图1 eGFP及PlCYP6∷eGFP在淡紫紫孢菌中的异源表达 A：使用共聚焦显微镜观察淡紫紫孢菌野生型（WT）,以及菌株Pl-G（异源表达eGFP）和Pl-GCYP6（异源表达PlCYP6∷eGFP）的eGFP 绿色荧光信号;B：使用PCR 扩增eGFP（约1 000 bp）和PlCYP6∷eGFP（约3 000 bp）;泳道M 为DNA marker,泳道1 为WT,泳道2 和泳道3 为菌株Pl-G,泳道4 和泳道5 为菌株Pl-GCYP6;C：对淡紫紫孢菌各菌株总蛋白中的eGFP 或PlCYP6∷eGFP 进行Western blot 检测;其中,使用eGFP-anti 作为一抗,羊抗鼠IgG 作为二抗;泳道M 为Precision Plus ProteinTM Standards,泳道1 为WT 总蛋白质,泳道2 和泳道3 为菌株Pl-G 总蛋白质,泳道4 和泳道5 为菌株Pl-GCYP6 总蛋白质

Fig. 1 Heterologous expressions of eGFP and PlCYP6∷eGFP in P. lilacinum A：Observation of the eGFP green fluorescence signal of P. lilacinum（WT）,strains Pl-G（heterologous expression of eGFP）and Pl-GCYP6（heterologous expression of PlCYP6∷eGFP）by confocal microscope. B：Amplification of eGFP（about 1 000 bp）and PlCYP6∷eGFP（about 3 000 bp）by PCR,lane M is a DNA marker;lane 1 is WT;lane 2 and lane 3 are strain Pl-G;lane 4 and lane 5 are the strain Pl-GCYP6. C：Detection of eGFP or PlCYP6∷eGFP in the total protein of P. lilacinum by Western blot. Among them,using eGFP-anti as the primary antibody and goat anti-mouse IgG as the secondary antibody;lane M is Precision Plus ProteinTM Standards,lane 1 is the total protein of WT;lane 2 and lane 3 are the total protein of strain Pl-G;lane 4 and lane 5 are the total protein of strain Pl-GCYP6

图2 免疫沉淀钓取PlCYP6互作蛋白 A：使用SDS-PAGE 凝胶电泳检测蛋白,M 为Precision Plus ProteinTM Standards,1 号及2 号泳道为Pl-G 菌株（异源表达eGFP）及Pl-GCYP6 菌株（异源表达PlCYP6∷eGFP）经过1 mol/L NaCl 处理0 min,15 min,30 min 和60 min 后提取的总蛋白,3 号及4 号泳道为总蛋白进行免疫沉淀后的洗脱产物,5 号及6 号泳道为免疫沉淀后去除非特异性蛋白时第一次清洗的上清液;B：使用Western blot 杂交免疫沉淀后的洗脱产物,Input 为总蛋白质溶液,使用eGFP-anti 作为一抗,羊抗鼠IgG 作为二抗。+ 代表含有,- 代表不含有

Fig. 2 Immunoprecipitation to catch interacting proteins of PlCYP6 A：Detection of proteins by SDS-PAGE gel electrophoresis;M is Precision Plus ProteinTM Standards;lane 1 and lane 2 are the extracted total protein of Pl-G strain（heterologous expression of eGFP）and Pl-GCYP6 strain（heterologous expression of PlCYP6∷eGFP）after treatment with 1 mol/L NaCl for 0,15,30 and 60 min;lane 3 and lane 4 are the eluted products of total protein after immunoprecipitation;lane 5 and lane 6 are supernatant that washed for the first time when non-specific proteins were removed after immunoprecipitation. B：Western blot was used to hybridize the eluted product after immunoprecipitation;Input was the total protein solution;eGFP-anti as the primary antibody and goat anti-mouse IgG as the secondary antibody;“+” indicates containing and “-” indicates not containing

图3 差异蛋白的分布 对照组：Pl-G 菌株（异源表达eGFP）,处理组：Pl-GCYP6 菌株（异源表达PlCYP6∷eGFP）。对照组和处理组免疫沉淀后的蛋白液经LC-MS/MS 分析后,分别鉴定蛋白质的数量和种类,数据库为UniProt 及基于基因组注释的蛋白数据库。与对照组相比,选择处理组中的特异蛋白为候选互作蛋白（482 个）

Fig. 3 Distribution of differential proteins Control group：Pl-G strain（heterologous expression of eGFP）. Treatment group：Pl-GCYP6 strain（heterologous expression of PlCYP6∷eGFP）. The protein solution after immunoprecipitation in the control group and the treatment group was analyzed by LC-MS/MS to identify the number and types of proteins respectively. The databases are UniProt and the protein databases based on genome annotation. Compared with the control group,the specific proteins in the treatment group were selected as candidate interacting proteins（482）

图4 候选互作蛋白的GO 功能注释及KEGG pathway 分析 基于GO 数据库及KEGG pathway 数据库对候选互作蛋白（482 个）进行蛋白功能注释,横坐标为蛋白数量,纵坐标为分类条目

Fig. 4 GO function annotation and KEGG pathway analysis of candidate interacting proteins Based on the GO database and the KEGG pathway database,the candidate interacting proteins（482）are annotated with protein function. The abscissa is the number of proteins,and the ordinate is the classification item

图5 酵母双杂交验证PlCYP6与ADH1蛋白的互作关系 A：依据PlCYP6 蛋白结构域进行截短分析原理图,WD40 repeat 及Cyclophilin-like 为PlCYP6 的预测蛋白结构域;B：酵母双杂交自激活验证;C：PlCYP6 及不同分段区域与ADH1 的酵母双杂交结果。BD：PGBKT7,AD：PGADT7-7。+ 代表AD 或BD 空载体。酵母可以在DDO 培养基（二缺培养基）上生长表示目标载体成功转化入酵母体内,酵母可以在QDO/X/A 培养基（四缺培养基）上生长表示目标蛋白之间存在直接互作关系

Fig. 5 Interaction relationship between PlCYP6 and ADH1 protein by yeast two-hybrid A：Principle diagram of truncation analysis based on PlCYP6 protein domain, WD40 repeat and Cyclophilin-like are the predicted protein domains of PlCYP6. B：Yeast two-hybrid self-activation verification. C：Yeast twohybrid results between PlCYP6 and different segmented regions with ADH1. BD：PGBKT7;AD：PGADT7-7;“+” refers to AD or BD empty carrier. If yeast grew on DDO medium（two-deficient medium）,the target vectors have been successfully transformed into the yeast. If yeast grew on QDO/X/A medium（four-deficient medium）,there will be direct interaction relationship between target proteins

图6 PlCYP6 与ADH1 在盐胁迫中的基因表达量分析 将野生型淡紫紫孢菌使用无菌水（Mock）和1 mol/L NaCl 处理60 min 后提取RNA,使用Actin 作为内参,测量PlCYP6 与ADH1 的基因表达量,使用t 检验进行样本间显著性差异分析,***表示P<0.001

Fig. 6 Analysis of the gene expression levels of PlCYP6 and ADH1 under salt stress The wild-type P.lilacinum was treated with sterile water（Mock）and 1 mol/L NaCl for 60 min,and then the RNA was extracted. Actin was used as an internal control to measure the gene expression levels of PlCYP6 and ADH1. The t test was used to determine significant differences between samples. “***” refers to P<0.001