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Application of phosphorylated bacterioprotein NopL

A phosphorylation, protein technology, applied in the field of molecular biology, can solve problems such as unclear mechanism

Inactive Publication Date: 2014-11-19
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are no related research results and reports. How NopL affects the premature aging of root nodule cells. The specific mechanism is still unclear. The research on the real mechanism of NopL affecting the premature aging of root nodule cells is still a technical problem that plagues us.

Method used

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  • Application of phosphorylated bacterioprotein NopL
  • Application of phosphorylated bacterioprotein NopL
  • Application of phosphorylated bacterioprotein NopL

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1 Construction of recombinant plasmid pBS- nopL -S12xA

[0047] 1. The present embodiment utilizes artificially synthesized DNA fragments and PCR to carry out site-directed mutagenesis to construct a recombinant plasmid pBS- nopL -S12xA. First synthetic mutant recombinant gene nopL -S12xA, the recombinant protein NopL-S12xA contains 12 mutated phosphorylation sites, all of which are point mutations from serine (Serine) to alanine (Alanine). The 12 mutation sites are: S7A / S12A / S17A / S36A / S73A / S89A / S139A / S148A / S187A / S198A / S240A / S245A.

[0048] 2. Among the above 12 mutation sites, the 5 point mutations S7A / S12A / S17A / S36A / S89A at the N-terminal were mutated by the method of artificially synthesizing nucleotide fragments. The artificially synthesized DNA fragment sequence (314 bp) is as follows: SEQ ID NO: 3, both ends contain Eco RV- Asc I restriction site for further molecular cloning.

[0049] 3. The remaining 7 point mutations were completed by PCR

...

Embodiment 2

[0089] Example 2 Construction of recombinant expression vector pPROEX- nopL -S12xA

[0090] The purpose of this example is to convert gene fragments nopL - S12xA was cloned into the pPROEX expression vector.

[0091] First design primers NopL-S12xA-F and NopL-S12xA-R, and introduce them at both ends of the primers Ehe I and Bam HI restriction site (underlined) to facilitate its insertion into the pPROEX vector.

[0092] The primer sequences are as follows:

[0093] NopL-S12xA-F: (as shown in SEQ ID NO: 16)

[0094] 5’-CGG GGCGCC ATGGATATCAATTCAACCGC-3';

[0095] NopL-S12xA-R: (as shown in SEQ ID NO: 17)

[0096] 5'- GCGGATCC TCAAATGTCAAAATCCACCG-3'.

[0097] Take pBS- nopL -S12xA is used as the template, and the above primers are used to carry out the PCR amplification reaction of exogenous fragments. The reaction system (total volume 50 μL) is as follows:

[0098] Template (50 ng) 1 μL

[0099] dNTP Mix (2.5 mM) 4 μL

[0100] Primer F (10 μM) 1 μL

[01...

Embodiment 3

[0110] Example 3 Induction, expression and purification of recombinant protein NopL-S12xA

[0111] 1. Induction and expression of recombinant protein NopL-S12xA

[0112] The recombinant engineered bacteria BL21 (DE3- nopL- S12xA) were streaked into LB solid medium containing ampicillin (50 μg / mL) and incubated at 37°C for 12-16 h.

[0113] A single colony was randomly selected and inoculated into 3 mL of LB liquid medium containing ampicillin (50 μg / mL), and cultured with shaking at 37 °C and 220 rpm for 12 h.

[0114] The cultured bacterial solution was inoculated into 300 mL of LB liquid medium containing ampicillin (50 μg / mL) at a ratio of 1:100, and incubated at 37°C with shaking to OD. 600 = 0.6 (about 3h), add IPTG to a final concentration of 1mM, and continue to shake at 37°C and 220rpm for 20h.

[0115] 2. Purification and recovery of recombinant protein NopL-S12xA

[0116] The above-mentioned bacterial solution induced by IPTG was centrifuged at 5500 g for 5 mi...

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Abstract

The invention discloses application of phosphorylated bacterioprotein NopL. According to the invention, NopL and a mutant thereof are successfully expressed and purified in vitro; a phosphorylation experiment is carried out, and it is discovered that NopL can be directly phosphorylated by SIPK, wherein NopL is the first discovered bacterioprotein which can be directly used as an SIPK substrate. Moreover, through construction of the mutant of a NopL-deleted phosphorylation site, it is found that NopL loses its original promotion effect on symbiosis of leguminous plants when NopL cannot be phosphorylated by protein kinase, so it is proved that the symbiosis promotion function of NopL is depended on phosphorylation. Such a discovery provides a novel direction for further research on the interaction mechanism of leguminous plants and rhizobium and has high theoretical and practical values.

Description

technical field [0001] The invention belongs to the technical field of molecular biology. More specifically, it relates to the use of NopL, a phosphorylated bacterial protein. Background technique [0002] A large number of studies have shown that limited nitrogen content in soil is an important factor limiting the growth and development of crops. Although nitrogen widely exists in the form of gas, the nitrogen that plants can directly absorb and use in the soil exists in the form of ammonium salt or nitrate. People can only produce chemical fertilizers through the extensive use of chemical nitrogen fixation to ensure the yield of crops. However, such a production method needs to consume a large amount of fossil fuels and increase the emission of greenhouse gas carbon dioxide, and in recent years, the excessive use of chemical fertilizers has also brought increasingly significant environmental problems, which is obviously not conducive to the sustainable development of ag...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/195C12N15/31C12N15/70C12Q1/48C12R1/41
CPCC07K14/195
Inventor 史德海林·奥斯丁·巴特瑟谢致平葛盈盈相其旺
Owner SUN YAT SEN UNIV
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