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Plant expression vector and application thereof in preparing phosphorylation modified rice starch

A plant expression carrier and phosphorylation technology, which is applied in the field of plant expression vectors and in the preparation of phosphorylated modified rice starch, can solve the problems of low phosphorus content and low phosphorylation level of modified starch, and achieve an increase in phosphorus content , promote expression, increase the effect of content

Active Publication Date: 2014-12-24
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are relatively few domestic studies on dextran hydration dikinase. Although it has been reported that dextran hydration dikinase is transferred to plants to modify starch, the phosphorus content of modified starch is relatively low (ie, the phosphorylation level is low). In order to further increase the phosphorus content of starch, it is often necessary to simultaneously transfer multiple enzyme genes to achieve this purpose, such as Chinese patent application 200780028601.1, which simultaneously transfers starch synthase II gene and glucan hydration dikinase gene in plants

Method used

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  • Plant expression vector and application thereof in preparing phosphorylation modified rice starch
  • Plant expression vector and application thereof in preparing phosphorylation modified rice starch
  • Plant expression vector and application thereof in preparing phosphorylation modified rice starch

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 Production of modified rice starch

[0038] 1. Construction of plant expression vector

[0039] (1) Cloning of potato glucan hydration dikinase gene

[0040] ① Design specific primers based on the sequence of the potato dextran hydration dikinase gene (StGWD). The base sequence of the primer is as follows:

[0041] StGWD-F: 5’-GGTCTTAAUTGCTGTACTTACCACTGATACCTC-3’;

[0042] StGWD-R: 5’-GGCATTAAUTCACATCTGTGGTCTTGTCTGAAC-3’.

[0043] ②Take the potato transcriptome RNA as the template, use StGWD-F / StGWD-R primers for RT-PCR amplification, recover the target band and connect it to pUCE, transfer it to Escherichia coli DH5α for sequencing, and compare the sequencing results Yes, it is known that the full length of the StGWD gene is 4167bp, and the nucleotide sequence is shown in SEQ ID No. 1, thus constructing the vector pUCE stGWD .

[0044] (2) Cloning of the promoter

[0045] ①Using barley (Jinnuo) genomic DNA as template and HorD-F / HorD-R as primers, high-fidelity enzyme p...

Embodiment 2

[0115] A process for producing glutinous rice starch phosphate by using genetically modified technology:

[0116] Referring to the method of Example 1, the vector pUCE D-HorD: GBSS-StGWD: NOS Transgenic glutinous rice Shaojing 08-31 was screened to obtain rice plants stably expressing potato glucan hydration dikinase, namely transgenic Shaojing 08-31.

[0117] RVA (Rapid Viscosity Analyzer) was used to determine the viscosity characteristics of glutinous rice wild type (Shaojing 08-31) and transgenic Shaojing 08-31.

[0118] Rice flour sample 3.00g, add 25.00ml distilled water.

[0119] The procedure of RVA is as follows: 50°C for 1 min, constant rate rise to 95°C (3.8min), hold at 95°C (2.5min), constant temperature drop to 50°C (3.8min), and hold at 50°C to 12.5min.

[0120] Table 7

[0121]

[0122] As shown in Table 7, the starch viscosity of the transgenic Shaojing 08-31 strain is higher than that of the waxy wild type.

Embodiment 3

[0124] A process for producing glutinous rice starch phosphate ester:

[0125] (1) With reference to the method of Example 1, a stable transgenic plant Zhonghua II was obtained.

[0126] (2) Planting the transgenic Zhonghua II and glutinous rice varieties (Shaojing 08-31) obtained in step (1) respectively.

[0127] (3) During the flowering period, select the sunny morning with Shaojing 08-31 as the female parent and transgenic Zhonghua II as the male parent to cross the hybrid rice F. 1 .

[0128] (4) Taking Shaojing 08-31 as the female parent, the above hybrid rice F 1 The male parent was backcrossed generation by generation, and the HPT gene and GWD gene marker-assisted selection were used to obtain stable genetic rice plants, which were named high-phosphate starch ester waxy rice lines.

[0129] (5) Extract the grain starch of the glutinous rice strain with high phosphoric acid starch ester, namely glutinous rice starch phosphate.

[0130] RVA (Rapid Viscosity Analyzer) was used to de...

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Abstract

The invention discloses a plant expression vector and the application thereof in preparing phosphorylation modified rice starch. The plant expression vector comprises a target gene inserting into an original vector and a promoter, wherein the target gene comprises a transit peptides gene segment of granule-bound starch synthase and a potato glucan-water dikinase gene which are sequentially connected, and the promoter is a barley endosperm specific promoter HorD. For the plant expression vector and the application thereof in preparing phosphorylation modified rice starch, the potato glucan-water dikinase gene is transferred into rice, and the starch of the rice can be phosphorylated through the expression of the potato glucan-water dikinase gene, so that the modification of the starch is realized. The potato glucan-water dikinase is led to amyloid through the transit peptides of the granule-bound starch synthase, and phosphorylation modification is performed on the starch. According to the invention, the expression of the potato glucan-water dikinase gene in the rice endosperm can be promoted through the barley endosperm specific promoter HorD, and the phosphorus content in the rice starch is greatly increased.

Description

Technical field [0001] The invention belongs to the technical field of starch modification, and particularly relates to a plant expression vector and its application in preparing phosphorylated modified rice starch. Background technique [0002] Starch is mainly composed of amylose and amylopectin. Amylose is a linear polysaccharide connected by α-1,4 glycosidic bonds, which accounts for about 30% of starch. Pullulan is a highly branched polysaccharide composed of α-1,4 glycosidic bonds and α-1,6 glycosidic bonds, accounting for about 70%. [0003] As a natural polymer compound, starch is widely used in many industrial sectors. However, due to the limitations of the physical and chemical properties of natural starch, it can no longer meet the needs of modern food, textile, paper and other industries. There are food additives with stable viscosity and good freeze-thaw stability. The paper industry hopes to have a new type of sizing agent to improve the wet strength and folding res...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C08B30/00A01H5/00
Inventor 包劲松孙潇
Owner ZHEJIANG UNIV
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