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Method for agrobacterium-induced genetic transformation of Ustilaginoidea virens

An Agrobacterium-mediated and genetic transformation technology is applied in the improvement field based on Agrobacterium tumefaciens-mediated genetic transformation of Aspergillus oryzae, which can solve the problems of unstable foreign genes and low transformation rate.

Inactive Publication Date: 2014-06-04
CHINA JILIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The technical effects of the present invention can overcome the above-mentioned defects, and provide a high-efficiency transgenic method for Aspergillus oryzae mediated by Agrobacterium tumefaciens, so as to make up for the defects that the conversion rate of the current fungal transgenic technology is generally low and the exogenous gene is unstable

Method used

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  • Method for agrobacterium-induced genetic transformation of Ustilaginoidea virens
  • Method for agrobacterium-induced genetic transformation of Ustilaginoidea virens
  • Method for agrobacterium-induced genetic transformation of Ustilaginoidea virens

Examples

Experimental program
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Effect test

Embodiment 1

[0040] Embodiment 1: Agrobacterium-mediated method for genetically transforming Aspergillus oryzae

[0041] (1) The Aspergillus nidulans promoter and the green fluorescent protein gene gfp were recombined into the pCAMBIA1300 plasmid containing the hygromycin B gene to obtain the pCAMBIA1300-TrpC-GFP recombinant plasmid (verified by sequencing, the recombinant plasmid contained the Aspergillus nidulans promoter , hygromycin B gene hpt and green fluorescent protein gene gfp), and transform Agrobacterium with recombinant plasmids.

[0042] Inoculate a single colony of Agrobacterium AGL-1 (provided by the Institute of Biotechnology, Zhejiang University) on an LB solid medium plate containing a final concentration of 50 μg / ml rifampicin, culture at 28°C for 40 hours, pick a single colony and transfer it to 25 ml containing In LB liquid medium with a final concentration of 50 μg / ml rifampicin, shake culture at 28°C, 150 rpm, and dark conditions until OD 600 If it is 0.8, take 0.1m...

Embodiment 2

[0061] Example 2 Detection of Sensitivity of Aspergillus oryzae to Hygromycin B

[0062] Add hygromycin B to the PSA solid medium so that the final concentration of hygromycin B is 100 μg / ml. The wild-type Aspergillus oryzae SX02-01 and the transformants obtained by the method in Example 1 (that is, Aspergillus oryzae SX0201 containing the plasmid pCAMBIA1300-TrpC-GFP) were respectively punched with a 5mm-diameter hole puncher to obtain mycelial blocks at the edge of the colony , and then inoculate the hyphae block onto the above-mentioned PSA solid medium containing hygromycin B, and culture at 26° C. for 8 days. see results figure 1 and figure 2 shown. The results show that the wild-type Aspergillus oryzae SX02-01 cannot grow on the medium containing the hygromycin B antibiotic because it does not contain the hygromycin gene, and the transformant obtained by the method of Example 1 (that is, contains the hygromycin B gene Aspergillus oryzae SX02-01) can grow on the medi...

Embodiment 3

[0065] Example 3 Effect of Metal Ion Activator on Agrobacterium Transformation Effect

[0066] CoCl in the improved AIM culture medium in step (2) of Example 1 2 ·6H 2 The final concentration of O was changed to 50, 100, and 150 mg / L, and the improved AIM culture solution without cobalt ions was used as a control. The co-cultivation temperature was 22 ° C, and the incubation time was 40 h. Other operations were the same as in Example 1 (transformation, pre-induction , co-cultivation, selective culture, re-screening and PCR detection), the results are shown in Table 3, metal cobalt ions have a significant role in promoting the conversion, and wherein the conversion rate of cobalt ion concentration is 100mg / L is the largest, which can reach 90.57%.

[0067] Table 3 Transformation effect of Agrobacterium adding metal ion activators

[0068]

[0069] Transformation rate = positive bacteria / total number of bacteria × 100%

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Abstract

The invention discloses a method for agrobacterium-induced genetic transformation of Ustilaginoidea virens. The method comprises the following steps of carrying out pre-induction culture on agrobacterium containing a hygromycin B gene and a marker gene, mixing an Ustilaginoidea virens spore liquid and the pre-induced agrobacterium liquid according to a volume ratio of 1: 1 to obtain a mixed bacterium liquid, uniformly coating the mixed bacterium liquid on an acetosyringone-containing modified AIM solid medium flat plate of which the surface is coated with a filter paper, a PVDF film or a nitrocellulose film, carrying out co-culture in the dark at a temperature of 20-24 DEG C for 36-48h, shearing the co-cultured film into small slices, paving the small slices on a modified selective medium CM, and carrying out culture at a temperature of 26 DEG C for 4-5 days to obtain hygromycin B gene-containing Ustilaginoidea virens. The method shortens converter time by 4-5d and has a conversion rate of 91.3%. The transgenic Ustilaginoidea virens has stable offspring, a low gene loss rate and converter stability of 95%.

Description

(1) Technical field [0001] The invention relates to a fungal genetic transformation method mediated by Agrobacterium tumefaciens, in particular to an improved method based on Agrobacterium tumefaciens mediated genetic transformation of Aspergillus oryzae. (2) Background technology [0002] Rice koji fungus (Ustilaginoidea virens (Cooke) Tak) belongs to Ascomyceles, Ergotaceae, and Pseudostilda. Rice smut is a fungal disease of rice caused by Aspergillus oryzae. Rice false smut has not been a serious damage to rice in history, but with global climate change, the large-scale promotion of high-yield and high-quality hybrid rice in my country, and the improvement of rice water and fertilizer levels, the damage of rice false smut has increased year by year. Oryza smut not only infects rice, but also infects corn and some weeds in the field such as crabgrass, Shuishe millet and wild rice. After rice false smut occurs, it not only reduces the yield and quality of rice, but also c...

Claims

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Application Information

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IPC IPC(8): C12N15/80C12N1/15C12R1/645
Inventor 邹克琴汪德凯胡东维李素芳朱诚
Owner CHINA JILIANG UNIV
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