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Conversion for Agrobacterium tumefaciens mediated plasmid to parietal sporamycin

A technology of Agrobacterium tumefaciens and Cephalosporium acremonium, applied to bacteria, using vectors to introduce foreign genetic material, recombinant DNA technology, etc., can solve the problems of low efficiency of Cephalosporium acremonium, achieve large industrial value, improve transformation rate, and plasmid The effect of widening the range

Inactive Publication Date: 2006-05-31
SHANGHAI INST OF PHARMA IND
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  • Abstract
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Problems solved by technology

[0004] The efficiency of existing carrier plasmid transforming Cephalosporium acremonium is very low, only 1~4cfu / μg plasmid DNA (plasmid can respectively reach 10 to escherichia coli and Streptomyces transformation rate. 6-9 and 10 5 cfu / μg plasmid DNA), which has become the biggest obstacle in genetic engineering research of Cephalosporium acremonium

Method used

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  • Conversion for Agrobacterium tumefaciens mediated plasmid to parietal sporamycin
  • Conversion for Agrobacterium tumefaciens mediated plasmid to parietal sporamycin
  • Conversion for Agrobacterium tumefaciens mediated plasmid to parietal sporamycin

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Experimental program
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Effect test

Embodiment 1

[0112] Construction of intermediate vector plasmid pYG306:

[0113] Plasmid pBI121 was double-digested with SacI and HindIII, separated by agarose gel electrophoresis, and a large fragment of 10.3 kb containing T-DNA was recovered with TheGene-clean II kit (available from Qbiogene Company), and then filled in with Klenow enzyme The 5′ end, the 3′ end was cut blunt by Mung bean nuclease, and then T 4 DNA ligase ligated to construct plasmid pYG305. There is an EcoRI single enzyme cutting point between the left and right borders of the T-DNA of pYG305. The plasmids pYG715vgb and pYG305 were respectively digested with EcoRI. After separation by agarose gel electrophoresis, the digested fragments of the two plasmids were recovered, among which the 6.3kb fragment of pYG715vgb After being dephosphorylated by CIAP alkaline phosphatase, it was ligated with the large fragment of pYG305 by T4DNA ligase to construct the intermediate vector plasmid pYG306. The flow chart of intermediate...

Embodiment 2

[0115] Enzyme digestion identification of plasmid pYG306:

[0116] The size of the intermediate vector plasmid pYG306 is 16.6kbp, the ligation product is transformed into E. coli, and Ap is screened r 、Km r 、Rif r 、Sm r The results of plasmid extraction, enzyme digestion and electrophoresis verification are shown in Figure 2.

[0117] The results of enzyme digestion showed that the size of the plasmid and the main restriction sites were correct.

Embodiment 3

[0119] Research on transformation of Agrobacterium tumefaciens by electroporation:

[0120] The intermediate vector pYG306 was transformed into Agrobacterium tumefaciens LBA4404 by electric shock transformation, and resistant transformants could be screened with 100 μg / ml Ap and 50 μg / ml Km.

[0121] Effect of electric field strength on the survival rate of Agrobacterium tumefaciens:

[0122] The electric field strength is an important parameter in electro-shock transformation, and the cell survival rate is directly related to the transformation rate. The effect of electric field strength on cell viability is shown in Figure 3. The lethality rate increases with the increase of the field strength, showing that the cell survival rate decreases with the increase of the field strength. The field strength reaches 15kv.cm -1 , the cell viability was only 17.5%.

[0123] Effect of electric field strength and electric pulse time on conversion rate:

[0124] Electric field strength ...

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Abstract

The invention is about the Cephalosporium acremonium transformation leaded by the plasmid Ti of the Agrobacterium tumefaciens. Compared to the 1-4 transformants it can get above 100 transformants from the 107 receptor cells. So the transforming efficient has improved greatly.

Description

technical field [0001] The invention relates to a method for transforming Cephalosporium acremonium, in particular to a method for transforming Cephalosporium acremonium mediated by Agrobacterium tumefaciens. Background technique [0002] The transformation method of traditional Cephalosporium acremonium still adopts the protoplast method to transform, compared with high-efficiency plasmid transformation escherichia coli (usually the transformation rate can reach 10 8 ~10 9 Transformants / μg plasmid DNA), the protoplast method commonly used in Cephalosporium acremonium plasmid transformation at present, its conversion rate is very low, only reaches 1-4 transformants / μg plasmid DNA. [0003] In plant genetic engineering research, a commonly used method is the binary plasmid transformation method mediated by Agrobacterium tumefaciens Ti plasmid (Bundock P, 1995). Recently, Marcel et al. (Marcel, 1998) applied this method to the transformation of filamentous fungi, and it has ...

Claims

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Application Information

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IPC IPC(8): C12N15/09C12N1/21C12N15/80
Inventor 朱春宝朱宝泉徐威赵文杰
Owner SHANGHAI INST OF PHARMA IND
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