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Application of gene promoter region sequence of glycerophospholipid peptidy transeferace in crop genetic improvement

A gene promoter region, glycerol phosphate acyl technology, applied in the direction of plant gene improvement, application, genetic engineering, etc., can solve the problems of inability to store organs - seed modification and improvement, neglect, limited promoter types, etc., to achieve high Stability and operability effects

Inactive Publication Date: 2012-05-23
HAINAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, from the point of view of actual application effects, the types of promoters that can be successfully applied to crop genetic improvement are still very limited, and it is impossible to effectively target the important storage organs of crops—seeds for directional modification and improvement
[0004] In the past, during the cloning process of seed endosperm-specific promoters, the research was mainly on enzymes related to carbohydrate and protein metabolism, but the cloning of promoters based on fat metabolism enzymes was ignored.

Method used

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  • Application of gene promoter region sequence of glycerophospholipid peptidy transeferace in crop genetic improvement
  • Application of gene promoter region sequence of glycerophospholipid peptidy transeferace in crop genetic improvement
  • Application of gene promoter region sequence of glycerophospholipid peptidy transeferace in crop genetic improvement

Examples

Experimental program
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Effect test

Embodiment 1

[0029] Example 1 Extraction of coconut genomic DNA

[0030] High-quality coconut genomic DNA was extracted from young leaves by the CTAB method. After the material is ground in liquid nitrogen, take 0.1g and add 600μL of CTAB extract (containing 4% β-mercaptoethanol), continue to grind to make it evenly suspended, place in 65℃ water bath for 45min; add 700μL of phenol:chloroform:isoamyl alcohol, 37℃ water bath Warm bath for 10 minutes. Centrifuge at 12000rpm for 10min; take the supernatant, add 60μL 5M NaCl, 1mL frozen absolute ethanol, freeze for 30min to precipitate DNA; centrifuge at 10000rpm for 5min, discard the alcohol and air dry. Add TE to the air-dried DNA, water bath at 37°C for 15-30min until the DNA is completely dissolved, add 700μL of chloroform: isoamyl alcohol (24:1), invert for 15min, centrifuge at 12000rpm for 5min; take the supernatant, add 30μL of 5M NaCl, 1mL freeze Precipitate with absolute ethanol at -20°C for 30 min, centrifuge at 10000 rpm for 3 min, di...

Embodiment 2

[0031] Example 2 Cloning and analysis of the upstream promoter sequence of LPAAT gene

[0032] The cloning of the upstream promoter sequence of the glycerophosphate acyltransferase gene was performed according to the operating manual of the Universal GenomeWalker Kit (Clontech). Genomic DNA was first digested with 4 blunt-end endonucleases (EcoRI, Pvu II, Dra I, Stu I). The digested product is purified and ligated with a specific linker. The ligated product is used in the first round of nested PCR reaction. The nucleotide sequence of the specific primer for the glycerophosphate acyltransferase gene is shown in SEQ ID NO: 2. The linker specific primer The nucleotide sequence is shown in SEQ ID NO:3. The PCR reaction program is: 94°C for 25s, 72°C for 3min, 7 cycles: 94°C for 25s, 67°C for 3min, 32 cycles; 67°C for 7min. In the second round of PCR, the product of the first round of PCR reaction was used as the substrate, and the same amplification procedure was applied. The nucle...

Embodiment 3

[0033] Example 3 Construction and genetic transformation of plant expression vectors

[0034] In order to verify the effect of the obtained promoter region sequence in tissue-specific expression control, three different types of promoter fragments deleted at the 5'end were used in the construction of plant expression vectors. The deletion fragments were inserted into the promoter verification expression vector pBI101.3, respectively named pBI101.3-L3, pBI101.3-L2, pBI101.3-L1. The 3 fragment deletion sites were -896, -817 and- 453. Both ends of the PCR amplification primers were added with 8-base restriction sites HindIII and BamHI. The relevant primer sequences are:

[0035] The nucleotide sequence of p101-1L is shown in SEQ ID NO: 6;

[0036] The nucleotide sequence of p101-2L is shown in SEQ ID NO: 7;

[0037] The nucleotide sequence of p101-3L is shown in SEQ ID NO: 8;

[0038] The nucleotide sequence of p101-R is shown in SEQ ID NO:9.

[0039] After the amplified product was rec...

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Abstract

The invention discloses a gene promoter region sequence of glycerophospholipid peptidy transeferace (LPAAT) and application in crop genetic improvement. The gene promoter region sequence of glycerophospholipid peptidy transeferace is shown as SEQ ID NO:1. The application method of the gene promoter of glycerophospholipid peptidy transeferace in crop genetic improvement comprises the following steps of: (1) inserting the gene promoter of glycerophospholipid peptidy transeferace into a promoter verifying expression vector pBI101.3 and starting the expression of a gus reporter gene; and (2) using the expression carrier for plant callus transgenosis research induced by agrobacterium tumefacien and obtaining the transgenosis plant by inducing, dip dyeing, coincubating and taking root of the plant callus. The gene promoter comes from coconut, can specially start expression of target gene in endosperm histiocyte, provides new promoter type in the future crop genetic improvement and has very high stability and operability without transgenic silencing.

Description

Technical field [0001] The present invention relates to the field of plant breeding, in particular to obtaining a glycerophosphate acyltransferase (LAPPT) gene promoter by using genetic engineering technology, and applying it to rice endosperm transgene operation and genetic improvement. Background technique [0002] Endosperm is an important storage tissue type for starch and protein in crop seeds. 60% of the energy and protein in the world's food production are derived from crop endosperm. In the process of crop transgenic research, compared with other expression organs and tissue types, the application of crop endosperm as a platform for heterologous gene or protein expression has many advantages such as low cost, easy control, large yield, and safe production process (Takaiwa et al, 2007, Endosperm tissue is a good production platform for artificial recombinant proteins intransgenic rice. Plant Biotechnol J, 5, 84-92.; Wu et al, 2007, Oral immunization with transgenic rice se...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/82C12N15/84C12P19/34
Inventor 李东栋林拥军郑育声
Owner HAINAN UNIVERSITY
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