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Constructing method of zein gene RNAi (Ribonucleic Acid Interference) carrier

A zein and gliadin technology, applied in the field of plant genetic engineering, can solve the problems of poor predictability, long transformation period, low efficiency and the like

Inactive Publication Date: 2012-07-04
FOOD CROPS RES INST YUNNAN ACADEMY OF AGRI SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The technical problem to be solved by the present invention is to overcome the prior art (using o2 The maize varieties with low prolamin content and high lysine content obtained through transgenesis of the gene and its modified gene) have long breeding cycle, low efficiency and poor predictability, and the high lysine maize varieties bred by the existing technology are due to o2 The chain effect of the gene leads to defects and deficiencies such as soft endosperm and poor disease resistance in the grain. The purpose is to provide the construction method of two zein gene RNAi vectors, pUC19-RNAi-22KD and pUC19-RNAi-19KD, to realize the inhibition The expression of zein, the purpose of increasing the content level of lysine

Method used

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  • Constructing method of zein gene RNAi (Ribonucleic Acid Interference) carrier
  • Constructing method of zein gene RNAi (Ribonucleic Acid Interference) carrier
  • Constructing method of zein gene RNAi (Ribonucleic Acid Interference) carrier

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0100] Example 1 Construction of Zein Gene RNAi Vector pUC19-RNAi-22KD

[0101] 1. Test material: corn ( Zea mays L.) The inbred line B73 is a maize germplasm widely used in maize genetic research and breeding practice. Buy.

[0102] 2. Experimental method:

[0103] 1. Extraction of B73 maize genomic DNA:

[0104] 1) Take 300 mg of corn leaves, add liquid nitrogen and grind them thoroughly;

[0105] 2) Add 700 μl 2% CTAB extract preheated at 65°C and mix gently;

[0106] 3) Place in a 65°C water bath for 30-60 minutes;

[0107] 4) Leave at room temperature for 2 minutes, add 300 μl chloroform-isoamyl alcohol (24:1), and shake fully for 1-2 hours;

[0108] 5) Centrifuge at 10,000 rpm for 10 min;

[0109] 6) Take the upper aqueous phase into a new centrifuge tube, add 600 μl isopropanol, and place at room temperature for 10 min;

[0110] 7) Centrifuge at 10,000 rpm for 1 min, discard the supernatant;

[0111] 8) Add 500 μl of 75% ethanol and let stand at room temper...

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 The application of the zein gene RNAi vector constructed by the method of the present invention in increasing the lysine content of corn varieties

[0245] Applying the zein gene RNAi vector pUC19-RNAi-22KD or pUC19-RNAi-19KD constructed by the present invention to transform maize varieties respectively can greatly increase the lysine content level of the grain, 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 differentia...

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Abstract

The invention discloses a constructing method of a zein gene RNAi (Ribonucleic Acid Interference) carrier. The method comprises the following steps of: constructing a recombinant carrier which is inserted into a forward target fragment and an endospem-specific expression promoter P-zp22 / 6 as well as a recombinant carrier which is inserted into a reverse target fragment and a terminator poly(A) by utilizing pUC19 as a frame carrier, and respectively carrying out digestion and connection on the two recombinant carriers to obtain a recombinant carrier pUC19-p22 / 6-22KD1-22KD2-poly(A); and inserting the GUS gene intron into the recombinant carrier to obtain the zein gene RNAi carrier of which the target fragment is partial cDNA (complementary Deoxyribonucleic Acid) fragment of a 22-KD alcohol-soluble protein gene. Another zein gene RNAi carrier is constructed by an identical method, and the target fragment of the zein gene RNAi carrier is partial cDNA fragment of a 19-KD alcohol-soluble protein gene. By adopting the method, the carrier constructing time is shortened and a transgenic plant with high lysine content can be successfully obtained.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering, and in particular relates to a method for constructing a zein gene RNAi vector suitable for genetic transformation of monocot plants. Background technique [0002] RNA interference (RNA interference, RNAi) refers to the specific degradation of mRNA of endogenous target genes mediated by endogenous or exogenous double-stranded RNA (double-stranded RNA, dsRNA), thereby inhibiting gene expression and producing corresponding functions The phenomenon of missing phenotypes. RNAi technology has become an important method for gene function research and crop quality improvement, and the construction of target gene RNAi vector is its core technology. In order to improve the efficiency of interference and promote the application of RNAi technology in plant genetic improvement, many studies have been carried out on the construction methods of RNAi vectors at home and abroad. There are two...

Claims

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Application Information

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IPC IPC(8): C12N15/82C12N15/66
Inventor 陈威番兴明刘丽王琨
Owner FOOD CROPS RES INST YUNNAN ACADEMY OF AGRI SCI
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