RNAi (RNA interfere) plant expression vector of soybean allergy protein gene Gly m Bd 28K

A plant expression vector and protein technology, applied in DNA/RNA fragments, plant products, recombinant DNA technology, etc., can solve the problems of limited utilization space, time-consuming, and difficulty in changing soybean quality traits at the same time, and achieve plant variety improvement, The effect of eliminating the harm of allergic people

Inactive Publication Date: 2010-12-08
NANJING AGRICULTURAL UNIVERSITY
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Problems solved by technology

Although traditional breeding techniques have successfully improved the nutritional value of soybeans, this process is not only time-consuming, but also has limited utilization space, and it is difficult to change multiple quality traits of soybeans at the same time. After all, the genetic resources that can be improved in soybeans are limited. , and the improvement of soybean traits is limited to the mutant materials that have been obtained

Method used

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  • RNAi (RNA interfere) plant expression vector of soybean allergy protein gene Gly m Bd 28K
  • RNAi (RNA interfere) plant expression vector of soybean allergy protein gene Gly m Bd 28K
  • RNAi (RNA interfere) plant expression vector of soybean allergy protein gene Gly m Bd 28K

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Embodiment Construction

[0055] 1. Transformation of pBI121 vector

[0056] 1) Transformation of the nos sequence of pBI121

[0057] ①Design primers P1 and P2, and use pBI121 plasmid as a template to amplify the nos terminator fragment containing the Asc I site,

[0058] Upstream primer P1: 5`-AGGCGCGCCGATCGTTCAAACATTTGGCA-3`

[0059] Downstream primer P2: 5`-GGAATTCGATCTAGTAACATAGATGACACC-3`

[0060] Amplification system: template pBI121 plasmid 1.0 μL (-1 ), dNTPmix 4.0μL (2.5mmol·L -1 ), PrimeSTAR TM HS DNA Polymerase 0.2 μL, ddH 2 O 37.8 μL;

[0061] Amplification program: pre-denaturation at 94°C for 4min, denaturation at 94°C for 30s, annealing at 51.9°C for 30s, extension at 72°C for 30s, extension at 72°C for 10min, 30 cycles;

[0062] The PCR product was connected into the cloning vector pMD19-T simple vector and verified by sequencing;

[0063] ② Design primer P3, and downstream primer P2, use the nos terminator fragment containing Asc I site as a template to amplify the nos fragment...

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Abstract

The invention relates to the construction of an RNAi (RNA interfere) plant expression vector of a soybean allergy protein gene Gly m Bd 28K, belonging to the filed of molecular biology. Plant vectors PBI (protein-bound iodine) 121 and pCAMBIA3301 are respectively transformed, and are connected to obtain a new vector pGSB. Intron segments of CHAS are amplified by PCR (polymerase chain reaction), and a GUS (glucuronidase) sequence in the vector pGSB is replaced by the amplified intron segments. Primers are designed for amplifying Gly m Bd 28K right interference segments and antisense interference segments and the amplified Gly m Bd 28K right interference segments and antisense interference segments are respectively introduced into the vector pGSBI to obtain the RNAi plant expression vector pYZ of the Gly m Bd 28 K. The Gly m Bd 28K is one of the main soybean allergy proteins, and the soybeans transformed from the RNAi plant expression vector can carry out gene silencing on allergic protein genes. The invention establishes a solid basis for ensuring the safety for eating soybeans by allergic people, and has important theoretical and practical meaning for improving the technological level of national soybean molecular breeding.

Description

1. Technical field [0001] The invention relates to the construction of an RNAi plant expression vector of soybean allergy protein gene Gly m Bd 28K, belonging to the fields of molecular biology and biotechnology. 2. Background technology [0002] Soybean [Glycine max (L.) Merr.] food allergy is a public health issue of close concern worldwide. The wide application of soybean protein in the food processing industry has brought certain food safety problems to people with soybean allergies. The survey found that about 2% of adults and 6% to 8% of children in the world suffer from food allergies, and soybean allergies are more common in children under the age of 5, mainly manifested as stomach discomfort or allergic dermatitis, the main symptoms It is perioral erythema, lip swelling, rash, itchy skin, diarrhea, etc., which can be life-threatening in severe cases. [0003] RNA interference (RNAi), also known as post-transcriptional gene silencing, is a new way to quickly turn o...

Claims

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Application Information

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IPC IPC(8): C12N15/66C12N15/113C12N15/82A01H5/00
Inventor 朱月林盖钧镒刘思辰杨立飞
Owner NANJING AGRICULTURAL UNIVERSITY
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