Method for improving quality of rice kernel amino acid

A rice grain and amino acid technology, applied in the field of plant genetic engineering, to achieve the effect of increasing the yield of a single plant, the method is easy to implement, and the improvement cycle is short

Inactive Publication Date: 2013-03-06
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no report on the use of spider silk protein gene to improve the nutritional quality of crops

Method used

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  • Method for improving quality of rice kernel amino acid
  • Method for improving quality of rice kernel amino acid
  • Method for improving quality of rice kernel amino acid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Embodiment 1: the construction of recombinant vector and the establishment of transformed Agrobacterium:

[0040] (1), according to figure 2 According to the technical route, the plasmid pUC18-Glb with the glutenin GluB-1 promoter (Glb) (according to the EMBL sequence X5433314 is PCR-amplified and cloned into the pUC18 vector (Takara company product), the method refers to Lee et al, 2001, Constitutive and seed-specific expression of a maize lysinefeedback-insensitivedihydrodipioolinate synthase gene leads to increased free lysine levels in rice seeds Molecular Breeding 8: 75-84) Digest with EcoR I and Hind III, separate and recover the target fragment, and use EcoR I and Hind III The pCAMBIA1300 (Cambia company product) after the double-digestion is ligated with T4 ligase to form an intermediate vector, and then a cDNA fragment of a spider dragline (Dragline) gene (sequence see figure 1) was cut with Kpn I and Sac I, separated and recovered the target fragment, and co...

Embodiment 2

[0044] Example 2: Agrobacterium-mediated genetic transformation:

[0045] (1), induction:

[0046] Ripe rice variety (the japonica rice variety disclosed by Dongjign Korea) seeds are shelled, then treated with 70% ethanol by volume for 1 minute, and 0.15% concentration of mercuric chloride (HgCl 2 ) Disinfect the surface of the seeds for 15 minutes; wash the seeds 4-5 times with sterilized water; place the seeds on the japonica rice induction medium; place the inoculated medium in a dark place for 4 weeks at a temperature of 25±1°C.

[0047] (2), Succession:

[0048] Select bright yellow, compact and relatively dry embryogenic calli, and place them on the japonica rice subculture medium for 2-3 weeks in the dark at a temperature of 25±1°C.

[0049] (3), pre-cultivation:

[0050] Select the compact and relatively dry embryogenic callus, put it on the japonica rice pre-medium and cultivate it in the dark for 4-5 days at a temperature of 25±1°C.

[0051] (4), Agrobacterium cu...

Embodiment 3

[0064] Extract DNA from the leaves of transgenic plants, and perform polymerase chain reaction (PCR). PCR program: 94°C pre-denaturation for 5 minutes; 33 cycles (94°C denaturation for 1 minute; 55°C annealing for 1 minute; 72°C extension for 2 minutes) , 72 DEG C to extend for 7 minutes;) Detect positive transformed plants, the single plant that can amplify the 1.2kb size band is the positive transformed single plant ( image 3 ). The RNA of the grains of the positive individual plants was extracted for Northern hybridization to detect the expression of the transferred fragments in the grains, and all the transferred fragments of the positive individual plants were expressed ( Figure 4 ). The large DNA samples of the extracted positive individual plants were digested with Sac I and Xba I (products of Takara Company) respectively, and Southern hybridization was performed to identify the fragment integration copy number of the positive transformed individual plants, and three...

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Abstract

The invention discloses a method for improving the quality of rice kernel amino acid, comprising the following steps of: A, syncretizing a cDNA (complementary deoxyribonucleic acid) segment of dragline silk protein gene separated from a spider with glutelin promoter, and constructing a recombinant carrier; B, introducing the recombined carrier to rice through agrobacterium tumefaciens mediated transgenosis method, and obtaining a converted plant; C, carrying out positive detection on T0 generation transgenic plant by using polymerase chain reaction (PCR), and obtaining positive monoclonal selfedseed; D, growing the selected T0 generation positive monoclonal seed into a family (T1), and carrying out positive detection on T1 generation monoclonal plant; E, continuously planting T1 generation positive monoclonal seed into T2 generation family, selecting pure monoclonal plant with improved amino acid quality, selfing and reserving seed; F, carrying out phenotypic selection on the selected pure monoclonal plant, and culturing new material. The method is simple and easy, and the operation is simple and convenient; the cDNA segment from spider dragline silk protein gene for expressing the rice grins improves the content of amino acid in the rice kernel, and improves nutrition of the kernels; the method improves quality of the rice kernels, and the efficiency is high; and the method is used for improving the crop quality.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering. More specifically, it relates to a method for improving the quality of amino acids in rice grains, which specifically includes using a transgenic method to transform a cDNA fragment of a spider silk protein gene into rice through Agrobacterium, so that the fragment is expressed in rice grains, so as to increase the amino acid content of rice grains , a method for creating new materials for rice quality improvement. Background technique [0002] Rice is one of the most important food crops in the world. However, the content of protein and amino acids, especially lysine and tryptophan, in its grains is very low. Since the content of protein and essential amino acids is a key factor reflecting its nutritional value, improving rice Grain amino acid content, especially essential amino acid content, has become an important task for rice quality improvement. [0003] At present, no m...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/63A01H4/00
Inventor 余四斌金冬红陈炳来李敏孙文强
Owner HUAZHONG AGRI UNIV
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