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Engineering bacterium capable of efficiently expressing chitinase and plant growth promoting application

A high-efficiency expression technology of chitinase, applied in plant growth regulators, plant growth regulators, applications, etc., can solve the problem of low expression and activity of chitinase, and achieve the effect of promoting germination and growth

Active Publication Date: 2020-06-16
HUBEI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In view of the deficiencies in the prior art, the present invention constructs an engineering bacterium that expresses chitinase efficiently through the gene sequence of chitinase derived from Bacillus licheniformis WX-02, thus solving the problem of the existing chitinase expression level and the problem of low activity

Method used

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  • Engineering bacterium capable of efficiently expressing chitinase and plant growth promoting application
  • Engineering bacterium capable of efficiently expressing chitinase and plant growth promoting application
  • Engineering bacterium capable of efficiently expressing chitinase and plant growth promoting application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Embodiment 1, construction of expression vector and protein expression

[0045] 1. Gene sequence amplification

[0046] A primer pair (ChiA-F, ChiA-R) was designed according to the nucleotide sequence shown in SEQ ID NO.1.

[0047] Table 1: Primers for amplification of chitinase-encoding gene

[0048]

[0049] See Table 1, the underlined part of the forward primer is the Cpo I restriction site, the underlined part of the reverse primer is the Not I restriction site, and the sequence design of this site conforms to T 4 Cohesive ends produced by DNA polymerase trimming.

[0050] PCR reaction system:

[0051]

[0052] PCR reaction conditions: pre-denaturation at 95°C for 5min, denaturation at 95°C for 30s, annealing at 56°C for 30s, extension at 72°C for 1min and 50s, 30 cycles of amplification, and finally extension at 72°C for 10min.

[0053] Detect the PCR product with 0.7% agarose gel electrophoresis, and use DNA purification kit (Omega company)

[0054] 2....

Embodiment 2

[0065] Example 2, construction of cofactor genes and related vectors

[0066] 1. Cofactor gene amplification

[0067] According to the HAC1, ERV29, SEC16, COG5 and TRM1 cofactor gene sequences derived from Pichia pastoris GS115, primers were designed for gene amplification, and these genes were respectively constructed between the Xho I and Xba I restriction sites of the pGAPZB vector.

[0068] See Table 2, the underlined part of the forward primer is the Xho I restriction site and its homologous sequence at the left end of the vector, and the underlined part of the reverse primer is the Xba I restriction site and its homologous sequence at the right end of the vector , the sequence design of this site conforms to T 5 A method for constructing vectors with exonucleases.

[0069] Table 2: Primers for Cofactor Gene Amplification

[0070]

[0071] PCR reaction system:

[0072]

[0073] PCR reaction conditions: 98°C pre-denaturation for 5 minutes, 98°C denaturation for 1...

Embodiment 3

[0078] Example 3, cofactor and 4 copies of chitinase co-expression recombinant strain

[0079] After the plasmids pGAPZB-HAC1, pGAPZB-ERV29, pGAPZB-SEC16, pGAPZB-COG5, and pGAPZB-TRM1 were linearized and digested with Avr II, the digested products were recovered from the solution. Then, under the condition of 1.5kv, electrotransform the GS115 competent with 4 copies of chitinase gene, spread the YPD plate containing 100 μg / mL Zeocin, and incubate at 28°C for 2 days. Then transfer the bacteria on the YPD (Zeocin) plate to the YPD plate and mark it. A yeast colony PCR was then performed to narrow the screen. An appropriate amount of the screened bacteria was used for methanol-induced expression in shake flasks. Subsequent expression steps of chitinase were carried out in accordance with point 5 in Example 2 for methanol-induced expression.

[0080] The result is as Figure 4 Shown: 1 is the control with 4 copies of chitinase, 2 / 3 / 4 / 5 / 6 are the chitin when overexpressing HAC1...

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Abstract

According to the engineering bacterium capable of efficiently expressing chitinase and the application of the engineering bacterium, the engineering bacterium takes pichia pastoris GS115 as an expression host, and strategies such as gene copy number, cofactor co-expression and high-density fermentation are combined, so that the obtained chitinase has the advantages of high expression quantity, high activity and the like. The chitinase expressed by the engineering bacterium can hydrolyze high-concentration colloidal chitin, the hydrolytic activity is good, the hydrolysate is mainly N-acetylglucosamine and then N, N-diacetyl chitobiose, and the chitinase can be used for promoting germination and growth of rice and wheat seeds.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to an engineering bacterium for efficiently expressing chitinase and its application in the germination and growth of plant seeds. Background technique [0002] Chitin (chitin) is a linear polymer connected by N-acetyl-β-D-glucosamine through β-1,4-glycosidic bonds, and is one of the most abundant natural polymer compounds. The product of chitin hydrolysis by chitinase is chitooligosaccharide, which has high application value in the fields of medicine, health care and agriculture. Chitosan oligosaccharides are effective plant elicitors, which have the functions of inducing plant resistance and promoting plant growth. Chitin is widely found in the shells of various arthropods such as shrimps, crabs, and insects. In nature, as many as 10 chitins are synthesized by organisms every year. 11 tons, is one of the most abundant polysaccharides in nature. Biodegradat...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N15/56C12N9/42C12N15/81C12P19/26C12P19/14A01N43/16A01P21/00C12R1/84
CPCA01N43/16C12N9/2442C12N15/815C12P19/14C12P19/26C12Y302/01014
Inventor 张桂敏宋文贺妮莎张诺刘天罡
Owner HUBEI UNIV
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