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The method of using artificial microrna to breed rough dwarf disease resistant corn and its special double-stranded rna

A rough dwarf disease and corn technology, applied in DNA/RNA fragments, recombinant DNA technology, botanical equipment and methods, etc., to achieve high biological safety, reduce uncertain phenotypes, and prevent and control a wide range of occurrences

Inactive Publication Date: 2016-07-06
山东省农业科学院高新技术研究中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] However, in the searchable prior art, the method of using artificial microRNA technology to improve maize resistance to rough dwarf disease has not been reported

Method used

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  • The method of using artificial microrna to breed rough dwarf disease resistant corn and its special double-stranded rna
  • The method of using artificial microrna to breed rough dwarf disease resistant corn and its special double-stranded rna
  • The method of using artificial microrna to breed rough dwarf disease resistant corn and its special double-stranded rna

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1 Cloning of endogenous microRNA (zma-MIR159a) in maize

[0027] According to the sequence information provided by maize zma-MIR159a (accession number: MI0001809) in the microRNA database (http: / / www.mirbase.org / ), it was submitted to NCBI for blastN, and the results showed that zma-MIR159a was contained in EST (EE295085). According to the sequence of EST and the position of zma-MIR159a in EST, primers were designed primer1: 5'-AGTTCCCTCACTCCCCAG-3' (SEQNO.38), primer2: 5'-GGACGCAACAAGCAAAAA-3' (SEQNO.39), primer3: 5 '-TCGATGCTTTGGGTTTGA-3' (SEQ NO. 40), primer4: 5'-GGAGGGGTGGATCAGGAT-3' (SEQ NO. 41).

[0028] The total RNA of Qi 319, an excellent inbred line of maize, was extracted and purified, and the method was as follows:

[0029] 1. Weigh 0.2g of fresh tissue and grind it fully in liquid nitrogen until it becomes powdery. During the grinding, add liquid nitrogen slowly to prevent the material from thawing.

[0030] 2. Quickly transfer the ground tissue t...

Embodiment 2

[0049] Example 2 Obtaining the dedicated double-stranded RNA sequence for transforming microRNA

[0050] Primer5 was designed according to the sequence of genome fragment S6 of rice black-streaked dwarf virus (RiceBlack-streakedDwarfVirus, RBSDV) reported in NCBI (accession number: AJ409148): 5'-AAGTTTTTTGAGTCTGAGATA-3' (SEQNO.42 ), primer6: 5'-GACATCAGCTGATTTGAGTC-3' (SEQ NO.43).

[0051]The corn diseased leaves harmed by rough dwarf disease were collected, and total RNA was extracted. The extraction and purification methods of total RNA were the same as those in Example 1. The total RNA was reverse-transcribed into cDNA, and the reverse transcription method was the same as in Example 1, except that the oligodT primer was replaced with a random primer. Use cDNA as a template, and use primer5 and primer6 as primers to perform a PCR program. The PCR reaction program is 94°C for 5 minutes; then 94°C for 50s, 54°C for 45s, 72°C for 3min, and 32 cycles; 72°C for 7min. The PCR pr...

Embodiment 3

[0060] The construction of embodiment 3 overexpression vector

[0061] The construction process of plant expression vector is as follows: figure 2 Shown:

[0062] According to the precursor sequence of maize zma-MIR159a and the sequence of dedicated double-stranded RNA, primer primer7: TGC was designed TCTAGA TCGATGCTTTGGGTTTGAAGCG-sequenceB-AGGGTCGTTCCGAAGGG (SEQ NO. 44), and primer8: CGC GGATCC GGAGGGGTGGATCAGGATG-sequenceA-GAGAGCGGCCAGCAAGGGGG (SEQ NO.45); wherein the underlined sequence is the restriction site, sequenceA and sequenceB are the special double-stranded RNA sequence and its reverse complementary sequence. Such as sequenceA: TCATACGAGCTAAATCGACAG, sequenceB: GTCGATTTAGCTCGTATGAGA.

[0063] Using maize zma-MIR159a as template, PCR reaction was carried out with primers primer7 and primer8. The PCR reaction program was 94°C for 5 min; then 94°C for 50 s, 54°C for 45 s, 72°C for 1 min, 30 cycles; 72°C for 7 min. The amplified sequence was ligated into a T ve...

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Abstract

The invention discloses a method for cultivating maize resisting a rough dwarf disease by utilizing a synthetic micro RAN (ribose nucleic acid) and a special double-stranded RNA for cultivating the maize. The special double-stranded RNA is a double-stranded RNA sequence consisting of a sequence A and a sequence B. The method comprises the following steps: firstly, cloning the micro RAN of maize endogenesis; taking the micro RAN as a template to carry out PCR (polymerase chain reaction) amplification on a pair of primers respectively containing the sequence A and the sequence B; connecting and transforming the amplified sequences into a plant expression vector with a strong promoter by enzyme digestion; and then guiding the plant expression vector into the maize by means of agrobacterium infection or by a gene gun method, so as to obtain the genetically modified maize resisting the rough dwarf disease. The maize with novel seed quality is cultivated by a synthetic micro RAN technology, off-target is unlikely to happen, the safety is high, and the disease-resistant character can be stably inherited.

Description

technical field [0001] The invention relates to a method for cultivating corn resistant to rough dwarf disease and its special double-stranded RNA, in particular to the use of artificial microRNA technology and its double-stranded special RNA, which belongs to the field of biotechnology. Background technique [0002] Corn is one of the most important food crops in my country. Since 2008, my country's corn planting area has stabilized at around 450 million mu, with a total output of about 160 million tons. However, with the development of my country's economy and the improvement of people's living standards, especially the extension and expansion of the industrial chain of animal husbandry and corn intensive processing, my country's demand for corn will continue to increase. At present, my country's corn production is facing huge pressure. There is a serious shortage of supply. In addition, corn pests and diseases are becoming more and more serious. Each year, plant diseases ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/82A01H5/00
Inventor 赵传志王兴军李爱芹毕玉平夏晗李长生孟昭东
Owner 山东省农业科学院高新技术研究中心
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