枯草芽孢杆菌NCD-2对棉花根系分泌物L-脯氨酸响应的转录-蛋白质组学联合分析

doi:10.3864/j.issn.0578-1752.2021.21.009

摘要/Abstract

摘要：

【目的】棉花根系分泌物L-脯氨酸是影响生防菌株定殖的关键因素之一,前期研究发现L-脯氨酸能够提高枯草芽孢杆菌（Bacillus subtilis）NCD-2生物膜形成能力。通过高通量测序技术分析L-脯氨酸调控枯草芽孢杆菌NCD-2生物膜形成和生防潜力相关的基因,为深入认识棉花根系分泌物与生防菌株的分子互作关系打下基础。【方法】以枯草芽孢杆菌NCD-2菌株为供试材料,外源添加浓度为10 mg·mL^-1的L-脯氨酸共培养24 h后,分别进行转录组（RNA-seq）和同位素标记相对定量蛋白质组技术（iTRAQ）分析,对所获得的转录组和蛋白质组数据进行生物信息学分析,利用实时荧光定量PCR（RT-qPCR）验证不同代谢通路中部分差异基因的表达情况。【结果】转录组分析发现,L-脯氨酸和NCD-2菌株共培养后,获得1 071个差异表达基因（DEG）,其中602个基因上调,469个基因下调。GO分析发现在生物过程、细胞组分和分子功能方面分别有49、14和30个功能条目显著富集。KEGG代谢通路主要富集在化合物代谢、鞭毛组装、细菌运动和趋化作用。蛋白质组学分析发现,筛选到211个差异表达蛋白（DEP）,其中118个蛋白上调,93个蛋白下调。GO分析发现在生物过程和分子功能方面分别有13和8个功能条目显著富集。KEGG代谢通路主要富集在氨基酸代谢、碳水化合物类代谢、鞭毛组装和ABC转运蛋白。进一步转录-蛋白质组学联合分析发现,在L-脯氨酸作用下,检测到共有的显著差异表达基因（或蛋白）112个,其中38个基因（或蛋白）下调,74个基因（或蛋白）上调。GO功能显著富集主要集中在营养库活性、催化活性、细胞膜、定位、细胞脂质代谢过程、氧化还原过程、sigma因子活性、转运活性、芽孢形成9个方面。KEGG代谢通路主要富集在能量代谢、ABC转运蛋白、抗生素生物合成、鞭毛组装、运动或趋化作用和双组分系统方面。RTqPCR验证了26个差异表达显著基因,结果发现在表达量上存在一定的差异,但表达趋势与RNAseq和iTRAQ组学分析结果基本一致。【结论】棉花根系分泌物L-脯氨酸与枯草芽孢杆菌NCD-2之间的互作存在一个复杂的生物过程,依赖于不同代谢通路网络中的多个基因。双组分系统、抗生素生物合成、能量代谢、运动或趋化作用、鞭毛组装和ABC转运蛋白通路中的差异基因（或蛋白）可能在棉花根系分泌物与枯草芽孢杆菌互作过程方面发挥着重要作用。

关键词: L-脯氨酸, 枯草芽孢杆菌, 转录组, 蛋白质组, 互作关系

Abstract:

【Objective】L-proline in root exudates of cotton is one of the key factors affecting the colonization of biocontrol microorganisms. Previous studies showed that L-proline could improve the ability of Bacillus subtilis NCD-2 biofilm formation. The objective of this study is to explore the regulatory genes related to the biofilm formation and biocontrol potential of strain NCD-2 through high-throughput sequencing technology, and to lay a foundation for further understanding the molecular interaction between cotton root exudates and biocontrol strain.【Method】Strain NCD-2 was co-cultured with L-proline at the concentration of 10 mg·mL ^-1 for 24 h, and it was analyzed by transcriptome (RNA-seq) and isotope labeled relative quantitative proteomics (iTRAQ). The expression of some differential genes in different metabolic pathways was verified by RT-qPCR.【Result】Transcriptome analysis showed that 1 071 differentially expressed genes (DEGs) were obtained after L-proline and NCD-2 co-culture, of which 602 genes were up-regulated and 469 genes were down-regulated. GO analysis showed that 49, 14 and 30 functional items were significantly enriched in biological process, cell component and molecular function, respectively. KEGG pathways were mainly enriched in compound metabolism, flagellum assembly, bacterial motility or chemotaxis. Proteomic analysis showed that a total of 211 differentially expressed proteins (DEPs) were detected compared to the control, of which 118 proteins were up-regulated and 93 proteins were down-regulated. GO analysis showed that 13 and 8 functional items were significantly enriched in biological process and molecular function, respectively. KEGG pathways were mainly enriched in amino acid metabolism, carbohydrate metabolism, flagellum assembly and ABC transporter. Further transcriptional-proteomics analysis revealed that 112 DEGs (or DEPs), including 38 down-regulated genes (or proteins) and 74 up-regulated genes (or proteins), were detected. GO functional items were significantly enriched in nine aspects, including nutrient reservoir activity, catalytic activity, cell membrane, localization, cellular lipid metabolic process, oxidation-reduction process, sigma factor activity, transport activity and spore formation. KEGG pathways were mainly enriched in energy metabolism, ABC transporter, antibiotic biosynthesis, flagellum assembly, motility or chemotaxis and two-component system. Twenty-six DEGs were verified by RT-qPCR. The results showed that there were some differences in the expression level, but the expression trend was basically consistent with that of RNA-seq and iTRAQ.【Conclusion】The interaction between L-proline in cotton root exudates and B. subtilis NCD-2 is a complex biological process, which depends on multiple genes in different metabolic pathway networks. It is cleared that DEGs (or DEPs) of the two-component system, antibiotic biosynthesis, energy metabolism, motility or chemotaxis, flagellum assembly and ABC transporter pathway may play an important role in the interaction between cotton root exudates and B. subtilis.

Key words: L-proline, Bacillus subtilis, transcriptome, proteome, interaction relationship

赵卫松,郭庆港,董丽红,王培培,苏振贺,张晓云,鹿秀云,李社增,马平. 枯草芽孢杆菌NCD-2对棉花根系分泌物L-脯氨酸响应的转录-蛋白质组学联合分析[J]. 中国农业科学, 2021, 54(21): 4585-4600.

ZHAO WeiSong,GUO QingGang,DONG LiHong,WANG PeiPei,SU ZhenHe,ZHANG XiaoYun,LU XiuYun,LI SheZeng,MA Ping. Transcriptome and Proteome Analysis of Bacillus subtilis NCD-2 Response to L-proline from Cotton Root Exudates[J]. Scientia Agricultura Sinica, 2021, 54(21): 4585-4600.

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基因 Gene		序列Sequence (5′ to 3′)	基因 Gene		序列Sequence (5′ to 3′)
fliF	F	ACCGAAAGCGGGAACTAC	opuCC	F	GCAAGGAAGCGGAGAAAG
fliF	R	CATCAGGCGGCTCTACCA	opuCC	R	AGCCGTAGGAATCAAACCA
gInQ	F	TATCGGGATGGTGTTTCA	gcvPB	F	TTATTTCCCGCTTAATGTTG
gInQ	R	GTCAGCCTTGTCTGGGAT	gcvPB	R	TCTTCCGCCTCGTATCTG
cheY	F	GGAGCACAAGCGGTAGAG	putC	F	AATCGTTTCAATCAACCCAG
cheY	R	CTGAATGGCATCAATAAC	putC	R	TATCGGCATCCGCCTCGT
fliY	F	CAGACCGTATTCCTGATG	rocA	F	TAGTTGAGCATCCGAAGAC
fliY	R	CACCGTTTCTTCTTCCTC	rocA	R	CAATTACCCGTTTGAGCC
opuCA	F	CCAGCAGAACATCTCACTC	yodQ	F	GGCAATACAGCCCTTCTT
opuCA	R	GCGGATAACGGTCTAAATAC	yodQ	R	GCCGCACTCTTCATCTAC
znuA	F	GGGCTTTCACCTGACCAA	ald	F	CCTCTTCTGACGCCAATG
znuA	R	CGAGCGTATCTGCGACCT	ald	R	CAACGCCTCCTCCGATAA
gapB	F	AAGAGGTTGTGGCTGGTG	yerA	F	GAGAAGGCTGGAACTGGG
gapB	R	TTGCTTCGACGACTATGT	yerA	R	ATTGTAGGCGTCGATTGC
mmgD	F	AATGGAAACGCTGGAACG	asnO	F	TGTCGGATGTGCCTGTTT
mmgD	R	TGTTTGCGGAAGGAGACC	asnO	R	CATTTGTGGTGCGTTGTG
acoC	F	TGGACCAGGCGGACGAAT	yisZ	F	AGGATCGGGACATGGTTA
acoC	R	TCGGGCAGCGATGACCTT	yisZ	R	ATGAAATCGGGTGAAAGC
thrD	F	ACTGATCGCCGCTTACTT	ggt	F	ACACTGTCCAGGATTTCG
thrD	R	ACCGCTCCGTGAGAATGT	ggt	R	GGAGGAGGAGTAGTAGCG
yjmD	F	CAGCGGAGGTGAAGAAGC	yoaD	F	CCCGAACTTTCATTTGTC
yjmD	R	GAGGGTAGCGAGCGGATT	yoaD	R	CTCCATCCTTCAGCCACT
epsA	F	TCGAATCTCAGTGACATCCA	epsC	F	AATCCAGAAGAGGCGGTCAA
epsA	R	AGATAGGTGCAATTCCGC	epsC	R	GCCGAAGCGAACAGCAAC
ssuB	F	ATTATCAATCGCACCAGG	scoB	F	TTGTCGCAAATGAGATACCC
ssuB	R	CAACAGTCAGCCAAGGAA	scoB	R	CGTCTTCCGTTCCTTCCA
gyrB	F	GAAGCACGGACAATCACC
gyrB	R	TCCAAAGCACTCTTACGG

处理 Treatment	原始序列 Raw reads	过滤序列 Clean reads	过滤序列大小 Clean reads bases (G)	Q20 比例Q20 percentage (%)	Q30比例Q30 percentage (%)	G和C占总碱基数量百分比 GC content (%)
CK	11104298±1620312	10870077±1617999	1.63±0.24	97.46±0.11	92.90±0.32	41.81±0.40
T	8978376±1390913	8783987±1431917	1.32±0.22	97.99±0.11	93.79±0.27	43.30±0.05

项目 Item	处理 Treatment	FPKM值 FPKM value
项目 Item	处理 Treatment	0-1	1-3	3-15	15-60	>60
基因数量 Number of genes	CK	616.33±7.64	130.33±10.41	493.67±31.13	984.67±33.01	2305.00±54.44
基因数量 Number of genes	T	690.00±22.27	165.67±4.73	569.33±18.58	1073.67±36.23	2031.33±74.22
基因表达比例 Gene expression ratio (%)	CK	13.60±0.17	2.87±0.23	10.90±0.69	21.74±0.73	50.88±1.20
基因表达比例 Gene expression ratio (%)	T	15.23±0.49	3.66±0.10	12.57±0.41	23.70±0.80	44.84±1.64

KEGG代谢途径 KEGG pathway	差异基因数目 Number of DEGs	所有基因数目 Number of all genes	差异表达基因比例 DEGs ratio (%)	P值 P-value	表达调控 Regulated
不同环境的微生物代谢 Microbial metabolism in diverse environments	40	169	23.67	0.0076	Up
链霉素生物合成 Streptomycin biosynthesis	6	9	66.67	0.0088	Up
碳代谢Carbon metabolism	25	94	26.60	0.0103	Up
硫代谢Sulfur metabolism	8	17	47.06	0.0118	Up
乙醛酸和二羧酸代谢 Glyoxylate and dicarboxylate metabolism	9	25	36.00	0.0276	Up
鞭毛组装 Flagellar assembly	26	33	78.79	0	Down
细菌趋化性 Bacterial chemotaxis	13	24	54.17	0	Down
核糖体 Ribosome	18	88	20.45	0.0248	Down
萜类化合物生物合成 Terpenoid backbone biosynthesis	5	14	35.71	0.0413	Down
错配修复Mismatch repair	6	20	30.00	0.0475	Down

KEGG代谢途径 KEGG pathway	注释代谢途径的差异表达蛋白数量 Number of DEPs with pathway annotation	注释代谢途径的蛋白数量 Number of proteins with pathway annotation	差异蛋白比例 DEPs ratio (%)	P值 P-value	上调数量Up-regulated number	下调数量Down-regulated number
酮体的合成与降解 Synthesis and degradation of ketone bodies	4	5	80.00	0.0004	4	0
乙醛酸和二羧酸代谢 Glyoxylate and dicarboxylate metabolism	9	29	31.03	0.0009	7	2
牛磺酸和亚牛磺酸代谢 Taurine and hypotaurine metabolism	3	7	42.86	0.0224	2	1
丙氨酸、天冬氨酸和谷氨酸代谢 Alanine, aspartate and glutamate metabolism	7	31	22.58	0.0226	4	3
鞭毛组装Flagellar assembly	2	3	66.67	0.0255	0	2
戊糖和葡萄糖醛酸的相互转化 Pentose and glucuronate interconversions	5	19	26.32	0.0284	4	1
精氨酸和脯氨酸代谢 Arginine and proline metabolism	5	19	26.32	0.0284	3	2
丁酸代谢Butanoate metabolism	5	19	26.32	0.0284	5	0
不同环境的微生物代谢 Microbial metabolism in diverse environments	22	151	14.57	0.0335	13	9
ABC转运蛋白ABC transporter	13	79	16.46	0.0486	7	6