Cloning and Expression Analysis of Pectate Lyase Gene CcPL20552 from Colletotrichum coccodes

doi:10.13560/j.cnki.biotech.bull.1985.2024-1075

Abstract

Abstract:

Objective Based on RNA-Seq data, the candidate effector protein gene CcPL20552 of pectate lyase is one of the pathogenic genes in the pathogenic process of Colletotrichum coccodes, and it has the characteristics of effector proteins. The study provides a basis for revealing the gene function and protein expression characteristics of CcPL20552 by studying its signal peptide function and also lays the foundation for revealing the molecular pathogenic mechanism of C. coccodes. Method Using potato stem cDNA infected with C. coccodes as a template, the full-length CDS coding region and signal peptide (-SP) sequence of CcPL20552 gene were cloned. Full-length fusion expression vector pET28a-PL20552 with His tag and fusion expression vector pET28a-SP 20552 without signal peptide were constructed, and expressed in Escherichia coli BL21 (DE3) through self-induction and IPTG induction expression, then the concentration and activity of purified protein were measured. Result The full-length coding region of CcPL20552 gene is 720 bp, the predicted molecular weight of the protein is 24.48 kD and it contains a signal peptide sequence consisting of 18 amino acids. But proteins mainly exist in the form of inclusion bodies in the sediment of lysed bacterial cells, the target protein is purified through denaturation and His labeling. Under the same induction conditions, the expression of full-length protein was significantly higher than that of protein without signal peptide. The optimal elution concentration is 100 mmol/L imidazole eluent.The full-length and designal peptide protein concentrations were 224.25 and 174.02 μg/mL, respectively, and the activities were 0.634 6 and 0.952 0 U/mL. Conclusion The results indicate that the CcPL20552 gene can be expressed through self induction and IPTG induction and the lower the temperature, the higher the protein expression level. The signal peptide has a greater impact on the protein expression, and the signal peptide has a secretory function.

Key words: Colletotrichum coccodes, pectate lyase gene, clone, signal peptide, prokaryotic expression

WANG Ting, WANG Yi-dan, REN Shu-jin, MA Ting, JIN Meng-jun, YANG Cheng-de. Cloning and Expression Analysis of Pectate Lyase Gene CcPL20552 from Colletotrichum coccodes[J]. Biotechnology Bulletin, 2025, 41(5): 290-299.

Figures/Tables 7

Fig. 1 PCR verification for cloning and colonyM: Marker (DL=2000)). A:(1: PL20552. 2: SP 20552). B: PL20552 colony PCR validation.C: SP 20552 colony PCR validation

Fig. 2 PCR validation of colony after introduction of signal peptide sequence into yeast YTK12A: pSUC2-SP20552 colony PCR validation. B: PSUC2 colony PCR validation. C:Avr1b colony PCR validation

Fig. 3 TTC color reaction

Fig. 4 Dual enzyme digestion of target gene and prokaryotic expression vector and colony PCR validationA: Dual enzyme digestion of target gene and prokaryotic expression vector, M:Marker (DL=10 000), 1: PL20552, 2: SP 20552, 3: pET28a, 4: pET28a unenzymatically digested; B: pET28a-PL20552 colony PCR validation; C: pET28a-SP 20552 colony PCR validation

Fig. 5 SDS-PAGE analysis of recombinant proteinA: Precipitation and supernatant after self induced fragmentation; M: protein marker;1:pET28a induced supernatant; 2: pET28a-SP20552 16 ℃ induced precipitation; 3: pET28aSP20552 16 ℃ induced supernatant; 4: pET28a-SP20552 37 ℃ induced precipitation; 5: pET28a-SP20552 37 ℃ induced supernatant; 6: pET28a-PL20552 16 ℃ induced precipitation; 7: pET28a-PL20552 16 ℃ induced supernatant; 8: pET28a-PL20552 37 ℃ induced precipitation; 9: pET28a-PL20552 37 ℃ induced supernatant. B: The supernatant and precipitate after induced fragmentation at 28 ℃; M: protein Marker; 1: pET28a induced supernatant; 2: pET28a-SP20552 uninduced precipitation; 3: pET28a-SP20552 uninduced supernatant; 4: pET28a-SP20552 induced precipitation; 5: pET28a-SP20552 induced supernatant; 6: pET28a-PL20552 uninduced precipitation; 7: pET28a-PL20552 uninduced supernatant; 8: pET28a-PL20552 induced precipitation; 9: pET28a-PL20552 induced supernatant. C: The supernatant and precipitate after induced fragmentation at 37 ℃; M: protein marker, 1: pET28a induced precipitation; 2: pET28a induced supernatant; 3: pET28a-SP20552 uninduced precipitation; 4: pET28a-SP20552 uninduced supernatant; 5: pET28a-SP20552 induced precipitation; 6: pET28a-SP20552 induced supernatant; 7: pET28a-PL20552 uninduced precipitation; 8: pET28a-PL20552 uninduced supernatant; 9: pET28a-PL20552 induced precipitation; 10: pET28a-PL20552 induced supernatant

Fig. 6 SDS-PAGE analysis of purified recombinant proteinA: Purification of recombinant protein pET28a-PL20552. B: Purification of recombinant protein pET28a-SP20552; 1-8: elution of 1-8 times purified protein eluate; M: protein marker

Fig. 7 Standard curve of BSA(A) and D-galacturonic acid(B)

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