Expression vector changing distribution of rice storage protein as well as preparation and use thereof

An expression vector and storage protein technology, which is applied in the field of expression vectors for changing the distribution of rice storage proteins, and can solve the problems of reducing and changing the expression sites of rice storage proteins.

Inactive Publication Date: 2008-08-27
SHANGHAI NORMAL UNIVERSITY
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  • Abstract
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Problems solved by technology

[0009] From the literature that has been reported so far, the waxy protein gene (waxy) promoter is mostly used to guide the waxy gene antisense fragment to reduce rice amylose content (Shimada H, et al, TheorAppl Genet, 1993, 86 (6): 665 ~672; Terada R, et al, Plant Cell Physiol, 2000, 41(7): 881~888; Wang Xinqi et al., Shanghai Agricultural Journal, 2002, 18 (Supplement): 69~73; Chen Xiuhua et al., Science Bulletin, 2002, 47(9):684~688; Liu Qiaoquan et al., Transgenic Res, 2003, 12:71~82; Li Jianyue et al., Science Bulletin, 2004, 49(24):2556~2561; Shen Gezhi et al., Plant Physiology and Molecular Biology Acta Sinica Sinica, 2004, 30(6): 637~643), there is no report on changing the expression site of rice storage protein by using the characteristic of the promoter expressed in the central part of rice

Method used

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  • Expression vector changing distribution of rice storage protein as well as preparation and use thereof
  • Expression vector changing distribution of rice storage protein as well as preparation and use thereof
  • Expression vector changing distribution of rice storage protein as well as preparation and use thereof

Examples

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Embodiment 1

[0048] This embodiment is to illustrate the method for constructing a rice glutenin gene expression vector that contains a resistance marker gene and is guided by a waxy gene promoter and the structure of the obtained vector. The construction process is as follows:

[0049] a) In pCAMBIA1301 (referred to as p1301, from researcher Wang Zongyang, Shanghai Institute of Plant Physiology and Ecology, Chinese Academy of Sciences) plasmid vector (see figure 2 Shown) PCR primers were designed at both ends of the NOS terminator, and HindIII restriction sites were introduced upstream and downstream; the PCR product was connected to the T vector to obtain the T-1 vector, and transformed into Escherichia coli competent cells; then HindIII was used for T- 1 The plasmid was digested, and the small fragment was recovered by electrophoresis, and the small fragment was connected with the pCAMBIA1301 plasmid HindIII digestion product to obtain the pCAMBIA1301-NOS vector (abbreviated as p1301-NO...

Embodiment 2

[0057] This embodiment is to illustrate the method for constructing a vector that does not contain a resistance marker gene and is guided by a waxy gene promoter and the structure of the obtained vector. The construction process is as follows:

[0058] According to the same method of embodiment 1, be that CN1298021A patent specification is searched for 232 indica type rice waxy gene promoter nucleotide sequences from publication number, design upstream primer at about 2.6kb place before translation initiation point ATG with reference to this sequence, in primer A KpnI restriction site was introduced at one end; using the same method as in Example 1, a downstream primer was designed at the 3' UTR behind the Gt1 gene coding region, and an XhoI restriction site was introduced at one end; a pair of new primers designed as above were used for the embodiment 1 The constructed p1301-Wx-Gt1 was subjected to PCR amplification reaction to obtain the DNA sequence of the rice glutelin gene...

Embodiment 3

[0060] This example is to illustrate the method for constructing a glycinin gene expression vector that does not contain a resistance marker gene and is guided by a waxy gene promoter and the structure of the obtained vector. The construction process is as follows:

[0061] a) Extract total RNA from the cotyledon of soybean (Glycine max) at the mid-mature stage, use a reverse transcription kit to obtain the first strand of cDNA, and then design a pair of specific PCR primers according to the glycinin Gy7 mRNA sequence published by Genbank AF319777, the upstream primer No enzyme cleavage site is introduced, the downstream primer introduces XhoI site and adds protective bases, using a high-fidelity DNA polymerase that does not contain A at the end of the PCR product, and using the first strand of cDNA as a template, PCR amplification obtains glycinin The cDNA sequence after the signal peptide of Gy7 gene;

[0062] b) According to the same method as in Example 1, search for the n...

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Abstract

The invention discloses an expression vector which changes the distribution of rice storage protein, a method for preparing the expression vector and the application. The expression vector comprises a gene promoter, a storage protein gene fragment, a terminator and an endonuclease sensitive sequence, wherein the gene promoter is the gene promoter which is located on the central position of the rice and can be expressed. The expression vector can be applied to transform the rice to obtain new varieties whose content and quality of storage protein are changed on the central position of the rice. The expression vector of the invention overcomes the defect that an existing technique only can increase the content and the quality of the protein which is near and around an aleurone layer of the rice, obtains the special effect that rice protein is expressed in the center of granules, and avoids the defect which is existed in an existing technique that the quality improvement affects the decline of production, and since the expression vector of the invention is transformed to obtain new rice quality, the protein content of progeny polished rice is increased by at least 10% on the basis of the protein content of control polished rice which is not transformed.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering, and in particular relates to an expression vector for changing the distribution of rice storage proteins, its preparation method and application. Background technique [0002] my country is a developing country with a large population. In order to meet the needs of our growing population for crops, increasing the planting area to achieve the purpose of increasing production is no longer in line with the current basic national policy of our country. Obtaining more nutritious crops with higher productivity is a more scientific and economical way to increase production. [0003] Rice is the most important food crop in my country, and the nutritional quality of rice has the closest relationship with people's life. The nutritional quality of rice mainly refers to the content of rice protein and essential amino acids. The human body's intake of protein is mainly obtained from plant s...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12N15/29A01H1/00A01H5/00
Inventor 李建粤王幻予张伟高菊芳沈忠伟石少华周永国徐申中
Owner SHANGHAI NORMAL UNIVERSITY
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