Method for culturing sugarcane mosaic disease-resistance varieties by artificial synthesis of MV4 sequence

A technology of artificial synthesis and resistance to mosaic disease is applied in the field of cultivating disease-resistant varieties of sugarcane, and the artificially synthesized MV4 sequence is used to cultivate sugarcane varieties that are resistant to mosaic disease, which can solve the problem of being unable to cope with the diversification, complexity and advantages of pathogenic strains in sugarcane areas. Changes in strains, etc., to achieve the effect of durable resistance and high biosecurity, high biosecurity, and high durability of resistance

Inactive Publication Date: 2015-08-19
FUJIAN AGRI & FORESTRY UNIV
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Problems solved by technology

Therefore, transgenic improvement targeting only a single gene of a single strain of mosaic virus is clearly unable to cope with the diversification, complexity, and changes in dominant strains of pathogenic strains in sugarcane fields

Method used

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  • Method for culturing sugarcane mosaic disease-resistance varieties by artificial synthesis of MV4 sequence
  • Method for culturing sugarcane mosaic disease-resistance varieties by artificial synthesis of MV4 sequence
  • Method for culturing sugarcane mosaic disease-resistance varieties by artificial synthesis of MV4 sequence

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Experimental program
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Effect test

Embodiment 1

[0040] Example 1: Construction of an RNAi vector resistant to sugarcane mosaic disease

[0041] The method for constructing the RNAi carrier of anti-sugarcane mosaic disease, comprises the following steps:

[0042] 1. Artificial synthesis of MV4 sequence:

[0043] The nucleic acid sequences of the collected ScMV virus strains and the nucleic acid sequences of the SrMV virus strains were compared for multiple sequences, and a 100% homologous sequence segment was selected from each of them, and then concatenated in the artificial synthesis method. Together, a total of 520bp is the target RNAi interference sequence; at the same time, Xba I and XhoI restriction sites are added to the 5' end of the sense strand of the artificially synthesized sequence, and Cla I and Kpn I restriction sites are added to the 5' end of the antisense strand , artificially synthesized to obtain the target fragment A; the target fragment A refers to the nucleotide sequence after adding enzyme cutting si...

Embodiment 2

[0046] Embodiment 2: The cultivation of a kind of anti-mosaic disease transgenic sugarcane

[0047] The cultivation of a mosaic disease-resistant transgenic sugarcane comprises the following steps:

[0048] 1. Material preparation: extract the constructed RNAi vector pG0229i-MV4 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;

[0049] 2. Genetic transformation of sugarcane by bombardment with gene gun:

[0050] 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 days, and transferred to MS+2mg / L 4h before the bombardmen...

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Abstract

The invention relates to a method for culturing sugarcane mosaic disease-resistance varieties by artificial synthesis of a MV4 sequence, which comprises artificial synthesis of MV4 sequence, RNAi interference carrier construction, cultivation of anti-mosaic disease transgene sugarcane material and disease resistance identification of disease-resistance transgene sugarcane. According to the invention, an RNAi technology is employed on an obtained plant expression vector, sugarcane antiviral breeding can get rid of dependence for antigenetic gene, and breeding period of sugarcane mosaic disease-resistance can be effectively shortened. The obtained transgene plant has the characteristics of broad-spectrum resistance, good disease resistance, durable resistance and high biological security.

Description

technical field [0001] The invention relates to a method for cultivating sugarcane disease-resistant varieties, in particular to a method for cultivating mosaic disease-resistant sugarcane varieties by using an artificially synthesized MV4 sequence, and belongs to the field of biotechnology. Background technique [0002] Sugarcane mosaic disease, also known as sugarcane mosaic disease, is a kind of viral disease that commonly occurs in various sugarcane-producing countries in the world. Some technological properties such as juice yield, reducing sugar, and sucrose content are also affected to varying degrees. At present, at least three pathogens have been identified to cause sugarcane mosaic disease: Sugarcane Mosaic Virus (ScMV), Sorghum Mosaic Virus (SrMV) and Sugarcane Streak Mosaic Virus. Among them, sugarcane mosaic virus and sorghum mosaic virus are widely distributed in sugarcane areas in my country. These two viruses are members of the genus Potyvirus (Potyvirus). T...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12N15/113A01H5/00
Inventor 郭晋隆高世武许莉萍阙友雄苏亚春吴期滨林庆良
Owner FUJIAN AGRI & FORESTRY UNIV
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