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Starch synthase gene promoter with seed-specific expression and application thereof

A starch synthase and promoter technology, applied in the fields of plant molecular biology and plant genetic engineering, can solve problems such as activity restriction

Inactive Publication Date: 2009-09-09
SICHUAN AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The existing seed-specific promoters are mainly derived from plant storage protein genes, such as prolamin gene promoter, peanut seed legumin legA gene promoter, rice glutenin gluA and gluB gene promoters and zein gene Promoters, etc., but an obvious defect of storage protein gene promoters is that the activity is generally limited by nitrogen supply

Method used

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  • Starch synthase gene promoter with seed-specific expression and application thereof
  • Starch synthase gene promoter with seed-specific expression and application thereof
  • Starch synthase gene promoter with seed-specific expression and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0118] Example 1: Analysis of maize dull1 gene expression site

[0119] 1. Extraction of total RNA from different tissues of corn in different periods

[0120] Take 50-100 mg of fresh tissue or tissues preserved in liquid nitrogen at -70°C, grind with liquid nitrogen, place in a 1.5 mL centrifuge tube, add 1 mL of Trizol (purchased from Invitrogen) and shake well, and let stand at room temperature for 5 minutes. Add 0.2mL chloroform, shake for 15sec, and let stand for 2min. Centrifuge at 12000r / min for 15min at 4°C and take the supernatant. Add an equal volume of ethylene glycol butyl ether, gently mix the liquid in the tube, and let it stand in an ice bath for 1 hour. Centrifuge at 10000r / min for 10min at 4℃, discard the supernatant. Add 1 mL of 75% ethanol and gently wash the precipitate. Centrifuge at 7500r / min at 4℃ for 5min, and discard the supernatant. Add 1 mL of 100% ethanol and gently wash the precipitate. Centrifuge at 7500r / min at 4℃ for 5min, discard the supernatant, d...

Embodiment 2

[0151] Example 2: Cloning of 5'flanking sequence of corn dull1

[0152] 1. Extraction of maize genomic DNA:

[0153] Weigh 1.0g of fresh corn leaves, cut them into 1cm pieces, put them in a pre-cooled mortar, and quickly grind them into uniform powder with liquid nitrogen. Before the tissue is dissolved, put all the powder into a 5mL centrifuge tube. Quickly add 2 mL of CTAB extraction buffer incubated at 65°C to the centrifuge tube, shake gently to mix, and then bath at 65°C for 30 min-1h, add an equal volume of chloroform: isoamyl alcohol (24:1), and mix gently. Shake for 15min. Centrifuge at 10000r / min for 8min at room temperature. Pipette the supernatant into a clean 5mL centrifuge tube. Add an equal volume of pre-cooled isopropanol (4°C) and centrifuge at 10000r / min for 5min at room temperature. The supernatant was discarded, and the precipitate was washed twice with 75% ethanol and once with absolute ethanol. Dry in a vacuum desiccator for 10 min, dissolve the DNA with an ap...

Embodiment 3

[0204] Example 3: Verification of dull1P promoter's promoter activity

[0205] 1. Construction of plant expression vector:

[0206] According to the SEQ ID No:1 sequence, design and synthesize a promoter primer containing restriction enzyme sites, use the 3.5Kb sequence linked to the sequencing vector pMD18-T as a template to clone the 1.8Kb dull1 gene promoter by PCR amplification Subsequence.

[0207] Pde 1: 5’---CACTCGTCGACGCCTTTGAGACTG---3’

[0208] Pde 2: 5’---TCTCCATGGCTAGAAGGGGAAGAAAAGAAG----3’

[0209] The underlined part of Pde1 is the SalI restriction site contained in the sequence itself, and the underlined part of Pde2 is the added NcoI restriction site.

[0210] The reaction system is as follows:

[0211] 10×LA PCR buffer 5μL

[0212] dNTP(10mmol / L) 5μL

[0213] MgCl 2 5μL

[0214] Pde1(10mmol / L) 2μL

[0215] Pde2(10mmol / L) 2μL

[0216] Die plate (10ng / μL) 0.5μL

[0217] LA Taq enzyme (5U / μL) 0.5μL

[0218] ddH 2 O 30μL

[0219] Total volume 50μL

[02...

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Abstract

In the invention, a Genomic walking method is employed to design a specific nested primer according to a cDNA sequence (GenBank Accession No.: AF036891) of a maize dull 1 gene, maize genome is taken as a template to separate a maize starch synthase gene dull 1 promoter from maize. The promoter has a spatiotemporal specific expression property, can drive target genes to efficiently express in plant seeds at grain filling stage, but is not expressed at leaves, leaf veins and roots, and has wide use in research of plant bioreactor. The promoter lays a foundation for researching an expression regulating and control mechanism of plant starch anabolism, creates a condition for starch biosynthesis regulation and control in gene engineering technology, and plays an important role in the research of plant starch improvement and the plant bioreactor.

Description

Technical field [0001] The present invention relates to the fields of plant molecular biology and plant genetic engineering, in particular to a seed-specifically expressed starch synthase gene promoter and its application. Background technique [0002] Seeds are the main source of food for humans and animals, as well as raw materials for many industries, and are closely related to human production and life. However, due to the limitations of plants themselves, the protein and other ingredients contained in seeds cannot fully meet human needs. With the rapid development of modern molecular biology, the application of genetically modified plants in theoretical research and plant breeding has become more and more extensive. Through DNA recombination technology, plants can not only obtain some insect resistance, disease resistance, and stress resistance that they do not possess. , It can also synthesize some essential amino acids that it does not have in the seeds to improve the nutr...

Claims

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Application Information

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IPC IPC(8): C12N15/29C12N15/63C12N5/10C12N1/15C12N1/19C12N1/21
Inventor 黄玉碧荣廷昭胡育峰张军杰李晓兵田孟良刘汉梅
Owner SICHUAN AGRI UNIV
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