Library construction, sequencing and analysis

[...

Embodiment 2

[0038] Example 2 Verification of the gene function of BnECH.A5 by CRISPR / Cas9-mediated gene knockout technology

[0039] 1. Screen target sequences and design primers

[0040] The nucleotide sequence (such as SEQ ID NO. 17) of the Brassica napus BnECH.A5 gene was obtained from the reference genome Darmor-bzh. Two targets, T1 and T2, were designed and screened (Table 1), and primers were designed for the target sequences (Table 2).

[0041] Table 1 Target sequence

[0042]

[0043] Table 2 target primer sequence

[0044]

[0045]

[0046] 2. Construction of the two-target gene editing vector pBnECH vector

[0047] (1) Target linker preparation: Dissolve the linker primers in Table 2 with ddH2O into a 10 μM stock solution. Add 1 μl of the left and right primers to 8 μl ddH2O, mix and dilute to 1 μM, and vortex to mix. Then put it into a water bath at 90°C, treat it for 30", and let it cool down to room temperature naturally.

[0048] (2) sgRNA expression cassette ...

Embodiment 3

[0134] Example 3 Rapeseed BnECH.A5 mutation can be used to increase seed oil content

[0135] After the mutant plants were obtained in Example 2, the homozygous mutant plants were obtained by selfing.

[0136] The oil content of mutant seeds was determined as follows.

[0137] Sample preparation:

[0138] 1) The mutant and wild-type seeds were dried in a constant temperature incubator at 37°C for 48 hours to ensure the consistency of seed moisture content. Each individual plant took out 50 mg seeds of uniform size and weighed and recorded the weight for subsequent use;

[0139] 2) Crush the weighed rape seeds with a mortar, place them in a clean 10ml glass test tube, and spin the seeds to the bottom of the tube by centrifugation;

[0140] 3) Add 1.5ml 2.5% sulfuric acid chromatographically pure methanol solution (0.01% BHT), 200μl standard sample (heptadecanoic acid triglyceride, 2mg / ml), 400μl chromatographically pure toluene to the above glass tube, and tighten the screw ...

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Abstract

The invention belongs to the technical field of biology, and particularly relates to a method for rapidly identifying a gene for regulating and controlling the oil content of rape based on a genomics approach and an application of the method. According to the technical key points, the method comprises the following steps: screening a high-oil-content inbred line 1L99 and a low-oil-content inbred line 1L363 through field multi-year multi-point tests, and then carrying out transcriptome sequencing analysis on seeds of the two inbred lines in six different development stages; differential genes obtained through transcriptome analysis are scanned at a qOC-A5QTL site, and it is found that the expression quantity of the BnECH.A5 gene in different rape seed development stages of a high-oil-content inbred line is low, the expression quantity of the BnECH.A5 gene in a low-oil-content inbred line is high, and the expression quantity of the BnECH.A5 gene in the key stage of seed oil content accumulation within 30-44 days after flowering changes drastically. The mutant plant of the gene is constructed, the result shows that the oil content of a function-deleted mutant plant is remarkably increased compared with that of a wild plant.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for quickly identifying genes regulating oil content of rapeseed based on a genomics approach and an application thereof. Background technique [0002] Rapeseed is an important oil crop and the main source of edible vegetable oil in my country. It is very important to increase the oil content of rapeseed to increase the yield of rapeseed oil. Studies have shown that every 1% increase in oil content is equivalent to a 2.3%-2.5% increase in rapeseed yield, and germplasm resources with high oil content are the key to breeding new varieties. Therefore, strengthening the analysis of the genetic basis of oil content metabolism in rapeseed is of great significance for creating new germplasm resources with high oil content. [0003] However, because oil content is greatly affected by the environment, genetic regulation is complex, and the genome of Brassica napus is re...

Claims

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Application Information

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IPC IPC(8): C12Q1/6869C12Q1/6895C12N15/82A01H5/10A01H6/20
CPCC12Q1/6869C12Q1/6895C12N15/8218C12N15/8247C07K14/415C12Q2600/158C12Q2600/13C12Q2535/122Y02A50/30
Inventor 黄会斌周永明赵青穆罕默德·沙希徳柯昌森蔡光勤王学敏范楚川
Owner HUAZHONG AGRI UNIV
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