Cloning, prokaryotic expression, and bioinformatics analysis of PaPR10-1 gene from Picea asperata Mast.
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摘要:
PR10是病程相关蛋白(PRs)家族的重要成员，能增强植物抵御外界胁迫侵扰的能力。为进一步研究PR10蛋白的生物学功能，在转录组测序的基础上，利用RT-PCR技术获得一个粗枝云杉(Picea asperata Mast.)基因PaPR10-1，利用生物信息学方法对其核苷酸和氨基酸序列进行结构和功能分析，进一步构建PaPR10-1融合蛋白原核表达系统，获得高纯度PaPR10-1融合蛋白，并采用底物法体外分析其核糖核酸酶活性。结果显示：PaPR10-1基因ORF长486 bp，编码161个氨基酸。PaPR10-1蛋白无跨膜结构和信号肽，为胞内蛋白，含有“P-Loop”和Bet _v1-like保守结构域。系统进化分析结果表明，PaPR10-1蛋白与海岸松(Pinus pinaster Aiton)PR10蛋白归为一个分支，亲缘关系较近。目的蛋白在30 ℃下以0.2 mmol/L的IPTG诱导1 h表达量最佳，且具有核糖核酸酶活性。
Abstract:PR10 is an important member of the pathogenesis-related protein (PR) family, which enhances the ability of plants to resist external stresses. In this study, a PR10 gene from Picea asperata Mast. PaPR10-1 was obtained by RT-PCR, with sequence characteristics then analyzed and prokaryotic expression system further constructed and optimized. Ribonuclease activity was then analyzed using the substrate method in vitro. Results showed that the open reading frame (ORF) of PaPR10-1 was 486 bp in length and encoded a protein with a 161 amino acid. Without a transmembrane structure and signal peptide, the PaPR10-1 protein was an intracellular protein with two conserved domains, “P-Loop” and Bet-v1-like, respectively. Phylogenetic analysis showed that PaPR10-1 was closely related to the PR10 protein of Pinus pinaster Aiton. Optimal expression conditions for the gene were 0.2 mmol/L IPTG at 30℃ for 1 h induction, and the expressed target protein exhibited ribonuclease activity.
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Keywords:
- Picea asperata /
- PR10 gene /
- Gene cloning /
- Bioinformatics /
- Prokaryotic expression
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图 2 PaPR10-1 基因 cDNA序列及其编码的氨基酸序列
方框为“P-Loop”保守基序(GXGGXG) ；横线为潜在的磷酸化位点。
Figure 2. PaPR10-1 gene cDNA sequence and its encoded amino acid sequence
Box is “P-Loop” conservative base order (GXGGXG); black bottom line is potential phosphorylation site.
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图 4 PaPR10-1蛋白二级结构预测与分析(A)以及蛋白三级结构预测(B)
A：蓝色表示α-螺旋；紫色表示无规则卷曲；红色表示延伸链；绿色表示β-转角；横向数值表示氨基酸位置。
Figure 4. Secondary structure prediction and analysis of PaPR10-1 protein (A) and tertiary structure prediction of (B)
A: Blue indicates α-helix; purple represents random coil; red indicates extended strand; green indicates β-turn; lateral value indicates amino acid position.
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图 5 PaPR10-1与6种植物PR10的多重序列比对
AAL50007.1：西部白松；ADJ53040.1：海岸松；QEQ43328.1：硬粒小麦；ACG68733.1：小麦；AAW83210.1：高粱；ABK22402.1：北美云杉。箭头表示β-折叠，字母TT表示β-转角，波浪线表示α-螺旋。
Figure 5. Multiple alignments of amino acid sequence of PaPR10-1 and six plant PR10s
AAL50007.1: Pinus monticola Dougl.; ADJ53040.1: Pinus pinaster Aiton; QEQ43328.1: Triticum durum Desf.; ACG68733.1: Triticum aestivum L.; AAW83210.1: Sorghum Bicolor (L.) Moench; ABK22402.1: Picea sitchensis (Bong.) Carr. Arrows indicate β-pleated sheets, TT indicates β-turns, squiggles indicate α-helix.
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图 6 PR10系统进化树分析
红色为双子叶植物，黄色为裸子植物，绿色为单子叶植物，蓝色为苔藓植物。
Figure 6. Phylogenetic tree analysis of PR10
Red indicates dicot, yellow indicates gymnosperms, green indicates monocot, blue indicates bryophyte.
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图 7 pET-32a-PaPR10-1 转入BL21( DE3)的条带检测
Figure 7. Band detection of pET-32a-PaPR10-1 transferred to BL21(DE3)
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图 8 pET-32a-PaPR10-1-BL21( DE3)诱导条件的优化
M：蛋白质分子量标准。A：最佳IPTG诱导浓度的筛选。1：不加IPTG诱导的pET-32a空载体；2：0.5 mmol /L 的 IPTG 诱导pET-32a空载体；3 ~ 7：IPTG诱导浓度分别为 0.2、0.4、0.6、0.8、1.0 mmol /L。B：最佳诱导时间的筛选。1：0.2 mmol /L IPTG 诱导pET-32a 空载体 4 h；2：不加IPTG诱导的pET-32a空载体；3：不加IPTG诱导的菌液; 4 ~ 8：0.2 mmol /L的IPTG分别诱导 1、2、3、4、5 h。C：最佳诱导温度的筛选。1：诱导全菌液；2 ~ 5：分别以 20℃、25℃、30℃和 37℃诱导上清中的蛋白表达；6 ~ 9：分别以20℃、25℃、30℃和 37℃诱导沉淀中的蛋白表达。
Figure 8. Optimization of induction conditions of pET-32a-PaPR10-1-BL21(DE3)
M: Protein marker. A: Screening of optimal IPTG-induced concentration. 1: pET-32a empty vector without IPTG; 2: IPTG (0.5 mmol/L)-induced pET-32a empty vector; 3–7: pET-32a-PICHI-BL21(DE3) was induced by 0.2, 0.4, 0.6, 0.8, 1.0 mmol/L IPTG. B: Screening of optimal induction time. 1: IPTG (0.2 mmol/L)-induced pET-32a empty vector; 2: pET-32a empty vector without IPTG; 3: pET-32a-PICHI-BL21(DE3) without IPTG; 4–8: pET-32a-PICHI-BL21(DE3) was induced by IPTG (0.2 mmol/L) in 1, 2, 3, 4, 5 h. C: Screening of optimal induction temperature. 1: pET-32a-PICHI-BL21(DE3) was induced at 37℃; 2–5: Supernatant of pET-32a-PICHI-BL21(DE3) was induced at 20℃, 25℃, 30℃, and 37℃; 6–9: Precipitation of pET-32a-PICHI-BL21(DE3) was induced at 20℃, 25℃, 30℃, and 37℃.
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图 9 PaPR10-1蛋白纯化(A)及RNase活性检测(B)
M：蛋白质分子量标准；a：纯化结果。 1：DEPC水；2：洗脱液；3：RNA酶清除剂 + PaPR10-1蛋白；4：PaPR10-1蛋白；5：RNA酶清除剂。
Figure 9. Purification of PaPR10-1 protein (A) and detection of ribonuclease activity (B)
M: Protein marker; a: Purification result. B: 1: DEPC water; 2: Eluent; 3: RNase scavenger + PaPR10-1 protein; 4: PaPR10-1 protein; 5: RNase scavenger.
表 1 引物序列
Table 1 Primer sequences
引物名称
Primer name引物序列 (5'→3')
Primer sequence (5'→3')PaPR10-1-F ATGGTGGCAGGGACGGTAACAAC PaPR10-1-R TTAGCAGTATAAGTCGGGGTTGGAG PaPR10-1-BamHⅠ-F GGAATTCATGGTGGCAGGGACGGTAACAAC PaPR10-1-HindⅢ-R CCGCTCGAGTTAGCAGTATAAGTCGGGGTTGGAG 
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