Part:BBa_K4653114
pMA5 - Veg promoter - SpoVG RBS - Epr - BvEP - 2x fd terminator
BvEP is an elicitor protein phosphopentomutase from Bacillus velezensis LJ02 which improves the tomato resistance to B. cinerea. High purity BvEP proteins induces the hypersensitivity response (HR) in Nicotiana tabacum. BvEP reduced the rotting rate and lesion diameter of tomato fruits caused by B. cinerea, and increased the expression of Pattern-triggered Immunity (PTI), Effector-triggered Immunity (ETI), systemic acquired resistance (SAR) related genes, ROS content, SOD and POD activities in tomato fruits, while there was no significant effect on weight loss and VC contents of tomato fruits. We designed Bacillus subtilis WB800 to express BvEP and secrete it into the environment to help tomato resist gray mold. We used pMA5 as a plasmid carrier, strong promoter Veg promoter, RBS, Epr signal peptide, BvEP expression gene, and 2x fd terminator.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 926
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 1414
Illegal BsaI.rc site found at 1482
Biology
BvEP is an elicitor protein phosphopentomutase from Bacillus velezensis LJ02 which improves the tomato resistance to B. cinerea. High purity BvEP proteins induces the hypersensitivity response (HR) in Nicotiana tabacum. BvEP reduced the rotting rate and lesion diameter of tomato fruits caused by B. cinerea, and increased the expression of Pattern-triggered Immunity (PTI), Effector-triggered Immunity (ETI), systemic acquired resistance (SAR) related genes, ROS content, SOD and POD activities in tomato fruits, while there was no significant effect on weight loss and VC contents of tomato fruits. We designed Bacillus subtilis WB800 to express BvEP and secrete it into the environment to help tomato resist gray mold. We used pMA5 as a plasmid carrier, strong promoter Veg promoter, RBS, Epr signal peptide, BvEP expression gene, and T7TE terminator.
Design
The fragme<12:11nt was amplified from the total DNA of <i><i B. Velezensis</d> LJ02 by specific primers. We retrieved the total genome of <I>Bacillus Velez. and entered the primer sequence after opening its FASTA file on Snapgene.
The fragments simulated by primers were used as BvEP sequences, which were blasted and showed high homology among the bacillus. However, we found that the fragment amplified by the primer was incomplete and lacked the start codon, so we performed blastp on the incomplete sequence in NCBI to obtain the complete amino acid sequence. Finally, the complete sequence of the protein was obtained on the whole genome.
Plasmid construction
We synthesized the nucleic acid fragment of Veg promoter-SpoVG RBS-Epr signal peptide-BvEP-T7TE Terminator and designed homologous arm primers required for seamless cloning. The plan is to link the fragment with plasmid vector pMA5 through seamless cloning, and transfer it to Bacillus subtilis WB800 to verify follow-up experiments.
References
[1] Li Z, Hu J, Sun Q, Zhang X, Chang R, Wang Y. A novel elicitor protein phosphopentomutase from Bacillus velezensis LJ02 enhances tomato resistance to Botrytis cinerea. Front Plant Sci. 2022 Nov 29;13:1064589. doi: 10.3389/fpls.2022.1064589.
[2] Daudi, A. and O’Brien, J. A. (2012). Detection of Hydrogen Peroxide by DAB Staining in Arabidopsis Leaves. Bio-protocol 2(18): e263. DOI: 10.21769/BioProtoc.263.
[3] Pruitt RN, Gust AA, Nürnberger T. Plant immunity unified. Nat Plants. 2021 Apr;7(4):382-383. doi: 10.1038/s41477-021-00903-3.
[4] Yuan M, Ngou BPM, Ding P, Xin XF. PTI-ETI crosstalk: an integrative view of plant immunity. Curr Opin Plant Biol. 2021 Aug;62:102030. doi: 10.1016/j.pbi.2021.102030. Epub 2021 Mar 5.
[5] Hu J, Chang R, Yuan Y, Li Z, Wang Y. Identification of Key Residues Essential for the Activation of Plant Immunity by Subtilisin From Bacillus velezensis LJ02. Front Microbiol. 2022 Aug 15;13:869596. doi: 10.3389/fmicb.2022.869596.
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