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Vegetable starch synthetase genetic promoter and use thereof

A technology of starch synthase and promoter, applied in the direction of plant gene improvement, application, genetic engineering, etc., can solve the problems of unsatisfactory strength and time

Inactive Publication Date: 2009-10-14
SICHUAN AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In previous studies on the regulation of starch biosynthesis by genetic engineering, endosperm-specific promoters such as cereal storage protein genes were mostly used. Compared with constitutive promoters, although these promoters can correctly drive foreign genes in specific tissues and organs of starch synthesis Expressed, but the intensity and timing of expression are not ideal

Method used

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  • Vegetable starch synthetase genetic promoter and use thereof
  • Vegetable starch synthetase genetic promoter and use thereof
  • Vegetable starch synthetase genetic promoter and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0110] Example 1: Analysis of the expression site of the maize zSs1 gene

[0111] 1. Extraction of total RNA from different tissues in different periods of maize

[0112] Take 50-100 mg of tissue fresh or stored at -70°C in liquid nitrogen, grind it with liquid nitrogen, put it in a 1.5 mL centrifuge tube, add 1 mL of Trizol (purchased from Invitrogen) to fully shake, and let it stand at room temperature for 5 min. Add 0.2mL chloroform, shake for 15sec, and let stand for 2min. Centrifuge at 12000r / min at 4°C for 15min, and take the supernatant. Add an equal volume of ethylene glycol butyl ether, mix the liquid in the tube gently, and let it stand in an ice bath for 1h. Centrifuge at 10000r / min at 4°C for 10min, and discard the supernatant. Add 1 mL of 75% ethanol and gently wash the pellet. Centrifuge at 7500r / min at 4°C for 5min, and discard the supernatant. Add 1 mL of 100% ethanol and gently wash the pellet. Centrifuge at 7500r / min at 4°C for 5min, discard the superna...

Embodiment 2

[0143] Cloning of embodiment 2, zSs1 gene 5' flanking sequence

[0144] 1. Extraction of maize genomic DNA.

[0145] Weigh 1.0 g of fresh corn leaves, cut them into 1 cm pieces, place them in a pre-cooled mortar, grind them into a uniform powder quickly with liquid nitrogen, and put all the powders into a 5 mL centrifuge tube before the tissue is dissolved. Quickly add 2mL of CTAB extraction buffer incubated at 65°C to the centrifuge tube, shake gently to mix, then bathe in water at 65°C for 30min-1h, add an equal volume of chloroform:isoamyl alcohol (24:1), mix well, gently Shake for 15 minutes. Centrifuge at 10000r / min for 8min at room temperature. Pipette the supernatant into a clean 5 mL centrifuge tube. Add an equal volume of pre-cooled isopropanol (4° C.), and centrifuge at 10,000 r / min for 5 min at room temperature. Discard the supernatant, wash the pellet twice with 75% ethanol and once with absolute ethanol. Dry in a vacuum desiccator for 10 min, dissolve DNA wit...

Embodiment 3

[0197] Embodiment 3, the application of starch synthase gene promoter in plant bioreactor

[0198] 1. Construction of plant expression vectors:

[0199] According to the sequence of SEQ ID No: 1, design and synthesize promoter primers containing restriction enzyme sites, and use the 1.6Kb sequence connected to the sequencing vector pMD18-T as a template to clone the 1.6Kb zSs1 gene promoter by PCR amplification method subsequence.

[0200] Pse1: 5'---CCAAGCTTTTGATGTTCTTTTTTTGTGTCTG---3'

[0201] Pse2: 5'---CGCCATGGCGGAGAGGGAGAGCAGACAG---3'

[0202] The underlined part of Pse1 is the added HindI restriction site, and the underlined part of Pse2 is the added NcoI restriction site.

[0203] The reaction system is as follows:

[0204] 10×LA PCR buffer 5μL

[0205] dNTP (10mmol / L) 5μL

[0206] MgCl 2 5μL

[0207] Pse1 (10mmol / L) 2μL

[0208] Pse2 (10mmol / L) 2μL

[0209] Mold (10ng / μL) 0.5μL

[0210] LA Taq enzyme (5U / μL) 0.5μL

[0211] wxya 2 O 30μL ...

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Abstract

The invention discloses a vegetable starch synthetase genetic promoter and use thereof. The promoter is separated from corn genome DNA with a nucleotide sequence which is showed as listed nucleotide sequence SEQ ID NO: 1; length of the nucleotide sequence is 1655bp. The vegetable starch synthetase genetic promoter of the invention can promote high expression of gene in leaf and seed without expression in root. The promoter is applicable to driving of gusA gene expression. The promoter plays an important role in vegetable starch quality improvement and vegetable biological reactor.

Description

technical field [0001] The present invention relates to the fields of plant molecular biology and genetic engineering, in particular to a promoter of a starch synthase gene isolated from a corn genome, as well as the spatiotemporal specificity of the promoter driving the expression of a target gene and its presence in plant starch. Improvement and application in plant bioreactors. Background technique [0002] Starch is not only the main source of food and feed, but also widely used in many fields such as medicine, chemical industry, textile, papermaking, construction and plastic industry, and plays a decisive role in economic development. [0003] In higher plants, the main sites of starch synthesis are amyloplasts and chloroplasts. The starch in the chloroplast is generally synthesized during the day as a temporary storage carbon source, and degraded at night for other metabolic uses, so it is called temporary starch; the starch synthesized in the amyloplast is the storag...

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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