Artificial tandem polypeptide ADPs for improving nitrogen fixation efficiency of rhizobium, expression vector and construction and application of recombinant rhizobium

An expression vector, nitrogen fixation efficiency technology, applied in application, fusion polypeptide, recombinant DNA technology and other directions, can solve the problem of less research, achieve the effect of improving efficiency, authentic and credible experimental results, and overcoming difficulties in transmembrane

Pending Publication Date: 2022-05-13
HUAZHONG AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

But how to apply the known NCR peptides, there are only very few studies

Method used

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  • Artificial tandem polypeptide ADPs for improving nitrogen fixation efficiency of rhizobium, expression vector and construction and application of recombinant rhizobium
  • Artificial tandem polypeptide ADPs for improving nitrogen fixation efficiency of rhizobium, expression vector and construction and application of recombinant rhizobium
  • Artificial tandem polypeptide ADPs for improving nitrogen fixation efficiency of rhizobium, expression vector and construction and application of recombinant rhizobium

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] 1. Entrust Nanjing GenScript Biotechnology Co., Ltd. to synthesize SEQ ID NO.5-SEQ ID NO.11-SEQ ID NO.6-SEQ ID NO.12-SEQ ID NO.7-SEQ ID NO.13-SEQ ID NO The sequence shown in .8, as a tandem expression cassette.

[0051] 2. Use restriction endonucleases XhoI and XbaI to digest the tandem expression cassette and the pHC60 vector, connect the two by T4 enzyme ligation, transform Escherichia coli DH5α competent cells, and screen on tetracycline-resistant LB plates , and verified by colony PCR (primers are SEQ ID NO.9 and SEQ ID NO.10, program: pre-denaturation at 95°C for 3min; denaturation at 95°C for 30s, annealing at 56°C for 30s, extension at 72°C for 1min, 27 cycles; re-denaturation at 72°C Extension 10min) and sequencing to identify the recombinant expression vector.

[0052] 3. Extract the recombinant plasmid from the successfully sequenced Escherichia coli, and transform it into rhizobia MAFF303099 competent cells, use TY medium containing fosfomycin and tetracycli...

Embodiment 2

[0054] The positive bacteria screened in Example 1 were cultured with fosfomycin (100 μg / ml) and tetracycline (10 μg / ml) resistant TY medium, cultivated overnight at 28° C., 180 r / min, and waited until OD 600 To 1.0, 4000rpm centrifuge 10min to collect rhizobia, dilute to OD with nitrogen-free medium 600 After reaching 0.05, it is evenly sprayed on the roots of japonicus japonicus, and the inoculation volume of each strain is 5mL.

[0055] 1. The effect of artificial tandem polypeptide ADPs on the morphology of rhizobia bacteria

[0056] 21 days after inoculation, the bacteroids in the root nodules were simply stained with crystal violet, the morphology of the bacteroids was observed with an optical microscope, and the length of the rhizobia was statistically analyzed using ImageJ.

[0057] The morphology of the bacteroids in the root nodules was observed under the optical microscope, such as figure 2 As shown, the length of bacteroids containing the NCR gene was significan...

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Abstract

The invention provides artificial tandem polypeptide ADPs for improving the nitrogen fixation efficiency of rhizobium, an expression vector and construction and application of recombinant rhizobium, and relates to the technical field of root nodule nitrogen fixation. A plurality of NCR polypeptides with similar isoelectric points are connected in series; the method comprises the following steps: constructing an artificial tandem polypeptide ADPs, inserting a coding gene of the artificial tandem polypeptide ADPs into pHC60 to form an expression vector, and then transforming Mesorhizobium japonicum MAFF303099 to obtain recombinant rhizobium, so that the artificial tandem polypeptide ADPs are expressed in the rhizobium. After the recombinant rhizobium is inoculated to lotus corniculatus, the thallus-like form can be obviously changed, and the nitrogenase activity can be improved.

Description

technical field [0001] The invention belongs to the technical field of nodule nitrogen fixation, and specifically relates to the construction and application of artificial tandem polypeptide ADPs, expression vectors and recombinant rhizobia for improving the nitrogen fixation efficiency of rhizobia. Background technique [0002] During the symbiosis between legumes and rhizobia, the roots of legumes form a special organ called a root nodule. According to the shape and structure of nodules, they can be divided into two types: stereotyped nodules and amorphous nodules. The early development process of these two types of nodules is basically the same, but there is terminal differentiation of bacteroids in late amorphous nodules. [0003] The terminal differentiation of bacteroids mainly refers to an irreversible differentiation state after rhizobium infects plants. Rhizobium undergoes morphological changes, such as cell elongation and genome amplification, changes in membrane p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/74C12N15/62C12N1/21A01G22/40A01G7/06C12R1/41
CPCC07K14/415C12N15/743A01G22/40A01G7/06C07K2319/00
Inventor 朱辉于俊杰余海翔曹扬荣肖爱芳
Owner HUAZHONG AGRI UNIV
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