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A plant expression vector and its application in the preparation of phosphorylated 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 phosphorylation level and low phosphorus content of modified starch, and achieve an increase in phosphorus content , promotion of expression, effect of changing thermodynamic properties

Active Publication Date: 2017-02-15
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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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  • A plant expression vector and its application in the preparation of phosphorylated modified rice starch
  • A plant expression vector and its application in the preparation of phosphorylated modified rice starch
  • A plant expression vector and its application in the preparation of phosphorylated modified rice starch

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] The production of embodiment 1 modified rice starch

[0038] 1. Construction of plant expression vectors

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

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

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

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

[0043] ②Take potato transcriptome RNA as a template, use StGWD-F / StGWD-R primers for RT-PCR amplification, recover the target bands and connect them to pUCE, then transfer them to Escherichia coli DH5α and perform sequencing. 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 a template and HorD-F / HorD-R as primers, high-fidelity enzyme p...

Embodiment 2

[0115] A process utilizing transgenic technology to produce glutinous rice starch phosphate:

[0116] Referring to the method of Example 1, the vector pUCE D-HorD：GBSS-StGWD：NOS The glutinous rice Shaojing 08-31 was transformed into a rice plant stably expressing potato glucan hydration dikinase, that is, the transgenic Shaojing 08-31.

[0117] Viscosity properties of wild type glutinous rice (Shaojing 08-31) and transgenic Shaojing 08-31 were determined by RVA (Rapid Viscosity Analyzer).

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

[0119] The program of RVA is as follows, 50°C for 1min, rising to 95°C at a constant rate (3.8min), maintaining at 95°C (2.5min), dropping to 50°C at a constant rate (3.8min), and maintaining at 50°C for 12.5min.

[0120] Table 7

[0121]

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

Embodiment 3

[0124] A kind of technique of producing glutinous rice starch phosphate:

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

[0126] (2) Transgenic Zhonghua II and glutinous rice varieties (Shaojing 08-31) obtained from the screening in step (1) were planted respectively.

[0127] (3) During the flowering stage, choose Shaojing 08-31 as the female parent and transgenic Zhonghua II as the male parent on a sunny morning to obtain the hybrid rice F 1 .

[0128] (4) With Shaojing 08-31 as the female parent, the above hybrid rice F 1 The male parent was backcrossed generation by generation, and stable genetic rice plants were obtained by assisted selection of HPT gene and GWD gene markers, named as high phosphate starch ester glutinous rice line.

[0129] (5) Extracting the grain starch of the glutinous rice strain with high phosphate starch ester, ie, glutinous rice starch phosphate.

[0130] Viscosity characteristics of wild...

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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 in particular relates to a plant expression carrier 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 linked by α-1,4 glycosidic bonds, accounting for about 30% of starch composition. Amylopectin is a highly branched polysaccharide connected by 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, but 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 an...

Claims

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Application Information

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