白粉菌诱导月季对甜菜夜蛾产生抗性的关键候选基因筛选及CYP71的生物信息学分析

刘思琪; 杨琦; 王科建; 李云仙; 杨发忠

doi:10.19303/j.issn.1008-0384.2022.012.012

白粉菌诱导月季对甜菜夜蛾产生抗性的关键候选基因筛选及CYP71的生物信息学分析

doi: 10.19303/j.issn.1008-0384.2022.012.012

西南林业大学西南地区林业生物质资源高效利用国家林业与草原局重点实验室，云南　昆明　650224

基金项目: 国家自然科学基金项目（32060695、31560517）；云南省农业联合专项面上项目（202101BD070001-107）

详细信息

作者简介:
刘思琪（2001−），女，主要从事生物分子学研究（E-mail：2586724449@qq.com）

通讯作者:
杨发忠（1974−），男，副教授，博士，主要从事生物分子学研究（E-mail：yangfazhong105@163.com）

中图分类号: S 436
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出版历程
- 收稿日期: 2022-10-08
- 修回日期: 2022-10-22
- 网络出版日期: 2022-12-28
- 刊出日期: 2022-03-28

Candidate Genes Selection in Powdery Mildew-induced Resistance of Rose Plant to Beet Armyworms and the Bioinformatics Analysis on CYP71

Key Laboratory of Ministry of Education for Forest Resources Conservation and Utilization in the Southwest Mountains of China, Southwest Forestry University, Kunming, Yunnan　650224, China

摘要

摘要: 目的通过检测白粉菌侵染月季后转录组基因的表达量变化，筛选白粉菌诱导月季产生对甜菜夜蛾抗性的关键候选基因，为月季抗虫基因的功能研究及抗虫品种的培育提供参考。方法以月季品种艳粉（Rosa chinensis Jacq.）为研究材料，利用二代测序技术对白粉菌侵染月季前后基因的表达量进行转录组测序。利用RSEM对基因的表达水平进行定量分析，DESeq2软件进行表达差异分析，利用KOBAS将得到的差异表达基因进行KEGG富集分析。对筛选出的可能参与三萜类物质生物合成的CYP71D差异表达基因（Differentially expressed genes，DEGs）进行生物信息学分析，并分析了CYP71D蛋白与抗虫性的关系。结果转录组测序共检测到差异表达基因1 646个，共643个差异表达基因被富集到81条KEGG通路中，其中31个差异表达基因与萜类合成相关。生物信息学分析结果表明了CYP71D亚家族的物种特异性。在植物中CYP71A主要参与类单萜的生物合成，CYP71D主要参与单萜类、倍半萜类、二萜类、三萜类等与抗病、虫有关的次生代谢物的生物合成。结论月季细胞色素P450超家族蛋白酶中CYP71簇的CYP71A、CYP71D亚家族可能与三萜化合物Dehydro (11,12) ursolic acid lactone（DUA）的生物合成有关，表明CYP71D家族蛋白参与修饰三萜化合物的底物识别位点，本研究为探索寄主植物介导的间接的病虫互作机理的研究提供了一定的思路。
- 月季 /
- 白粉菌 /
- 三萜类化合物 /
- 甜菜夜蛾 /
- CYP71 /
- 生物信息学
Abstract: Objective The transcriptomes of rose plants artificially infected by powdery mildew were obtained to identify the genes responsible for the resistance to infestation by beet armyworms. Method Transcriptomes of Yanfen rose (Rosa chinensis Jacq.) before and after being infected by powdery mildew were obtained using the second-generation sequencing technology. RSEM was used to quantitatively analyze the gene expressions, DESeq2 software for the expression differences, and KOBAS for the KEGG enrichment analysis on the differentially expressed genes (DEGs). Bioinformatics of the DEGs of CYP71D susceptible of associating with triterpenoids biosynthesis was secured to study the relationship between the CYP71D and the insect resistance. Result A total of 1 646 DEGs were detected by the transcriptome sequencing. Of which, 643 were enriched into 81 KEGG pathways with 31 DEGs related to terpenoid synthesis. The bioinformatics on them showed a species specificity of CYP71D subfamily. In plants, CYP71A were mainly involved in the biosynthesis of monoterpenes, while CYP71D in that of the disease and insect resistance-related secondary metabolites, such as monoterpenes, sesquiterpenes, diterpenes, and triterpenoids. Conclusion The CYP71A and CYP71D subfamilies of the cytochrome P450 enzyme in the rose might relate to the synthesis of dehydro (11,12) ursolic acid lactone (DUA). It suggested a possible association of CYP71D with the formation of triterpenoids. The results provided information for the study on the relationship between the genes in host plants and infecting insects.
- Rosa chinensis Jacq. /
- powdery mildew /
- triterpenoids /
- beet armyworm /
- CYP71 /
- bioinformatics

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图 1 差异表达基因排名前十的KEGG代谢通路

Figure 1. KEGG enrichment of DEGs top 10

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图 2 CPY71基因结构及在染色体上的分布

Figure 2. Structure and chromosome distribution of CPY71 gene

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图 3 CYP71蛋白的跨膜区域与信号肽预测

Figure 3. Predicted transmembrane region and signal peptide of CYP71 proteins

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图 4 CPY71家族蛋白的二级与三级结构预测

Figure 4. Predicted secondary and tertiary structures of CPY71 family proteins

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图 5 CYP71的保守结构域

Figure 5. Conserved domain of CYP71 protein

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图 6 3个CYP71亚家族的系统进化树

Figure 6. Phylogenetic tree of 3 CYP71 subfamilies

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图 7 CYP71D蛋白和烟草、大麻的CYP71D蛋白多序列比对

Figure 7. Multiple sequence alignment of CYP71D protein with CYP71D proteins of tobacco and cannabis

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表 1 转录组测序数据

Table 1. Transcriptome sequences

样本名称 Sample	总条目数 Raw reads	质控总条目数 Clean reads	质控总数据量 Clean data/Gb	错误率 Error rate/%	Q20/%	Q30/%
健康样本1 CK1	46403668	46118398	6.88	0.0244	98.30	94.63
健康样本2 CK2	51906926	51578808	7.68	0.0244	98.31	94.68
健康样本3 CK3	54877772	54487558	8.12	0.0248	98.17	94.32
感病样本1 Ta1	52578650	52226040	7.78	0.0245	98.29	94.61
感病样本2 Ta2	51390748	51052798	7.59	0.0247	98.20	94.39
感病样本3 Ta3	48584868	48273580	7.18	0.0246	98.25	94.52
Q20、Q30分别指测序质量在99%和99.9%以上的碱基占总碱基的百分比。 Q20 and Q30 refer to the percentage of bases with sequencing quality above 99% and 99.9%, respectively.

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表 2 Reads与参考基因组的比对结果

Table 2. Comparison between Reads and reference genome （单位：%）

样本 Sample	Clean reads数目总量 Total mapped	编码区 CDS	基因区间 Intergenic	内含子区 Introns	3′非编码区 3′UTR	5′非编码区 5′UTR
健康样本1 CK1	86.53	86.85	0.78	1.91	5.81	4.65
健康样本2 CK2	86.82	87.20	0.73	1.78	5.52	4.77
健康样本3 CK3	86.60	87.29	0.70	1.68	5.71	4.63
感病样本1 Ta1	86.74	86.90	0.74	1.91	5.88	4.56
感病样本2 Ta2	86.40	86.54	0.75	1.99	5.75	4.96
感病样本3 Ta3	86.30	85.84	0.77	2.04	6.56	4.78

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表 3 CPY71蛋白的理化性质及亚细胞定位

Table 3. Physicochemical properties and subcellular localization of CPY71 protein

基因ID Gene ID	蛋白质登录号 Protein accession	氨基酸长度 Amino acid length/aa	分子质量 Molecular weight /kDa	理论等电点 Theoretical pI	不稳定性系数 Instability index	平均亲水性系数 GRAVY	亚细胞定位预测 Subcellular localization
LOC112176060	XP_024169643.1	510	58.18	5.74	46.17	−0.022	内质网 Endoplasmic reticulum
LOC112195754	XP_024191713.1	510	58.33	8.78	41.88	−0.139	内质网 Endoplasmic reticulum
LOC112195786	XP_024191752.1	512	58.01	7.01	42.50	−0.200	内质网 Endoplasmic reticulum
LOC112198434	XP_024195327.1	513	58.64	7.26	46.55	−0.166	内质网 Endoplasmic reticulum
LOC112177305	XP_040369681.1	515	58.38	7.71	36.81	−0.187	内质网 Endoplasmic reticulum
LOC112166540	XP_024159180.1	512	58.54	8.76	41.76	−0.212	内质网 Endoplasmic reticulum

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表 4 CYP71簇的亚家族分类及其功能特征

Table 4. Classification and functional characteristics of CYP71 subfamilies

亚家族 Subfamily	代谢指向性 Metabolism	功能/代谢产物的类别 Function/class of metabolites
CYP71A	专一	类单萜 Monoterpenoid
CYP71AR	专一	类单萜 Monoterpenoid
CYP71AV	专一	倍半萜 Sesquiterpenoid
CYP76B	专一	环烯醚萜 Iridoid，类单萜 Monoterpenoid，解毒异生素 Xenobiotics Detoxification
CYP71BA	专一	倍半萜 Sesquiterpenoid
CYP71BL	专一	倍半萜 Sesquiterpenoid
CYP71D	专一	类单萜 Monoterpenoid，倍半萜 Sesquiterpenoid，二萜 Diterpenoid，吲哚生物碱 Indole Alkaloid，类黄酮 Flavonoid
CYP71Z	专一	二萜 Diterpenoid
CYP76M	专一	二萜 Diterpenoid
CYP82G	专一	萜类衍生物 Terpenoid-derived
CYP93E	专一	三萜 Triterpenoid
CYP99A	专一	二萜 Diterpenoid
CYP701A	一般	二萜类植物激素 GA Diterpenoid Phytohormone GA
CYP701A	专一	二萜 Diterpenoid
CYP705A	专一	三萜 Triterpenoid
CYP706B	专一	倍半萜 Sesquiterpenoid

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