BbRho5对球孢白僵菌生长速率的作用研究

doi:10.13560/j.cnki.biotech.bull.1985.2021-0210

摘要/Abstract

摘要：

为阐明BbRho5对球孢白僵菌生防潜能的作用,构建了Bbrho5单基因敲除菌株ΔBbrho5,以野生型菌株WT作为对照,在不同培养基上测定菌落生长速率,并测定了菌株对多菌灵胁迫耐受性及对大蜡螟幼虫体壁侵染能力。进一步获取和分析了ΔBbrho5和WT细胞内基因转录组数据。结果表明,BbRho5蛋白功能缺陷显著抑制球孢白僵菌菌丝生长速率,同时微弱影响其多菌灵胁迫抗逆性及生防能力。相较于WT,ΔBbrho5中具有770个差异表达基因（DEGs）,其中上调基因395个,下调基因375个。GO分析显示,ΔBbrho5 VS WT中DEGs主要富集于氧化还原酶活力（oxidoreductase activity）和单加氧酶活力（monooxygenase activity）功能。KEGG通路富集结果显示,DEGs主要富集于氮代谢及多种氨基酸代谢通路。在氮代谢通路中富集到7个功能基因,其中有5个上调,2个下调,说明敲除菌株可能采用增强氮源利用及谷氨酸合成以应对Bbrho5缺陷引起的生长迟缓。以上研究结果揭示了球孢白僵菌中小GTP酶BbRho5对球孢白僵菌生长速率具有重要影响,且氮代谢和氨基酸代谢可能为其重要的响应代谢通路。

关键词: 小GTP酶, BbRho5, 球孢白僵菌, 生长速率, 转录组

Abstract:

In order to clarify the role of BbRho5 in the biocontrol potential of B. bassiana,a Bbrho5 single gene knockout strain ΔBbrho5 was constructed. The colony growth rate was measured on different media while the wild-type strain WT was used as the control. In addition,carbendazim-tolerance of WT and ΔBbrho5 and their virulence to Galleria mellonella larval body wall were also tested. The transcriptome data of ΔBbrho5 and WT were further obtained and analyzed. The results showed that the functional defect of BbRho5 protein significantly inhibited the hyphal growth rate of B. bassiana;meanwhile BbRho5 showed mild effect in carbendazim-tolerance and virulence in B. bassiana. Compared with WT,there were 770 differentially expressed genes（DEGs）in ΔBbrho5,of which 395 were up-regulated and 375 were down-regulated. GO analysis showed that DEGs in ΔBbrho5 VS WT were mainly enriched in oxidoreductase activity and monooxygenase activity. KEGG pathway enrichment showed that DEGs were mainly enriched in nitrogen metabolism and various amino acid metabolism pathways. Seven functional genes were enriched in the nitrogen metabolism pathway,of which 5 were up-regulated and 2 were down-regulated,indicating that ΔBbrho5 enhanced nitrogen source utilization and glutamate synthesis to cope with the growth retardation caused by Bbrho5 deficiency. The above results reveal that the small GTPases BbRho5 has an important effect on the growth rate of B. bassiana,and nitrogen metabolism and amino acid metabolism may be important metabolic pathways.

Key words: small GTPase, BbRha5, Beauveria bassiana, growth rate, transcriptome

关怡, 李新, 王定一, 杜茜, 张龙斌, 叶秀云. BbRho5对球孢白僵菌生长速率的作用研究[J]. 生物技术通报, 2022, 38(2): 132-140.

GUAN Yi, LI Xin, WANG Ding-yi, DU Xi, ZHANG Long-bin, YE Xiu-yun. Functional Study of BbRho5 on the Growth Rate of Beauveria bassiana[J]. Biotechnology Bulletin, 2022, 38(2): 132-140.

图/表 10

表1 Bbrho5单基因缺失及回补菌株构建引物

Table 1 Primers for constructing single gene deletion mu-tant or complementary strains of Bbrho5

引物名称 Primer	序列 Sequence（5'-3'）
rho5-up-F	AAAAAGGAATTCCGTCGCCAACATCTCCATC
rho5-up-R	AAAAACGGGATCCGTCGTCGGTGGTGGTGAG
rho5-dn-F	AACGTCGACCCATGGCTCGAGTGGTATCCGCC- CCCTTTCT
rho5-dn-R	CGTTAACACTAGTCAGATCCGGTTAGGGAGGC- TTCGTA
rho5-comp-F	ATCCGTCGACCTGCAGCCACGTCGCCAACATCT- CCAT
rho5-comp -R	ACACTAGTCAGATCTTCTAGCAGTGGTGCTCAG- CAGACAA

图1 Bbrho5敲除及回补菌落PCR鉴定 1：回补菌株;2：野生菌株;3：敲除菌株;M：Marker

Fig. 1 PCR verification for Bbrho5 deletion or compleme-ntary 1：Complementary strain. 2：WT strain. 3：Gene deletion strain. M：Marker

图2 野生菌株、敲除菌株和回补菌株的生长速率测定

Fig. 2 Colony diameters of WT,Bbrho5 deletion or com-plementary stains

图3 球孢白僵菌对多菌灵的耐药性（A）及对大蜡螟的生物防治能力（B）

Fig.3 Fungal tolerance to carbendazim（A）and the virulence for G. mellonella larve infection（B）

表2 WT和ΔBbrho5的转录组数据质量统计

Table 2 Quality statistics of the WT and ΔBbrho5 transcriptome data

Sample	Clean reads	Clean bases	Error rate/%	Q20/%	Q30/%	GC content/%
WT-1	54944640	8177521548	0.023	98.86	96.11	55.56
WT-2	54680452	8142825538	0.0233	98.76	95.82	55.61
WT-3	53371986	7953327629	0.0234	98.73	95.75	55.71
ΔBbrho5-1	50282602	7496483802	0.0233	98.74	95.81	56.01
ΔBbrho5-2	49584208	7395969389	0.0234	98.75	95.77	55.71
ΔBbrho5-3	52574486	7847183298	0.0232	98.82	96	55.94

表3 WT及ΔBbrho5序列比对分析结果统计

Table 3 Alignment and analysis of WT andΔBbrho5 sequence

Sample	Total reads	Total mapped	Multiple mapped	Uniquely mapped
WT-1	54944640	52334433（95.25%）	242381（0.44%）	52092052（94.81%）
WT-2	54680452	52109660（95.3%）	225669（0.41%）	51883991（94.89%）
WT-3	53371986	50353743（94.34%）	210188（0.39%）	50143555（93.95%）
ΔBbrho5-1	50282602	47863412（95.19%）	202423（0.4%）	47660989（94.79%）
ΔBbrho5-2	49584208	47574991（95.95%）	239083（0.48%）	47335908（95.47%）
ΔBbrho5-3	52574486	50041097（95.18%）	302786（0.58%）	49738311（94.61%）

图4 ΔBbrho5 vs WT差异表达基因散点图

Fig. 4 Scatter diagram of DEGs between ΔBbrho5 and WT

图5 ΔBbrho5 vs WT差异基因GO富集分析柱形图

Fig. 5 GO analysis of DEGs between ΔBbrho5 and WT

图6 ΔBbrho5 vs WT显著差异基因功能富集分析

Fig. 6 KEGG analysis of DEGs between ΔBbrho5 and WT

图7 ΔBbrho5 vs WT的iPath代谢通路分析

Fig. 7 iPath analysis of DEGs between ΔBbrho5 and WT

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