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Peanut seed embryo specificity promoter and cloning and application thereof

A promoter and specific technology, applied in the field of plant transgenic and molecular genetics, can solve the problems affecting the normal development of plants and energy waste

Inactive Publication Date: 2013-07-17
山东省农业科学院高新技术研究中心 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, in plant genetic engineering research, mostly constitutive promoters are used, such as CaMV35S promoter, corn Ubiquitin promoter, etc. These promoters can drive foreign genes to be expressed efficiently in all tissues and organs of plants, resulting in energy waste , the expression of some exogenous genes also affects the normal development of plants

Method used

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  • Peanut seed embryo specificity promoter and cloning and application thereof
  • Peanut seed embryo specificity promoter and cloning and application thereof
  • Peanut seed embryo specificity promoter and cloning and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1 Cloning of Peanut AhLEC1A Gene Promoter

[0028] 1.1 Extraction of peanut genomic DNA

[0029] The DNA of young peanut leaves was extracted with the method provided by the plant DNA extraction kit (product of Beijing Tiangen Biochemical Technology Co., Ltd.), and dissolved in an appropriate amount of TE (pH 8.0) buffer.

[0030] 1.2 Cloning of peanut AhLEC1A gene promoter

[0031] 2.5 μg of peanut genomic DNA was digested with DraI, EcoRV, PvuII, StuI endonucleases, extracted and purified with phenol, and dissolved in 20 μl of TE (pH7.5) buffer. Take 4 μl of digested complete DNA, connect to Adaptor according to the requirements of BD Genome Walker Universal Kit (sequence: 5′GTAATACGACTCACTATAGGGCACGCGTGGTCGACGGCCCGGGCTGGT 3′, as shown in Seq ID No:5), and construct 4 peanut genomic DNA libraries (LD, LE , LP, LS). According to the obtained genome sequence of the peanut AhLEC1A gene, two nested 3′ end-specific primers LEC1AGSP1-3 (5′TGGGGATGGATAGAGAAACCAACG...

Embodiment 2

[0046] Example 2 Construction and Transformation of Plant Expression Vectors

[0047] 2.1 Fusion of peanut AhLEC1A gene promoter sequence and GUS reporter gene

[0048] Select pCAMBIA3301 as the plant expression vector, digest the target fragment obtained by PCR amplification, electrophoresis and gel recovery. At the same time, the expression vector was digested with restriction endonucleases HindⅢ and NcoI, and the vector fragment was recovered. The promoter fragment and the vector fragment were connected, and pCAMBIA3301-AhLEC1A promoter recombinant vectors containing promoters of different lengths were screened by plasmid restriction map identification. The specific process can refer to the attached image 3 :

[0049] 1. Use primers P1 with restriction sites and P2-P8 with different restriction sites to amplify the different length promoter sequences of peanut AhLEC1A gene, and separate and recover the target fragments by gel electrophoresis.

[0050] 2. Recover the targ...

Embodiment 3

[0074] Example 3 Histochemical staining of GUS activity on seeds of different tissues and different developmental stages of transgenic plants

[0075] 3.1 GUS active histochemical staining method:

[0076] 1. Place the sample in an ice-water mixture for 2 minutes;

[0077] 2. Dry the sample with filter paper and put it into a centrifuge tube, add 90% acetone to soak the sample for 10 minutes;

[0078] 3. After washing the sample quickly with GUS staining buffer, add GUS staining solution and soak for 5 minutes, and pour off the dyeing solution;

[0079] 4. Add an appropriate amount of dye solution to immerse the sample, and pump air twice, 5 minutes each time;

[0080] Store overnight at 5.37°C constant temperature;

[0081] 6. Add absolute ethanol to decolorize, then transfer to water. Observe and take pictures with the naked eye or under an anatomical microscope.

[0082] 3.2 GUS staining buffer formula:

[0083] 100ml buffer

[0084]

[0085] wxya 2 O was adjuste...

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Abstract

The invention belongs to the technical field of biology and discloses a novel promoter, of peanut LEC1 gene, obtained by cloning. The gene sequence of the promoter is shown as SeqIDN.4, and the promoter promotes the gene to specifically express in an embryo or endosperm at a specific stage of seed development. The promoter and six plant expression carriers of GUS reporter genes promoted by the promoter and with 5' end serial deletion are constructed, and arabidopsis is converted. Specific expression in the endosperm of a 5' untranslated region and the GUS gene promoted by the promoter and at upstream of the region at an early development stage is finally confirmed, and cloning and deletion analysis of the promoter have important theoretical significance and actual application value on studying plant seed development and reserve substance accumulation and improving the content of reserve substances like starch, fat and protein in seeds.

Description

technical field [0001] The invention relates to the field of molecular genetics, in particular to the field of plant transgenic technology, in particular to the cloning of a new peanut gene-specific promoter. Background technique [0002] Seeds play an important role in agricultural production and national economy. Statistics show that 80% of human food is directly taken from plant seeds. Rice, wheat and other seeds are rich in starch, which is the staple food for human survival; soybeans, peanuts, rapeseed, sesame and other seeds have high oil content and are edible. main source of oil. The amount of stored substances in the seeds directly affects the yield of crops. Therefore, the root of improving the yield of food crops and oil crops is to increase the accumulation of storage substances in their seeds. Although the excellent crop varieties cultivated by conventional breeding techniques have made significant contributions to solving the world's food security problems. ...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/10C12N15/82C12N15/84A01H5/00
Inventor 单雷唐桂英徐平丽柳展基陈营
Owner 山东省农业科学院高新技术研究中心
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