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Method for culturing transgenic rice GRH and black gold rice and detecting target gene of the transgenic rice GRH

A technology of transgenic rice and cultivation method, applied in the field of rice breeding, can solve the problem of high price

Active Publication Date: 2018-08-24
武汉天问生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the general price is relatively high, ranging from tens to hundreds of yuan for a bottle; in addition, most Chinese people are not used to taking health care products, and the capsule form of health care products can easily make people feel like taking medicine
At present, there is no relevant report on the cultivation method of rice for eye protection and health care

Method used

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  • Method for culturing transgenic rice GRH and black gold rice and detecting target gene of the transgenic rice GRH
  • Method for culturing transgenic rice GRH and black gold rice and detecting target gene of the transgenic rice GRH
  • Method for culturing transgenic rice GRH and black gold rice and detecting target gene of the transgenic rice GRH

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0097] Construction of plant expression vector and Agrobacterium-mediated genetic transformation of Kongyu 131:

[0098] Artificially synthesized phytoene synthase gene psy (2765bp) modified with glutenin promoter and HindIII restriction site at the 5' end, nos terminator and Sal I restriction site at the 3' end and modified at the 5' end Phytoene dehydrogenase gene crtI gene (5760bp) with glutenin promoter and EcoRI restriction site, 3' end modified with nos terminator and Kpn I restriction site;

[0099] Construct the final expression vector pC1300dT-psy / crtI with pCAMBIA1300 containing double T-DNA region as the expression vector: first digest psy with HindIII and Sal I, and then connect to the same double T-containing double T -The intermediate vector pC1300dT-psy is formed on the pCAMBIA1300 vector of the DNA region; then, use EcoR I and Kpn I to cut crtI, and then connect to the pC1300dT-psy vector that also uses EcoR I and Kpn I to obtain the final expression The struc...

Embodiment 2

[0121] PCR detection of transgenic plants and screening of marker-free families:

[0122] The 2 cm leaf tissue of the T0 generation plant was divided into individual plants, and a small amount of rice genomic DNA was extracted by the CTAB method; specific primers for gene amplification were designed, and the primer sequences are shown in Table 1:

[0123] Table 1 Primers for PCR detection of transgenic plants

[0124]

[0125]

[0126] Utilize PCR to detect, and PCR product is detected by 0.8% agarose gel electrophoresis, screens out three kinds of primers to amplify positive T0 generation regenerated plants, obtains 17 positive T0 generation transformation seedlings altogether at last, utilizes SEQ ID NO:1 ~2 primers were used to carry out PCR detection on all the offspring of the T1 generation plants, and the PCR detection results were as follows figure 2 As shown in a, it was found that one of the T1 generation plants had no amplified band, and the plant was named G...

Embodiment 3

[0128] Hybridization of GRH and Black Rice Variety Heishai

[0129] The homozygous GRH family rich in β-carotene was crossed with the Heishai (HS) variety rich in anthocyanin in the seed coat, and then backcrossed with Heishai as the recurrent parent, and then self-crossed to be homozygous. The plant height, number of effective panicles, number of solid grains per panicle, thousand-grain weight, seed setting rate and yield per plant selected from the cross and backcross progeny BC3F2 of GRH and HS were consistent with those of HS, and the exogenous genes psy and crtI were detected by PCR. single plant. The seeds of the selected plants have black seed coats and yellow endosperms. These plants were further homozygously selfed to obtain a family named "Black Gold Rice". The cultivation process of black golden rice is as follows: image 3 as shown in a.

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Abstract

The invention provides a method for culturing transgenic rice GRH and black gold rice and detecting a target gene of the transgenic rice GRH, and belongs to the field of seed breeding of rice. The method for culturing the transgenic rice GRH comprises the following steps of (1) building a final expression vector; (2) transforming the heredity ofperforming genetic transformation on the final expression vector, so as to obtain a T0-generation transformed plants; (3) screening the plants with positive target plants in the T0-generation transformed plants, and selfing to fruit, so as to obtain a T1-generation transformed plants; (4) screening the plants without hpt expression in the T1-generation transformed plants, so as to obtain the transgenic rice GRH. The transgenic rice GRH and the blackrice are used as the parents to culture the black gold rice which is rich in anthocyanin, lutein and beta-carotenoid, so as to obtain the certain eye-protection and health-care effects. The method has the advantages that the method is simple and reliable, the transformation rate is high, the deactivating of the endogenous gene of the rice due to insertion of the target genes is avoided, and the good commercializing potential is realized.

Description

technical field [0001] The invention belongs to the field of rice breeding, and in particular relates to a method for cultivating transgenic rice GRH and black gold rice and detecting a GRH target gene. Background technique [0002] Secondary metabolites of some plants have important health care effects on human health. For example, anthocyanins have strong antioxidant capacity and can protect the human body from free radical damage. Studies have shown that anthocyanins can prevent certain chronic diseases such as diabetes, obesity, certain cancers, and cardiovascular diseases (Zhang et al. 2014). Beta-carotene is an important plant-derived provitamin A that is converted into vitamin A in the human body. Vitamin A deficiency can lead to night blindness, dry eye syndrome and, in severe cases, blindness and death (Schaub et al. 2017). Lutein can protect the macula in the retina and prevent blindness caused by age-related macular degeneration (Madaan et al. 2017). In additi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12N15/66A01H1/02A01H5/00A01H6/46
CPCA01H1/02C12N15/8261
Inventor 陈浩林拥军
Owner 武汉天问生物科技有限公司
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