, 孙运军, 余子全, 黄伟涛, 胡益波, 丁学知, 夏立秋 省部共建淡水鱼类发育生物学国家重点实验室 湖南师范大学 生命科学学院 微生物分子生物学湖南省重点实验室，湖南 长沙 410081
收稿日期：2019-04-17；接收日期：2019-06-03；网络出版时间：2019-06-03
基金项目：湖南省重大项目(No. 2017NK1030)，国家高技术研究发展计划(863计划) (No. 2011AA10A203)，国家重点基础研究发展计划(973计划) (No. 2012CB722301)，国家自然科学基金(No. 31770106)，湖南省生物发育工程及新产品研发协同创新中心项目(No. 20134486)，湖南省教育厅项目(No. 10CY013)资助
摘要：fcl基因编码的GDP-岩藻糖合成酶(GDP fucose synthetase，GFS)，能催化由GDP-D-甘露糖合成GDP-L-岩藻糖过程中的两步差向异构酶和还原酶反应；还参与氨基糖和核糖的生物合成，是调控生物体糖代谢、核苷酸代谢的关键酶之一。通过前期基因组测序表明须糖多孢菌Saccharopolyspora pogona中存在fcl基因。利用基因工程技术构建了fcl基因的过表达菌株S. pogona-fcl和敲除菌株S. pogona-Δfcl。结果表明该基因对菌株生长发育、蛋白表达及其转录水平、杀虫活性、丁烯基多杀菌素的生物合成均存在影响。经HPLC分析显示，S. pogona-Δfcl的丁烯基多杀菌素产量增加为野生型菌株的130%，S. pogona-fcl的丁烯基多杀菌素产量降低了25%。生测结果显示，与野生型菌株相比S. pogona-Δfcl对棉铃虫的杀虫活性明显增强，而S. pogona-fcl的杀虫活性降低。利用扫描电镜观察发现，S. pogona-Δfcl菌丝体表面出现褶皱，呈现短棒状，S. pogona-fcl菌丝形态与野生型菌株一致。以上结果表明，fcl基因的敲除影响菌丝体的生长发育，能促进丁烯基多杀菌素的生物合成和增强杀虫活性，该基因的过表达抑制了丁烯基多杀菌素的生物合成和降低了杀虫活性。SDS-PAGE结果表明，三株菌株在96 h时蛋白表达差异最为明显。对差异蛋白通过实时荧光定量聚合酶链式反应结果显示，三菌株蛋白的转录水平存在显著表达差异。通过研究结果构建了网络代谢调控图，分析fcl 基因对须糖多孢菌生长发育及丁烯基多杀菌素生物合成代谢调控网络途径的影响，初步构建了fcl基因调控的代谢途径，为揭示丁烯基多杀菌素生物合成的调控机制及相关后续研究提供了实验依据。
关键词：须糖多孢菌丁烯基多杀菌素fcl基因生物合成生长发育
Effect of fcl gene for butenyl-spinosyn biosynthesis and growth of Saccharopolyspora pogona
Shengnan Peng, Haocheng He, Shuangqin Yuan, Jie Rang, Shengbiao Hu
, Yunjun Sun, Ziquan Yu, Weitao Huang, Yibo Hu, Xuezhi Ding, Liqiu Xia State Key Laboratory Breeding Base of Microbial Molecular Biology, College of Life Sciences, Hunan Normal University, State Key Laboratory of Development Biology of Freshwater Fishes, Changsha 410081, Hunan, China
Received: April 17, 2019; Accepted: June 3, 2019; Published: June 3, 2019
Supported by: Major Program of Hunan Province (No. 2017NK1030), National Basic Research Program of China (863 Program) (No. 2011AA10A203), National Basic Research Program of China (973 Program) (No. 2012CB722301), National Natural Science Foundation of China (No. 31770106), Hunan Province Biological Development Engineering and New Product Development Collaborative Innovation Center (No. 20134486), Hunan Education Department Project (No. 10CY013)
Corresponding author: Shengbiao Hu. Tel: +86-731-88872905; E-mail: shengbiaohu@hunnu.edu.cn;
Liqiu Xia. Tel: +86-731-88872298; E-mail: xialq@hunnu.edu.cn.
Abstract: The fcl gene encodes GDP-fucose synthase, which catalyzes two-step differential isomerase and reductase reactions in the synthesis of GDP-L-fucose from GDP-D-mannose. It also participates in the biosynthesis of amino sugar and ribose sugar, and is one of the key enzymes to regulate the metabolism of sugar and nucleotides in organisms. The presence of fcl gene in Saccharopolyspora pogona was found through sequencing result of genome. The mutant S. pogona-fcl and S. pogona-Δfcl were constructed by gene engineering technology. The results showed that the gene had an effects on growth and development, protein expression and transcriptional level, insecticidal activity, and biosynthesis of butenyl-spinosyn of Saccharopolyspora pogona. The results of HPLC analysis showed that the yield of butenyl-spinosyn in S. pogona-Δfcl was 130% compared with that in S. pogona, which reduced by 25% in S. pogona-fcl. The results of determination of insecticidal activity showed that S. pogona-Δfcl had a stronger insecticidal activity against Helicoverpa armigera than that of S. pogona, while the S. pogona-fcl had a lower insecticidal activity against Helicoverpa armigera compared with S. pogona. Scanning electron microscopy (SEM) was used to observe the morphology of the mycelia. It was found that the surface of the S. pogona-Δfcl was wrinkled, and the mycelium showed a short rod shape. There was no significant difference in mycelial morphology between S. pogona-fcl and S. pogona. Aboved all showed that deletion of fcl gene in S. pogona hindered the growth and development of mycelia, but was beneficial to increase the biosynthesis of butenyl-spinosyn and improve insecticidal activity. Whereas the fcl gene over-expression was not conducive to the biosynthesis of butenyl-spinosyn and reduced their insecticidal activity. SDS-PAGE results showed that the difference of protein expression among the three strains was most obvious at 96 hours, which was identified by real-time fluorescence quantitative polymerase chain reaction, the results showed that there were significant differences of related genes in transcriptional levels among the three strains. Based on the results of the study, a network metabolic control map was constructed to analyze the effect of fcl gene on growth and the regulation pathway of butenyl-spinosyn biosynthesis, which provided an experimental basis for revealing the regulation mechanism of butenyl-spinosyn biosynthesis and related follow-up studies.
Keywords: Saccharopolyspora pogonabutenyl-spinosynfcl genebiosynthesisgrowth and development
须糖多孢菌Saccharopolyspora pogona属于放线菌糖多孢菌家族中成员，是一种革兰氏阳性好氧型菌[1]。丁烯基多杀菌素(Butenyl-spinosyn)是由须糖多孢菌经过有氧发酵后获得的一类次级代谢产物，其分子结构为类似于多杀菌素的大环内酯类化合物，由丁烯基取代多杀菌素结构上的乙基而成[2-3]。在须糖多孢菌中，丁烯基多杀菌素的合成受到基因簇的严格调控，包括编码合成糖苷配基、福乐糖胺、鼠李糖和使鼠李糖甲基化的23个基因[4-5]。作为一种绿色、安全、高效的天然杀虫剂，丁烯基多杀菌素的理化性质[6-8]，生物合成途径[9]、发酵工艺[10-12]及杀虫机理[13-15]等均有研究和报道。相比于多杀菌素和其他化学农药而言，丁烯基多杀菌素具有更广的杀虫谱和对环境更为友好等优点[16-17]。例如，针对多杀菌素防治效果不佳的苹果毒蛾和烟青虫，丁烯基多杀菌素的防治效果则很好，因而被期望用于大范围的害虫防治[6, 18]。但野生型须糖多孢菌产生丁烯基多杀菌素的含量极低，使其在推广应用上受到极大的限制[19-20]。因此，通过对须糖多孢菌基因组进行定向遗传改造[21-23]，促进对丁烯基多杀菌素的生物合成，已成为近年研究的热点之一。
Becker等的研究表明，细胞质中GDP-岩藻糖的生物合成途径主要分为从头合成途径和补救合成途径[24]。Silvia等研究表明，岩藻糖与细胞间的识别和粘附有关[25]。Zhou等对人源的GDP-岩澡糖合成酶(FX蛋白)的研究结果表明，FX蛋白在结构上由两个同二聚体组成，且与大肠杆菌的GFS存在一定差别[26]。Somers等的研究表明，GFS在发挥催化作用时结合NADPH，并保留Ser-Tyr-Lys催化三联体结构[27]。在细菌细胞中，GDP-甘露糖主要参与两个方面的代谢过程：一是在GDP-甘露糖-4, 6-脱水酶和GDP-岩藻糖合成酶的作用下合成GDP-岩藻糖，进入氨基糖和核酸糖的代谢过程二是转化为GDP-D-鼠李糖，进而合成丁烯基多杀菌素[28-29]。GFS在放线菌中也可能发挥着重要作用，但当下对放线菌中的GFS研究甚少。本文利用遗传修饰技术获得fcl基因的过表达菌株和敲除菌株，研究该基因对须糖多孢菌的生长发育和丁烯基多杀菌素的生物合成等方面产生的影响，为促进丁烯基多杀菌素高效生物合成和构建高产工程菌株提供了新的技术途径。
1 材料与方法1.1 菌株和质粒本研究中使用的菌株、质粒及引物见表 1。
表 1 菌株、质粒及引物Table 1 Strains, plasmids and primers used in this study
| Related description | Source | |
| Strains | ||
| E. coli DH5α | Host for general cloning | Lab store |
| E. coli S17 | Donor strains for conjugation | Lab store |
| S. pogona | The producer strains of butenyl-spinosyn | Lab store |
| S. pogona-fcl | S. pogona harboring pOJ260-PermE-fcl | This work |
| S. pogona-Δfcl | S. pogona harboring pOJ260-fcl | This work |
| Plasmids | ||
| pOJ260 | E. coli-cloning vector, containing pUC18 replicon, oriT, AprR | Lab store |
| pOJ260-cm-PermE | Containing PermE sequence | Lab store |
| pOJ260-PermE-fcl | PermE -fcl inserted into pOJ260 by Hind Ⅲ and EcoR Ⅰ | This work |
| pOJ260- fcl | Homologous arm inserted into pOJ260 by Hind Ⅲ and EcoR Ⅰ | This work |
| Primers | ||
| PermE-F | CCCAAGCTTCTGGACTTCTAGAGCTAGCC | |
| PermE-R | GCATGCCGGTCGACTCTA | |
| fcl-F | GGTAGGATCCTCTAGAGTCGACCGGCATGCGGAGATCCTCGTGCCCTGAC | |
| fcl-R | CCGGAATTCCACTGCGATGACATCAGCGTAC | |
| Apr-F | AGCCCTAACGGCAAGTTTGCAAGCAGCAGATTACG | |
| Apr-R | GGTACTGTCCGTGCTATCCGTCGACCTGCATACTA | |
| fcl-up-F | CCGGAATTCGCACATCCAGCGAACCTG | |
| fcl-up-R | TCTGCTGCTTGCAAACTTGCCGTTAGGGCTTTCTC | |
| fcl-down -F | TGCAGGTCGACGGATAGCACGGACAGTACCCTCGTT | |
| PermE-F | CCCAAGCTTCTGGACTTCTAGAGCTAGCC | |
| fcl-down-R | ACCAAGCTTTACGCATCGGTCGCCAAAT | |
| 16S-F | CGTCAGCTCGTGTCGTGAGA | |
| 16S-R | GTGAAGCCCTGGGCATAAGG | |
| rpoE-F | TGACCCAGGAGACCTTCATCC | |
| rpoE-R | CGAGGAGGGTGTCGTTGAAGA | |
| groEL-F | GGTTCAGGTCCGCGTTCTC | |
| groEL-R | GCCACCCTGGTCGTCAACA | |
| rpoB-F | GACATTCGCCAGTGGTCGTAC | |
| rpoB-R | CTCGCAGATGATGCCCTTG |
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1.2 培养基大肠杆菌Escherichia coli接种于LB培养基中，37 ℃培养过夜；须糖多孢菌S. pogona接种于种子活化培养基(CSM)中，280 r/min、30 ℃培养2 d；接合转移实验采用R6培养基，工程菌株的发酵采用半合成发酵培养基，280 r/min、30 ℃培养10-12 d；产孢能力实验采用CSM、TSB、BHI和R6培养基。
1.3 fcl基因重组载体的构建1.3.1 过表达载体pOJ260-PermE-fcl的构建及鉴定过表达载体pOJ260-PermE-fcl的构建流程如图 1A所示。以pOJ260-cm-PermE为模板，以引物对PermE-F/PermE-R进行PCR扩增，得到300 bp左右的PermE基因片段。以须糖多孢菌为模板，引物对fcl-F/fcl-R进行常规扩增，得到约1.5 kb的fcl基因。然后通过重叠延伸PCR得到1.8 kb左右的PermE-fcl融合片段(图 1B)。该重组质粒经过Hind Ⅲ/EcoRⅠ单双酶切验证，结果表明载体pOJ260- PermE-fcl构建成功(图 1C)。
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| 图 1 过表达载体的构建与鉴定 Fig. 1 Construction and identification of overexpression vectors. (A) The vector map of pOJ260-PermE-fcl. (B) PermE and PermE-fcl frangment. M: DL 5000; 1: PermE; 2: PermE-fcl. (C) Enzyme digestion of recombinant plasmid. M: DL 10000; 1: The pOJ260-PermE-fcl digested with Hind Ⅲ; 2: The pOJ260-PermE-fcl with EcoRⅠ; 3: The pOJ260-PermE-fcl with Hind Ⅲ/EcoRⅠ. |
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1.3.2 敲除载体pOJ260-fcl的构建及鉴定敲除载体pOJ260-fcl的构建流程如图 2A所示。以须糖多孢菌为模板，引物对fcl-up-F/ fcl-up-R，常规PCR扩增得到约1 kb的fcl基因上游臂片段；引物对fcl-down-F/fcl-down-R，常规PCR扩增得到约1 kb的fcl基因下游臂片段；引物对Apr-F/Apr-R，常规PCR扩增得到约1.5 kb的aac(3) Ⅳ (图 2B)。然后通过重叠延伸PCR得到3.5 kb左右的敲除融合片段。该重组质粒经过Hind Ⅲ/EcoRⅠ单双酶切验证，结果表明敲除载体pOJ260-fcl构建成功(图 2C)。
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| 图 2 fcl基因敲除载体的构建与鉴定 Fig. 2 Construction and identification of knocout vectors. (A) The vector map of pOJ260-fcl. (B) aac(3) IV gene and homologous arm fragments of fcl gene. M: DL 2000; 1: aac(3)IV; 2: fcl-up; 3. fcl-down. (C) Enzyme digestion of pOJ260-fcl. M: DL 10 000; 1: the pOJ260-fcl digested with Hind Ⅲ; 1: the pOJ260-fcl digested with EcoRⅠ; 3: the pOJ260-fcl digested with Hind Ⅲ/EcoRⅠ. |
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1.4 fcl基因工程菌株的构建和鉴定1.4.1 fcl基因过表达菌株的构建和鉴定以含目标质粒的E. coli S17作为供体菌，以须糖多孢菌S. pogona作为受体菌，进行属间接合转移，根据同源重组原理将重组质粒整合到须糖多孢菌基因组上。提取S. pogona和S. pogona-fcl基因组作为模板，以引物对PermE-F/fcl-R PCR扩增大小为约1.8 kb的PermE-fcl融合片段；以引物对Apr-F/Apr-R PCR扩增aac(3)Ⅳ片段。结果表明，过表达菌株S. pogona-fcl构建成功(图 3)。
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| 图 3 fcl基因过表达菌株的构建及PCR鉴定 Fig. 3 Construction and PCR identification of S. pogona-fcl. (A) Schematic diagram of S. pogona-fcl. (B) PermE-fcl. M: DL 5000; 1, 2: S. pogona-fcl genome; 3, 4: S. pogona genome. (C) aac(3)Ⅳ. M: DL 5000; 1, 2: S. pogona genome; 3, 4: S. pogona-fcl genome. |
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1.4.2 fcl基因敲除菌株的构建和鉴定以含重组质粒的E. coli S17作为供体菌，以须糖多孢菌S. pogona作为受体菌，进行属间接合转移试验，根据同源重组原理将质粒整合至须糖多孢菌基因组上。提取S. pogona和S. pogona-Δfcl基因组作为模板，以引物对fcl-F/fcl-R扩增长度约为1.5 kb的fcl基因，以引物对Apr-F/Apr-R PCR扩增aac(3)Ⅳ基因片段。结果表明，成功获得敲除菌株S. pogona-Δfcl (图 4)。
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| 图 4 fcl基因敲除菌株的构建及PCR验证 Fig. 4 Construction and PCR identification of S. pogona-Δfcl. (A) Schematic diagram of S. pogona-Δfcl. (B)fcl gene. M: DL 5000; 1: S. pogona-Δfcl genome; 2: S. pogona genome. (C) aac(3)Ⅳ. M: DL 5000. 1, 2: S. pogona-Δfcl genome. 3, 4: S. pogona genome. |
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1.5 fcl基因对菌株生长发育及菌丝体形态特征的影响生长曲线测定：以半合成发酵培养基为空白对照，每12 h测定培养基中OD600值，按稀释倍数计算各瓶培养基中的实际OD600值。实验重复3次，绘制生长曲线。
产孢能力和菌丝体形态的观察：取100 μL野生型菌株和工程菌株S. pogona-fcl、S. pogona- Δfcl菌液分别涂布于BHI、CSM、TSB以及R6培养基固体平板中间区域。每24 h观察不同平板上孢子的产生情况并记录。同时各取1 mL培养96 h的发酵液，超纯水灭菌后清洗菌体沉淀15次以上，在液氮中固定，低温、真空条件下镀金，利用日立SU8010冷场扫描电子显微镜观察。此外，对菌体沉淀加入戊二醛溶液进行固定，用不同浓度的乙醇溶液进行梯度脱水，低温下镀金，利用日立SU8010超高分辨率扫描电子显微镜进行菌丝体形态观察。
1.6 fcl基因对菌株发酵液杀虫活性的影响各取1 mL三株菌发酵10 d的发酵液，分别与20 mL棉铃虫Helicoverpa armigera饲料进行混匀后倒入24孔板中，待凝固后，每个孔内放置1条生长状态相同的棉铃虫，置于26-28 ℃恒温培养6 d，期间每24 h观察并记录棉铃虫存活数。
1.7 fcl基因对菌株丁烯基多杀菌素生物合成产量的影响超声破碎细胞法对第10天的发酵液(等体积乙酸乙酯萃取)进行破碎，经真空冷冻浓缩完全冻干等处理后，加入50 μL甲醇溶解沉淀。利用Agilent 1290超高效液相色谱仪(UHPLC)检测丁烯基多杀菌素含量，检测条件如文献[3]所述，并收集目的峰进行质谱鉴定。
1.8 fcl基因对须糖多孢菌菌体蛋白的影响参照文献[10]进行菌株第2、4、6、8、10天蛋白的提取，经SDS-PAGE检测发现第4天蛋白差异最大。
1.9 转录水平的qRT-PCR测定利用Trizol试剂提取菌株S. pogona、S. pogona-fcl、S. pogona-Δfcl的RNA。分别以野生型菌株及工程菌株第4天的反转录cDNA作为模板，16S RNA作为内参，利用7500实时荧光定量PCR系统仪器(Applied Biosystems，USA)，对基因rpoE、groEL、rpoB进行转录水平验证。
2 结果与分析2.1 fcl基因对须糖多孢菌生长发育的影响为确定fcl基因对须糖多孢菌生长的影响，利用分光光度计每12 h对发酵液OD600进行测定。结果发现，直至72 h时，过表达菌株S. pogona-fcl的生长变化与野生型菌株S. pogona基本一致；野生型菌株从96 h开始进入稳定期，过表达菌株从84 h开始进入稳定期；相较于野生型菌株而言，S. pogona-fcl的OD600值略有降低，但与S. pogona生长趋势基本一致；两株菌株均于156 h进入衰退期，216 h衰退稳定。敲除菌株S. pogona-Δfcl生长缓慢，108 h进入稳定期，132 h进入衰退期，228 h衰退稳定，OD600值明显下降。说明fcl基因的敲除抑制了须糖多孢菌的生长，使细胞密度下降；该基因过表达后，菌株细胞密度与野生型菌株基本一致(图 5)。
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| 图 5 fcl基因工程菌株的生长曲线 Fig. 5 Growth curve of fcl gene engineered strains. |
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2.2 fcl基因对须糖多孢菌体形态和产孢能力的影响为研究fcl基因对菌体形态的影响，利用超高分辨率扫描电镜和冷场扫描电镜对发酵96 h的野生型菌株S. pogona、过表达菌株S. pogona-fcl与敲除菌株S. pogona-Δfcl的菌丝体形态进行观察。结果表明，S. pogona-fcl与野生型菌株菌丝体表面光滑无褶皱，分支较多；S. pogona-Δfcl菌丝表面褶皱明显，菌丝体呈现短棒状；说明fcl的过表达对菌体生长无明显影响，敲除后对菌丝体的生长发育具有抑制作用(图 6、7)。
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| 图 6 fcl基因工程菌株超高分辨场扫描电子显微镜观察 Fig. 6 Scanning electron microscopy observation of fcl gene engineered strains. (A) S. pogona. (B) S. pogona-fcl. (C) S. pogona-Δfcl. |
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| 图 7 fcl基因工程菌株冷场扫描电子显微镜观察 Fig. 7 Observation of fcl gene engineered strains by scanning electron microscope. (A-C). S. pogona. (D-F) S. pogona-fcl. (G-I) S. pogona-Δfcl. |
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为研究fcl对须糖多孢菌在不同固体培养基上产孢能力的影响，分别观察野生型菌株和工程菌株在4种培养基上的产孢情况。结果发现，3株菌株均从第48 h开始产生孢子；120 h时，S. pogona-fcl和野生型菌株在CSM和TSB培养基上的产孢能力最强；S. pogona-Δfcl在R6培养基上的产孢能力最弱，三菌株在BHI培养基上产孢能力相同。三株菌株自身在4种培养基上产孢能力也存在差异，由强到弱依次为CSM、TSB、BHI、R6。可能是由于培养基营养成分的不同，从而导致须糖多孢菌同一菌株在上述4种培养基中的产孢能力出现明显差异(图 8)。
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| 图 8 fcl基因工程菌株在固体培养基上产孢能力比较 Fig. 8 Comparison of sporulation ability of fcl gene engineered strains on solid media. (A) Sporulation ability at 48 h. (B) Sporulation ability at 120 h. |
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2.3 fcl基因对丁烯基多杀菌素产量影响的比较为确定fcl基因对丁烯基多杀菌素生物合成的影响，利用HPLC分析野生型菌株S. pogona、过表达菌株S. pogona-fcl以及敲除菌株S. pogona- Δfcl发酵液中丁烯基多杀菌素的产量。利用质谱对13 min的色谱峰进行鉴定，结果发现该时间点的色谱峰含有分子量为189.1的三甲基鼠李糖碎片峰(图 9)，证明该物质是丁烯基多杀菌素组分[30]。HPLC结果显示，S. pogona在保留时间为13 min处的丁烯基多杀菌素峰面积为606.2 mAU。S. pogona-fcl在该时间段的峰面积为455.4 mAU，丁烯基多杀菌素的产量降低为野生型菌株的75%。S. pogona-Δfcl在该段时间内的峰面积为786 mAU，丁烯基多杀菌素的产量提高至野生型菌株的130% (图 10)。表明fcl基因的敲除能促使丁烯基多杀菌素产量的提高，过表达使产量降低。
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| 图 9 fcl基因工程菌株丁烯基多杀菌素的质谱鉴定 Fig. 9 MS identification of butenyl-spinosyn from fcl gene engineered strain. (A) Determined by LC-MS. (B) Determined by LC-MS/MS. |
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| 图 10 fcl基因工程菌株丁烯基多杀菌素产量的比较分析 Fig. 10 The butenyl-spinosyn yield analysis of fcl gene engineered strains. (A) S. pogona. (B) S. pogona-fcl. (C)S. pogona-Δfcl. |
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2.4 fcl基因对菌株发酵液杀虫活性的影响为研究fcl基因工程菌株发酵液对棉铃虫H. armigera杀虫活性的影响，利用三菌株第10天的发酵液对棉铃虫进行生物杀虫活性测定。结果显示，与野生型菌株相比，S. pogona-fcl的杀虫活性最弱，半致死时间较野生型菌株推迟约0.7 d。S. pogona-Δfcl发酵液的杀虫活性明显增强，半数致死时间较野生型菌株提前约0.6 d (表 2)。以上结果表明，fcl基因的敲除有利于菌株杀虫活性的增强，过表达后杀虫活性减弱(图 11)。
表 2 fcl基因工程菌株对棉铃虫杀虫活性的测定Table 2 Insecticidal activity of fcl gene engineered strains against H. armigera
| Strain | Slope | Relative coefficient (R2) | LT50 value (d) | 95% confidence interval |
| S. pogona | 9.571 4 | 0.979 3 | 4.346 | 4.005-4.741 |
| S. pogona-fcl | 9.400 0 | 0.990 3 | 5.069 | 4.710-5.529 |
| S. pogona-Δfcl | 9.629 0 | 0.981 5 | 3.690 | 3.370-4.018 |
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| 图 11 fcl基因工程菌株对棉铃虫杀虫变化柱状图 Fig. 11 Insecticidal changing histogram of fcl gene engineered strains against H. armigera. |
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2.5 fcl基因工程菌株蛋白SDS-PAGE分析及差异蛋白鉴定为研究fcl基因对须糖多孢菌蛋白表达的影响，利用SDS-PAGE分析野生型菌株S. pogona、敲除菌株S. pogona-Δfcl和过表达菌株S. pogona- fcl蛋白，结果发现第4天的菌株蛋白差异最大。第4天时，S. pogona-fcl中蛋白条带A、B和C表达量降低；S. pogona-Δfcl中蛋白条带A和C表达量明显升高，蛋白条带B表达量降低(图 12)。将差异蛋白条带胶内酶解后进行质谱鉴定，发现它们分别为35 kDa的ECF亚家族RNA聚合酶σ-24亚基(RopE)、57 kDa的分子伴侣(GroEL)以及144 kDa的DNA指导的RNA聚合酶亚基β(RopB)。利用在线蛋白分析软件Uniprot和KEGG等对其进行功能分析(表 3)。
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| 图 12 fcl基因工程菌株蛋白的SDS-PAGE分析 Fig. 12 SDS-PAGE analysis of the protein of fcl gene engineered strains. M: protein marker. (A) S. pogona and S. pogona-fcl for 4 d. 1: S. pogona-fcl; 2: S. pogona. (B)S. pogona and S. pogona-Δfcl for 4 d. 1: S. pogona; 2: S. pogona-Δfcl. |
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表 3 fcl基因工程菌株差异蛋白的质谱鉴定Table 3 MS identification of differential proteins of fcl gene engineered strains
| Number | Protein | Gene | MW(kDa) | Function |
| A | ECF subfamily RNA polymerase sigma-24 subunit | rpoE | 35 | DNA-dependent RNA polymerase catalyzes the transcription of DNA into RNA using the four ribonucleoside triphosphates as substrates. |
| B | Molecular chaperonin | groEL | 57 | Prevents misfolding and promotes the refolding and proper assembly of unfolded polypeptides generated under stress conditions. |
| C | DNA-directed RNA polymerase subunit beta | rpoB | 144 | DNA-dependent RNA polymerase catalyzes the transcription of DNA into RNA using the four ribonucleoside triphosphates as substrates. |
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2.6 转录水平的qRT-PCR测定为了对差异蛋白的表达量进行验证，利用荧光定量PCR(qRT-PCR)对其转录水平进行测定。结果发现，第4天时，敲除菌株S. pogona-Δfcl中rpoB和rpoE转录水平相对于野生型菌株分别上调5.422、2.189倍；groEL的转录水平下调为野生型菌株的1.828倍。过表达菌株S. pogona-fcl中，rpoB、rpoE及groEL的转录水平相对于野生型菌株分别下调4.329、1.529、2.336倍(图 13)，证实了这些基因在转录水平和蛋白表达水平变化趋势一致。
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| 图 13 fcl基因工程菌株中基因转录水平的qRT-PCR检测 Fig. 13 qRT-PCR detection of gene transcription level in fcl gene engineered strains. |
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3 结论fcl基因编码的GDP-岩藻糖合成酶在氨基糖和核酸糖的代谢中起着至关重要的作用，是影响链霉菌生长发育的重要调控基因。丁烯基多杀菌素的推广应用受到野生型菌株产量低的限制，如何提高丁烯基多杀菌素的产量是当前亟待解决的问题。通过对须糖多孢菌基因进行基因工程改造后获得fcl基因的过表达菌株S. pogona-fcl和敲除菌株S. pogona-Δfcl。
已有关于fcl基因及其编码GDP-岩藻糖合成酶的研究表明，细胞的fcl基因具有重要调控作用。Mabeau等通过从海带和褐藻小叶海胆等多种生物体的细胞壁中分离出岩藻糖后发现，岩藻糖有助于增强细胞壁的稳定性[31]。Beatriz等在盘基网柄菌中的研究结果表明，当HL250菌株不能将GDP-甘露糖转化为GDP-岩藻糖时，作为孢子外壳的一组蛋白质的糖基化受阻，使得孢子外壳的完整性被破坏，导致孢子的产生和萌发受到抑制[32]。此外，还有研究报道岩藻糖具有促进细胞抗病毒和抗氧化等功能[33-34]。本研究发现fcl基因的敲除对须糖多孢菌的生长发育和产孢能力都有一定的抑制作用。与同一培养时间点的野生型菌株相比，S. pogona-Δfcl的细胞密度减小，菌株在R6培养基上产孢能力最弱；但对fcl基因进行过表达后，S. pogona-fcl的细胞密度无明显变化，在CSM和TSB培养基上产孢能力与野生型菌株相同，均表现为最强。Black等的研究表明，岩藻糖有助于防止细胞发生脱水[35]。本研究中，S. pogona-fcl菌丝体形态与野生型菌株一致，S. pogona-Δfcl的菌丝体变短，表面出现皱缩。结合相关研究报道及本文研究结果表明，fcl基因对须糖多孢菌的生长发育具有关键调控作用。
Michela等的研究表明，在草履虫小球藻病毒中，GDP-甘露糖能够在GDP-甘露糖-4, 6-脱水酶和GDP-4-酮基-6-脱氧-D-甘露糖差向异构酶/还原酶的催化作用下被转化为GDP-鼠李糖[36]。多杀菌素的生物前体是dTDP-鼠李糖，包括d (A、T、C、G) DP-鼠李糖4种。通过KEGG代谢通路分析可知，GDP可以转化为dGDP，因而GDP-鼠李糖也是参与丁烯基多杀菌素合成代谢的重要物质，能够促进丁烯基多杀菌素的生物合成。利用LC-MS对3个差异蛋白RNA聚合酶亚基β、ECF亚家族RNA聚合酶σ-24亚单位以及分子伴侣进行鉴定，运用KEGG、Uniprot和NCBI等软件对差异蛋白进行了功能分析。分析结果显示，rpoB基因编码DNA指导的RNA聚合酶亚基β和rpoE编码ECF亚家族RNA聚合酶σ-24亚单位在RNA的生物合成中发挥重要的作用，RNA作为模板大量翻译出与丁烯基多杀菌素生物合成的酶及其他相关蛋白。groEL基因编码分子伴侣可以通过影响氮代谢途径中核糖体蛋白因子，进而影响蛋白质的翻译和组装，并且参与RNA的降解过程。实验结果表明，与野生型菌株相比，S. pogona-Δfcl丁烯基多杀菌素的产量增加为野生型菌株的130%；S. pogona-fcl丁烯基多杀菌素的产量下降到野生型菌株的75%。S. pogona-Δfcl中rpoB、rpoE以及groEL基因的转录水平表现为上调，虽然groEL基因的转录水平也出现一定的下调，但由于其下调倍数较小，对于细胞中RNA的整体含量而言没有较大影响，因而丁烯基多杀菌素的产量因前体物质和相关酶类含量的增多而得到增加，对棉铃虫的杀虫活性相应显著提高。S. pogona-fcl中rpoB和rpoE以及groEL基因的转录水平均表现为下调，丁烯基多杀菌素的产量因前体物质和相关酶类含量的减少而降低，对棉铃虫的杀虫活性相应降低。
综上所述，fcl基因可以通过影响丁烯基多杀菌素的前体物质和RNA的合成过程、细胞脱水以及细胞壁的稳定性等方面，影响到菌丝体的生长发育、形态变化以及丁烯基多杀菌素的生物合成。通过对须糖多孢菌中GDP-岩藻糖合成酶编码基因fcl进行敲除和过表达，观察野生型菌株与工程菌株在表型和丁烯基多杀菌素生物合成之间存在的差异。结果表明fcl基因不仅在须糖多孢菌生长发育中发挥重要作用，而且与次级代谢产物丁烯基多杀菌素的生物合成密切相关，依据上述基础构建出了fcl基因参与的代谢调控网络图(图 14)，为后续如何进一步促进须糖多孢菌丁烯基多杀菌素的生物合成和提高产量提供了重要基础。
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| 图 14 须糖多孢菌中fcl基因的代谢调控网络图 Fig. 14 Metabolic regulatory network map of fcl gene in S. pogona. |
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