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Corn virus resistant small RNA

A corn and virus technology, applied in the direction of DNA/RNA fragment, recombinant DNA technology, determination/inspection of microorganisms, etc.

Active Publication Date: 2021-06-22
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There is no report on the application of using miRNA sequences to regulate crops' own genes to prevent and control maize chlorotic mottle virus

Method used

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  • Corn virus resistant small RNA
  • Corn virus resistant small RNA
  • Corn virus resistant small RNA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 Effect of silencing miR167 in corn plants on MCMV accumulation 1. Propagation of CMV virions carrying STTM167 and STTM167m in Nicotiana benthamiana 1, infiltration of tobacco plants with Agrobacterium carrying CMV-STTM167 and CMV-STTM167m carriers ( 1) The preparation method of recombinant cucumber mosaic virus (CMV) Beijing isolate silencing miR167 vector and its control vector is as follows:

[0032] pC201-2b N81 (ZMBJ-CMV RNA2b N81 ) as a template, PCR amplification was performed with primers composed of STTM167-F and STTM167-R and a template of STTM-48nts to obtain STTM167. STTM167m was obtained by PCR amplification with primers composed of STTM167m-F and STTM167m-R and a 48nt template (see figure 2 ).

[0033] STTM167-F:

[0034] 5'-ATGTCCGAGTCTGAGTAGATCATGCTCTAGGCAGCTTCAGTTGTTGTTGTTATGG-3'; (SEQ ID NO. 11)

[0035] STTM167-R:

[0036] 5'-AAGGGGAGGTTCTAGTGAAGCTGCCTAGAGCATGATCTAATTCTTCTTCTTTAGACCA-3'; (SEQ ID NO.12)

[0037] STTM-48nts:

[0038] G...

Embodiment 2

[0083] Example 2 Effects of transient silencing and overexpression of miR167 on MCMV accumulation in protoplasts

[0084] 1. Cultivate etiolated seedlings infected by MCMV

[0085] 1. MCMV crude extract preparation

[0086] Take the poison source preserved in the laboratory, add 0.01M phosphate buffer saline and a little corundum at a ratio of 1:1 (W / V), and grind it into a homogenous slurry in a pre-cooled mortar. The obtained homogenate was centrifuged at 5000 rpm for 3 min, and the supernatant was taken for later use.

[0087] 2. Acupuncture inoculation with MCMV

[0088] Select flat corn seeds, soak them in deionized water for 40 minutes, place the embryos upside down on absorbent paper soaked in water. Acupuncture inoculation was carried out using the acupuncture inoculation method of CMV mentioned above, and the inoculated seeds were placed at 25° C. for dark cultivation and kept moist. After 2 days, the germinated seeds were planted in nutrient soil and continued to...

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Abstract

The invention provides a corn virus resistant small RNA, and belongs to the fields of genetic engineering and crop molecular biology. The small RNA provided by the invention is miR167 and is derived from corn, and the nucleotide sequence of the small RNA is shown as any one of SEQ ID NO.1-10. According to the application disclosed by the invention, the maize chlorotic mottle virus MCMV is inoculated after the miR167 is silenced in the maize, mosaic symptoms caused by virus infection are obviously enhanced, and the accumulation level of the MCMV is obviously improved; the MCMV accumulation is increased due to silence of the miR167 in the corn protoplast, and the MCMV accumulation in the corn protoplast overexpressing the miR167 is reduced, so that the miR167 plays a role in resisting the MCMV in the corn. The invention shows that the miR167 can be used for culturing an anti-MCMV corn variety, can also be used in the fields of improvement of disease-resistant plant germplasm resources and genetic breeding, and has a good market application prospect.

Description

technical field [0001] The present invention relates to the fields of crop molecular biology and molecular breeding, and more specifically relates to a small RNA resistant to corn virus, namely miR167, and its application. Background technique [0002] Maize is the main food crop in the world. Maize lethal necrosis (MLN) caused by virus infection is the main disease of maize, which poses a huge threat to the safe production of maize. The disease is mainly caused by co-infection of maize chlorotic mottle virus (MCMV) and sugarcane mosaic virus (SCMV). [0003] MCMV is a Machlomovirus belonging to the Tombusviridae family and can be transmitted by seeds, sap, vector beetles and thrips. The main host is maize, and it can also infect monocotyledonous plants such as sorghum and barley in the field. The typical symptoms of MCMV infecting corn are leaf chlorosis accompanied by mosaic leaves, which can cause leaf necrosis and plant dwarfing in severe cases, and it has been include...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12Q1/6895C12N15/82A01H5/00A01H6/46
CPCC12N15/1131C12Q1/6895C12N15/8218C12N15/8283C12N2310/141C12Q2600/13
Inventor 周涛刘学东刘思佳陈曦范在丰
Owner CHINA AGRI UNIV