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Method for cultivating sugarcane resistant to mosaic disease by using RNAi silencing translation initiation factor gene

A technology of translation initiation factor and resistance to mosaic disease, applied in DNA/RNA fragments, genetic engineering, plant genetic improvement, etc., can solve problems such as loss of resistance and insufficient resistance

Inactive Publication Date: 2019-07-12
FUJIAN AGRI & FORESTRY UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

RNAi technology has been applied to the cultivation of transgenic sugarcane materials resistant to mosaic disease, but the main object of its interference is the CP gene of the virus. Because the polymorphism of the CP gene is very high, the obtained transgenic materials are only immune to the virus strain derived from the CP gene. Other strains of the same virus have insufficient or lost resistance

Method used

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  • Method for cultivating sugarcane resistant to mosaic disease by using RNAi silencing translation initiation factor gene
  • Method for cultivating sugarcane resistant to mosaic disease by using RNAi silencing translation initiation factor gene
  • Method for cultivating sugarcane resistant to mosaic disease by using RNAi silencing translation initiation factor gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1: Cloning of sugarcane translation initiation factor genes SceIF4E1, SceIF4E2, SceIF4E3 genes and verification of their interaction with SCSMV-, SCMV-, SrMV-VPg:

[0050] The SceIF4E1, SceIF4E2 and SceIF4E3 genes of sugarcane were cloned by homologous cloning technique. Using the leaves of sugarcane Badila, a tropical species susceptible to SCSMV, as experimental materials, RNA was extracted by Trizol method and reverse-transcribed into cDNA. Compare the open reading frame (open reading frame, ORF) sequence of eIF4E1, eIF4E2, eIF4E3 gene of sorghum and maize, design three pairs of specific PCR primers, use cDNA as template, and obtain sugarcane SceIF4E1, SceIF4E2 and SceIF4E2 by PCR amplification and RACE. ORF of the SceIF4E3 gene. The nucleotide sequence of the SceIF4E1 gene is the nucleotide sequence shown in SEQ ID NO:1 in the sequence listing, with a full length of 663bp, and its encoded amino acid sequence is the amino acid sequence shown in SEQ ID NO:2 i...

Embodiment 2

[0054] Example 2: Constructing an RNAi interference carrier resistant to sugarcane mosaic disease

[0055] The method for constructing the RNAi interference carrier of anti-sugarcane mosaic disease may further comprise the steps:

[0056] 1. Obtaining the RNAi interference target sequence: using the RNAi vector design library in GenBank to screen the intermediate target segment of the SceIF4E1 gene, and selected the 220th to 436th base segment of the SceIF4E1 gene as the target segment of RNAi, The intermediate target segment of the SceIF4E2 gene was screened, and the 200th to 439th base segment of the SceIF4E2 gene was selected as the target segment of RNAi. A pair of specific primers Sc4E1-F (upstream primer) and Sc4E1-R (downstream primer) are designed for the target segment of the RNAi of the SceIF4E1 gene, and Xba I and Xho I enzyme cleavage sites are introduced into Sc4E1-F, targeting A pair of specific primers Sc4E2-F (upstream primer) and Sc4E2-R (downstream primer) w...

Embodiment 3

[0061] Example 3: Cultivation of a transgenic sugarcane material with broad-spectrum resistance to mosaic disease

[0062] A kind of transgenic sugarcane breeding with broad-spectrum resistance to mosaic disease comprises the following steps:

[0063] 1. Material preparation: extract the constructed RNAi interference vector pG0229-4E1+2 plasmid DNA, measure its concentration and purity with a nucleic acid protein analyzer, and quantify it to 1 μg / μL; the recipient sugarcane variety is ROC22;

[0064] 2. Genetic transformation of sugarcane by bombardment with gene gun:

[0065] The pretreatment of acceptor material: at the top part of sugarcane plant, get the young heart leaf within 10cm above the growth point in the hypertrophy band of white heart leaf, after being sterilized with 75% ethanol with volume concentration, and it is cut into thickness different. Discs larger than 3mm were inoculated on MS+3.0mg / L 2,4-D+30g / L sucrose+6g / L agar powder pH 5.8 induction culture for 7...

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Abstract

The invention relates to a method for cultivating mosaic disease resisting sugarcane by using RNAi for silencing the translation initiation factor gene. The method comprises the steps of cloning of the SceIF4E1 gene and the SceIF4E2 gene, structuring of an RNAi interference vector, genetic transformation, cultivation of a transgenetic sugarcane material resistant to the mosaic disease and evaluation of disease resistance. The SceIF4E1 and the SceIF4E2 serve as an interference target sequence of the RNAi, the transgenetic seedlings obtained after genetic transformation produce the broad spectrum resistance to different strains of three pathogens, namely, the sugarcane mosaic virus, the sorghum mosaic virus and the sugarcane streak mosaic virus, of the sugarcane mosaic disease and have the advantages of being good in disease resistance, long in resistance and high in biological safety, the cultivation of the transgenetic sugarcane gets rid of the dependency on anti-genetic genes, and the breeding period of the sugarcane resistant to the mosaic disease can be effectively shortened. The mosaic disease resistant transgenetic sugarcane cultivated through the method is superior to the transgenetic sugarcane obtained by transforming a virus sequence on transgenetic organism safety.

Description

technical field [0001] The invention relates to a method for cultivating sugarcane disease-resistant varieties, in particular to a method for cultivating varieties resistant to sugarcane mosaic disease by using RNAi silencing translation initiation factor gene, that is, using RNAi technology to interfere with eukaryotic translation initiation factor gene SceIF4E1 in sugarcane A method for cultivating transgenic sugarcane resistant to sugarcane mosaic disease with SceIF4E2 belongs to the field of biotechnology. Background technique [0002] Sugarcane (Saccharum sp.hybrid) is the most important sugar crop and energy crop in my country and even in the world. Sugarcane sugar accounts for 92% of the total sugar in my country and 74% of the world's total sugar. Sugarcane ethanol accounts for the world's biomass fuel ethanol 60% of. Sugarcane mosaic disease is one of the viral diseases that seriously endanger sugarcane production, and it commonly occurs in sugarcane areas in Guangx...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/29C12N15/11C12N15/82A01H5/00A01H6/46
Inventor 徐景升杨永庆程光远高世武郭晋隆翟玉山彭磊邓宇晴郑艳茹许莉萍
Owner FUJIAN AGRI & FORESTRY UNIV
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