Application of a gene that promotes the accumulation of linolenic acid in plant seeds

A plant seed and linolenic acid technology, applied in the field of gene application, can solve the problems of no obvious increase in the total amount of target fatty acids and a decrease in total oil content

Active Publication Date: 2020-10-30
HUAZHONG UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

A few studies reported that overexpression of LPAAT increased the percentage of target fatty acids in triglycerides but decreased the total oil content, and the total amount of final target fatty acids did not increase significantly

Method used

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  • Application of a gene that promotes the accumulation of linolenic acid in plant seeds
  • Application of a gene that promotes the accumulation of linolenic acid in plant seeds
  • Application of a gene that promotes the accumulation of linolenic acid in plant seeds

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Cloning of BnLPAAT2 Gene in Brassica napus

[0034] (1) RNA extraction from developing seeds of Brassica napus

[0035] The pipette tips, centrifuge tubes and solutions involved in the following operations are free from RNase contamination, and all operations are performed with gloves on.

[0036] (1) Preparation of extract: take 500 μl of RL lysate, then add 5 μl of β-mercaptoethanol and mix well.

[0037] (2) Homogenization treatment: remove the pods from the developing seeds in liquid nitrogen, quickly grind them into powder, add 500 μl RL lysate, and vortex vigorously to mix. Centrifuge at 12000rpm for 5min, and absorb the supernatant.

[0038] (3) Transfer all the solution to the filter column CS, centrifuge at 12000rpm for 2min, carefully pipette the supernatant in the collection tube into a new RNase-Free centrifuge tube, and try to avoid contacting the cell debris in the collection tube with the tip.

[0039] (4) Add 0.5 times the supernatant volume of absolu...

Embodiment 2

[0057] Overexpression vector construction

[0058] (1) Construction of plant expression vectors and Agrobacterium transformation

[0059] The cloned gene sequence was used as a template to amplify with the high-fidelity enzyme KOD plus from TOYOBO Company, and the obtained gene fragment was recovered by agarose gel for use; take 40 μl of the plasmid of the plant expression vector pBinGlyRed3 and add 4 μl of EcoR I enzyme and 5 μl of digestion buffer After mixing, enzyme digestion was performed at 37° for 30 minutes, and the carrier was recovered by agarose gel for use. Take 2 μl of gene and plasmid recovery fragments respectively, add 1 μl of infusion kit mix to mix, and ligate at 50° for 20 minutes. The ligation product was transformed into Escherichia coli and positive plaques were obtained, and then the plasmid was extracted for use. The recombinant plasmid carries the glycinin promoter to drive the specific expression of LPAAT2 gene in seeds ( figure 2 ). The recombin...

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Abstract

The invention discloses application of a gene for facilitating accumulation of linolenic acid in plant seeds. A gene segment has a nucleotide sequence or a complementary sequence shown as any one of SEQ ID No. 1 to SEQ ID No. 4, or a derivative nucleotide sequence which is not less than 95 percent in homology and has the same function as a nucleotide sequence shown as any one of SEQ ID No. 1 to SEQ ID No. 4 due to addition, deletion or substitution of one or more nucleotides. The function and application of Brassica napus lysophosphatidic acid acyltransferase 2 (BnLPAAT2) in facilitating the accumulation of the linolenic acid of the plant seeds are discovered for the first time, and the accumulation of the linolenic acid in the plant seeds can be facilitated effectively by introducing a gene segment with a specific nucleotide sequence and a protein with a specific amino acid sequence. The linolenic acid content of overexpression strain seeds is increased remarkably through a cloned BnLPAAT2 gene, and the oil content of the seeds is increased to a certain degree.

Description

technical field [0001] The invention belongs to the field of plant genetic engineering, and more specifically relates to the application of a gene for promoting the accumulation of linolenic acid in plant seeds. Specifically, the present invention uses molecular biology methods to isolate the gene BnLPAAT2 (Brassica napus LysophosphatidicAcid Acyltransferase 2), which can preferentially catalyze the esterification of linolenic acid to form phosphatidic acid, from the developing seeds of Brassica napus, which encodes an acyltransferase. Through the overexpression analysis in Arabidopsis, it was proved that BnLPAAT2 catalyzed the esterification of linolenic acid at the sn-2 position of triglyceride with preference, and finally increased the content of linolenic acid and oil content in seeds. Background technique [0002] In higher plants, oils mainly exist in the form of triglycerides, and triglycerides are mainly composed of a glycerol backbone and three fatty acids linked by...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/10C12N15/54A01H5/00A01H6/20
CPCC12N9/1025C12N15/8247
Inventor 栗茂腾尹永泰郭祯怡陈康刘思
Owner HUAZHONG UNIV OF SCI & TECH
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