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Zein gene RNAi (Ribonucleic Acid interference) vector

A zein and gene technology, applied in the field of plant genetic engineering, can solve problems such as poor disease resistance, achieve the effect of shortening the breeding cycle, shortening the time, and improving the silencing efficiency

Inactive Publication Date: 2013-04-10
FOOD CROPS RES INST YUNNAN ACAD OF AGRI SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, the present invention regulates the synthesis of zein through RNA interference technology, which solves the problem of using zein in conventional breeding. o2 Key issues such as soft endosperm and poor disease resistance of genetically selected high-lysine maize varieties

Method used

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  • Zein gene RNAi (Ribonucleic Acid interference) vector
  • Zein gene RNAi (Ribonucleic Acid interference) vector
  • Zein gene RNAi (Ribonucleic Acid interference) vector

Examples

Experimental program
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Effect test

Embodiment 1

[0097] Example 1 Construction of Zein Gene RNAi Vector pUC19-RNAi-22KD

[0098] 1. Test material: corn ( Zea maysL.) The inbred line B73 is a maize germplasm widely used in maize genetic research and breeding practice. Buy.

[0099] 2. Experimental method:

[0100] 1. Extraction of B73 maize genomic DNA

[0101] 1) Take 300 mg of corn leaves, add liquid nitrogen and grind them thoroughly;

[0102] 2) Add 700 μl 2% CTAB extract preheated at 65°C and mix gently;

[0103] 3) Place in a 65°C water bath for 30-60 minutes;

[0104] 4) Leave at room temperature for 2 minutes, add 300 μl chloroform-isoamyl alcohol (24:1), and shake fully for 1-2 hours;

[0105] 5) Centrifuge at 10,000 rpm for 10 min;

[0106] 6) Take the upper aqueous phase into a new centrifuge tube, add 600 μl isopropanol, and place at room temperature for 10 min;

[0107] 7) Centrifuge at 10,000 rpm for 1 min, discard the supernatant;

[0108] 8) Add 500 μl of 75% ethanol and let stand at room temperatu...

Embodiment 2

[0216] Example 2, Construction of Zein Gene RNAi Vector pUC19-RNAi-19KD

[0217] The construction of the zein gene RNAi vector pUC19-RNAi-19KD is the same as that of Example 1 except for the following steps, which will not be repeated here.

[0218] 1. The method for obtaining the endosperm-specific expression promoter P-zp22 / 6 is the same as in Example 1.

[0219] 2. Construct the recombinant vector pUC19-p22 / 6 containing the endosperm-specific expression promoter P-zp22 / 6, the method is the same as that of Example 1.

[0220] 3. Acquisition of partial cDNA fragment 19-KD-P of 19-KD gliadin gene

[0221] 1. Design primers for the partial cDNA fragment 19-KD-P of the 19-KD gliadin gene

[0222] Find the mRNA sequence of the 19-KD gene in the database MaizeGDB, and use this mRNA as the motif to find the corresponding 19-KD genomic DNA sequence on Genebank (accession number: AC196717.3). This sequence and the recombinant vector pUC19- For the analysis of multiple cloning si...

Embodiment 3

[0244] Example 3 Application of the Zein Gene RNAi Vector of the Present Invention in Increasing the Lysine Content of Corn Varieties

[0245] Applying the zein gene RNAi carrier pUC19-RNAi-22KD or pUC19-RNAi-19KD of the present invention to transform maize varieties respectively can greatly increase the lysine content level of the grains and achieve the purpose of improving the quality of maize.

[0246] 1. Test materials

[0247] corn( Zea mays L.) Hybrid H99×HiⅡB (purchased from Beijing Zhongnong Dakang Technology Development Co., Ltd.). Eighteen days after artificial self-pollination, the ears were sterilized with 70% ethanol, 2.5% sodium hypochlorite and sterilized water in sequence, and young embryos with a length of about 10-1.2 mm were picked under aseptic conditions.

[0248] 2. Culture medium

[0249] The media used for the induction, subculture, biolistic transformation, selection of transformed callus, and differentiation of regenerated plants of maize embry...

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Abstract

The invention discloses a zein gene RNAi (Ribonucleic Acid interference) vector. The provided zein gene RNAi (Ribonucleic Acid interference) vector pUC19-RNAi-22KD contains an endosperm specific expression promoter P-zp22 / 6, positive and negative target gene 22-KD zein gene part cDNA ( complementary Deoxyribonucleic Acid) fragments 22-KD-P, a GUS (Glucuronidase) gene intron and a terminator poly(A). The invention further provides another zein gene RNAi vector pUC19-RNAi-19KD, and the vector contains an osperm specific expression promoter P-zp22 / 6, positive and negative target gene 19-KD zein gene part cDNA fragments 19-KD-P, a GUS gene intron and a terminator poly(A). After the vector is introduced into a corn variety, a corn transgenic plant with high lysine content can be obtained successfully, the lysine content is increased by 15-60 percent, and the transformation period is only 1-2 years.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering, and in particular relates to a zein gene RNAi vector containing plant GUS gene introns suitable for genetic transformation of monocot plants. Background technique [0002] RNA interference (RNA interference, RNAi) is an important tool for studying biological development and gene function. It induces endogenous The mRNA of the target gene is degraded, which in turn inhibits its corresponding gene expression. RNAi has the advantages of specificity, stability, high efficiency, fast speed, and does not change the genetic composition of the genome. It provides a powerful means for plant functional genomics research and plant genetic improvement. At present, RNA interference technology has been applied to improve the quality of oil and starch, the increase of nutrients and the reduction of harmful substances, the resistance to browning, and the storability of fruits and other crop qu...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/63C12N15/66
Inventor 陈威番兴明刘丽王琨
Owner FOOD CROPS RES INST YUNNAN ACAD OF AGRI SCI