Analysis of the antennal transcriptome and differentially expressed genes of female and male Monolepta signata

doi:10.6048/j.issn.1001-4330.2024.04.023

Abstract

Abstract:

【Objective】 To establish the transcriptome database of adult Monolepta signata antennae, screen the differentially expressed genes in the antennae of female and male M. signata and analyze the physiological functions of these genes. 【Methods】 In this study, Illumina high-throughput sequencing technology was used to conduct transcriptome sequencing on the antennae of male and female M. signata and analyze the functional annotation and pathway enrichment of unigenes, and after that the expression level of odorant binding protein genes in antennae was detected by RT-qPCR. 【Results】 A total of 73,050 unigenes were obtained from the antennae transcriptome sequencing of female and male of adult M. signata, with the percentage of Q30 bases above 93.23, and 34,233 unigenes were annotated in at least one database (46.86%). A total of 395 differentially expressed genes were detected in the antennae of male M. signata compared with that of female M. signata. Among them, 288 genes were up-regulated and 107 genes were down-regulated.【Conclusion】 In the GO database, the most significant function of enrichment of these differentially expressed genes were odorant binding, olfactory receptor activity, and sensory perception of smell. RT-qPCR results showed that the relative expression levels of odorant binding protein genes in antennae of female and male M. signata were consistent with the transcriptome sequencing results. The transcriptome database of the antenna of adult M. signata is successfully obtained.

Key words: Monolepta signata; antennae transcriptome; functional annotation; differentially expressed genes

摘要：

【目的】建立双斑长跗萤叶甲成虫触角转录组数据库,筛选雌虫、雄虫触角中的差异表达基因并解析其涉及的生理功能,为进一步研究双斑长跗萤叶甲的基因功能分析及嗅觉感受机制奠定分子基础。【方法】采用Illumina高通量测序技术对双斑长跗萤叶甲雌虫、雄虫触角进行转录组测序,对获得的unigenes进行功能注释、通路富集等生物信息学分析,利用RT-qPCR技术检测气味结合蛋白基因在触角中的表达水平。【结果】双斑长跗萤叶甲雌、雄成虫触角转录组测序共获得73 050条unigenes,Q30碱基百分比均在93.23%以上,34 233条unigenes至少在1个数据库中获得注释。与双斑长跗萤叶甲雌虫触角相比,雄虫触角中检测到395个差异表达基因,其中288个基因上调表达,107个基因下调表达。【结论】在GO注释库中,差异表达基因富集最显著的功能为气味结合、嗅觉受体活性和嗅觉感知。气味结合蛋白基因在雌虫、雄虫触角中的相对表达水平趋势与转录组测序结果相符合,获得了双斑长跗萤叶甲成虫触角转录组数据库。

关键词: 双斑长跗萤叶甲, 触角转录组, 功能注释, 差异表达基因

CLC Number:

S433

HE Wanjie, MENG Hanying, ZHI Mengting, CHEN Jing. Analysis of the antennal transcriptome and differentially expressed genes of female and male Monolepta signata[J]. Xinjiang Agricultural Sciences, 2024, 61(4): 984-995.

何婉洁, 孟涵颖, 支梦婷, 陈静. 双斑长跗萤叶甲雌虫、雄虫触角转录组及差异表达基因分析[J]. 新疆农业科学, 2024, 61(4): 984-995.

Figures/Tables 13

References 45

[1]	中国科学院动物研究所昆虫分类区系室叶甲组, 河北省张家口地区坝下农业科学研究所植保组, 河北省蔚县农业局植保站西合营公社技术站. 双斑萤叶甲研究简报[J]. 昆虫学报, 1979, 22 (1) : 115-117.
	Research Group of Leaf Beetle, Division of Insect Taxonomy Institute of Agriculture of Baxia, Changchiakou District, Hopei Province, Technical Station, Plant Protection Station of Xiheying People’s Commune, Agricultural Bureau of Yu County, Hopei Provinge. A preliminary study of the bionomics of the galerucid beetle, Monolepta hieroglyphica (Motschulsky)[J]. Acta Entomologica Sinica, 1979, 22 (1) : 115-117.
[2]	虞佩玉, 王书永, 杨星科. 中国经济昆虫志,第五十四册,鞘翅目,叶甲总科(二)[M]. 北京: 科学出版社, 1996.
	YU Peiyu, WANG Shuyong, YANG Xingke. Economic Insect Fauna of China. Coleoptera: Chrysomeloidea (II)[M]. Beijing: Science Press, 1996.
[3]	Wagner T, Bieneck S. Galerucine type material described by victor motschulsky in 1858 and 1866 from the Zoological Museum Moscow (Coleoptera: Chrysomelidae, Galerucinae)[J]. Entomologische Zeitschrift mit Insekten-Börse, 2012, 122 (5) : 205-216.
[4]	梁日霞, 王振营, 何康来, 等. 基于线粒体COⅡ基因序列的双斑长跗萤叶甲中国北方地理种群的遗传多样性研究[J]. 昆虫学报, 2011, 54(7): 828-837.
	LIANG Rixia, WANG Zhenying, HE Kanglai, et al. Genetic diversity of geographic populations of Monolepta hieroglyphica (Motschulsky) (Coleoptera: Chrysomelidae) from North China estimated by mitochondrial COⅡ gene sequences[J]. Acta Entomologica Sinica, 2011, 54(7): 828-837.
[5]	陈光辉, 尹弯, 李勤, 等. 双斑长跗萤叶甲研究进展[J]. 中国植保导刊, 2016, 36(10): 19-26.
	CHEN Guanghui, YIN Wan, LI Qin, et al. Research progress on Monolepta hieroglyphica (Motschulsky)[J]. China Plant Protection, 2016, 36(10): 19-26.
[6]	李菁, 张小飞, 徐玲玲, 等. 中国南方双斑长跗萤叶甲地理种群遗传结构及Wolbachia感染[J]. 昆虫学报, 2021, 64(6): 730-742.
	LI Jing, ZHANG Xiaofei, XU Lingling, et al. Genetic structure and Wolbachia infection in geographical populations of Monolepta hieroglyphica(Coleoptera: Chrysomelidae)in South China[J]. Acta Entomologica Sinica, 2021, 64(6): 730-742.
[7]	赵秀梅, 郑旭, 郭井菲, 等. 齐齐哈尔市玉米田双斑长跗萤叶甲成虫发生规律[J]. 应用昆虫学报, 2021, 58(4): 979-984.
	ZHAO Xiumei, ZHENG Xu, GUO Jingfei, et al. Occurrence of Monolepta hieroglyphica adults in cornfields in Qiqihar[J]. Chinese Journal of Applied Entomology, 2021, 58(4): 979-984.
[8]	陈静, 张建萍, 张建华, 等. 双斑长跗萤叶甲的嗜食性[J]. 昆虫知识, 2007, 44(3): 357-360.
	CHEN Jing, ZHANG Jianping, ZHANG Jianhua, et al. Food preference of Monolepta hieroglyphica[J]. Chinese Bulletin of Entomology, 2007, 44(3): 357-360.
[9]	He Q, Song X M, Ma H W, et al. The complete mitochondrial genome of Monolepta hieroglyphica (Motschulsky) (Coleoptera: Chrysomelidae)[J]. Mitochondrial DNA Part B, 2021, 6(7): 2019-2021.[LinkOut]
[10]	田永浩, 张建萍, 陈静, 等. 新疆棉花新害虫双斑长跗萤叶甲的发生特点及防治策略[J]. 安徽农学通报, 2007, 13(10): 120-121, 230.
	TIAN Yonghao, ZHANG Jianping, CHEN Jing, et al. Occurring characteristic and preventions and control strategy of Monolepta hieroglyphica(motschulsky)-a new pest of the cotton field in Xinjiang[J]. Anhui Agricultural Science Bulletin, 2007, 13(10): 120-121, 230.
[11]	杜建军, 云雷. 双斑长跗萤叶甲发生危害特点及防治措施[J]. 陕西农业科学, 2009, 55(3): 202-203.
	DU Jianjun, YUN Lei. Characteristics and control measures of occurrence and damage of Lepidoptera bimaculata[J]. Shaanxi Journal of Agricultural Sciences, 2009, 55(3): 202-203.
[12]	李广伟, 陈秀琳. 新疆棉区双斑长跗萤叶甲生活习性及消长动态调查研究[J]. 中国植保导刊, 2010, 30(6): 8-10.
	LI Guangwei, CHEN Xiulin. Studies on biological characteristics and population dynamics of Monolepta hieroglyphica in cotton in Xinjiang[J]. China Plant Protection, 2010, 30(6): 8-10.
[13]	Zhang X L, Zhang R Y, Li L, et al. Negligible transcriptome and metabolome alterations in RNAi insecticidal maize against Monolepta hieroglyphica[J]. Plant Cell Reports, 2020, 39(11): 1539-1547.
[14]	Zheng F, Jiang H, Jia J L, et al. Effect of dimethoate in controlling Monolepta hieroglyphica (Motschulsky) and its distribution in maize by drip irrigation[J]. Pest Management Science, 2020, 76(4): 1523-1530. DOI PMID
[15]	戴建青, 韩诗畴, 杜家纬. 植物挥发性信息化学物质在昆虫寄主选择行为中的作用[J]. 环境昆虫学报, 2010, 32(3): 407-414.
	DAI Jianqing, HAN Shichou, DU Jiawei. Progress in studies on behavioural effect of semiochemicals of host plant to insects[J]. Journal of Environmental Entomology, 2010, 32(3): 407-414.
[16]	陈秀琳, 李琳琳, 陈玉鑫, 等. 梨小食心虫气味受体GmolOR10基因克隆及表达谱分析[J/OL]. 植物保护: 2023, 1-13 [2023-04-28].
	CHEN Xiulin, LI Linlin, CHEN Yuxin, et al. Cloning and expression pattern of odorant receptor GmolOR10 in Grapholita molesta Busck[J/OL]. Plant Protection: 2023, 1-13 [2023-04-28].
[17]	Pham H T, McNamara K B, Elgar M A. Age-dependent chemical signalling and its consequences for mate attraction in the gumleaf skeletonizer moth, Uraba lugens[J]. Animal Behaviour, 2021, 173: 207-213.
[18]	Segura D F, Belliard S A, Vera M T, et al. Plant chemicals and the sexual behavior of male tephritid fruit flies[J]. Annals of the Entomological Society of America, 2018, 111(5): 239-264.
[19]	赵航, 吴国星, 汤永玉, 等. 叉角厉蝽触角转录组及嗅觉相关基因分析[J]. 环境昆虫学报, 2022, 44(5): 1205-1217.
	ZHAO Hang, WU Guoxing, TANG Yongyu, et al. Analysis of the antennal transcriptome and olfaction-related genes of Eocanthecona furcellata[J]. Journal of Environmental Entomology, 2022, 44(5): 1205-1217.
[20]	张聪, 王振营, 何康来, 等. 双斑长跗萤叶甲触角感器的扫描电镜观察[J]. 应用昆虫学报, 2012, 49(3): 756-761.
	ZHANG Cong, WANG Zhenying, HE Kanglai, et al. Scanning electron microscopy studies of antennal sensilla of Monolepta hieroglyphica[J]. Chinese Journal of Applied Entomology, 2012, 49(3): 756-761.
[21]	刘敏, 刘爱萍, 韩海斌. 寄生蜂转录组学研究进展[J]. 植物保护, 2021, 47(2): 11-19.
	LIU Min, LIU Aiping, HAN Haibin. A review of transcriptome sequencing in parasitoid wasps[J]. Plant Protection, 2021, 47(2): 11-19.
[22]	Güell M, van Noort V, Yus E, et al. Transcriptome complexity in a genome-reduced bacterium[J]. Science, 2009, 326(5957): 1268-1271. DOI PMID
[23]	刘伟, 郭光艳, 秘彩莉. 转录组学主要研究技术及其应用概述[J]. 生物学教学, 2019, 44(10): 2-5.
	LIU Wei, GUO Guangyan, BEI Caili. A summary of the main research techniques of transcriptology and their applications[J]. Biology Teaching, 2019, 44(10): 2-5.
[24]	李资聪, 刘磊, 杨斌, 等. 黄野螟化学感受基因的鉴定与分析[J]. 植物保护, 2021, 47(6): 34-48.
	LI Zicong, LIU Lei, YANG Bin, et al. Identification and analysis of chemosensory genes in Heortia vitessoides[J]. Plant Protection, 2021, 47(6): 34-48.
[25]	白鹏华, 王冰, 张仙红, 等. 昆虫气味受体的研究方法与进展[J]. 昆虫学报, 2022, 65(3): 1-22.
	BAI Penghua, WANG Bing, ZHANG Xianhong, et al. Research methods and advances of odorant receptors in insects[J]. Acta Entomologica Sinica, 2022, 65(3): 1-22.
[26]	詹文会, 张苏芳, 耿红卫, 等. 天牛嗅觉感受相关蛋白研究进展[J]. 河南农业科学, 2018, 47(3): 1-6. DOI
	ZHAN Wenhui, ZHANG Sufang, GENG Hongwei, et al. Research progress on olfactory recognition proteins of longhorn beetle[J]. Journal of Henan Agricultural Sciences, 2018, 47(3): 1-6.
[27]	Rondoni G, Roman A, Meslin C, et al. Antennal transcriptome analysis and identification of candidate chemosensory genes of the harlequin ladybird beetle, Harmonia axyridis (Pallas) (Coleoptera: Coccinellidae)[J]. Insects, 2021, 12(3): 209.
[28]	庄翔麟, 吉帅帅, 赵昱杰, 等. 灭字脊虎天牛CSP基因的鉴定、表达谱及XquaCSP7基因的功能研究[J]. 植物保护, 2020, 46(3): 30-39.
	ZHUANG Xianglin, JI Shuaishuai, ZHAO Yujie, et al. Identification and expression pattern of chemosensory protein genes from Xylotrechus quadripes Chevrolat (Coleoptera: Cerambycidae) and functional studies of XquaCSP7 gene[J]. Plant Protection, 2020, 46(3): 30-39.
[29]	Wang X, Wang S, Yi J K, et al. Three host plant volatiles, hexanal, lauric acid, and tetradecane, are detected by an antenna-biased expressed odorant receptor 27 in the dark black chafer Holotrichia parallela[J]. Journal of Agricultural and Food Chemistry, 2020, 68(28): 7316-7323.
[30]	Yuvaraj J K, Roberts R E, Sonntag Y, et al. Putative ligand binding sites of two functionally characterized bark beetle odorant receptors[J]. BMC Biology, 2021, 19(1): 16.
[31]	Hong B, Chang Q, Zhai Y J, et al. Functional characterization of odorant binding protein PyasOBP2 from the jujube bud weevil, Pachyrhinus yasumatsui (Coleoptera: Curculionidae) 2022.
[32]	Grabherr M G, Haas B J, Yassour M, et al. Full-length transcriptome assembly from RNA-Seq data without a reference genome[J]. Nature Biotechnology, 2011, 29(7): 644-652. DOI PMID
[33]	Zhang Y N, Kang K, Xu L, et al. Deep sequencing of antennal transcriptome from Callosobruchus chinensis to characterize odorant binding protein and chemosensory protein genes[J]. Journal of Stored Products Research, 2017, 74 : 13-21.
[34]	黄聪, 李有志, 杨念婉, 等. 入侵昆虫基因组研究进展[J]. 植物保护, 2019, 45(5): 112-120, 134.
	HUANG Cong, LI Youzhi, YANG Nianwan, et al. Progresses in invasive insect genomics[J]. Plant Protection, 2019, 45(5): 112-120, 134.
[35]	Gu X C, Zhang Y N, Kang K, et al. Antennal transcriptome analysis of odorant reception genes in the red turpentine beetle (RTB), Dendroctonus valens[J]. PLoS One, 2015, 10(5): e0125159.[PubMed]
[36]	杨雪娇, 张蔓, 江宇航, 等. 七星瓢虫触角转录组及嗅觉相关基因分析[J]. 昆虫学报, 2020, 63(6): 717-726.
	YANG Xuejiao, ZHANG Man, JIANG Yuhang, et al. Analysis of the antennal transcriptome and olfaction-related genes of Coccinella septempunctata (Coleoptera: Coccinellidae)[J]. Acta Entomologica Sinica, 2020, 63(6): 717-726.
[37]	巩雪芳, 谢寿安, 杨平, 等. 花椒窄吉丁触角转录组及嗅觉相关基因分析[J]. 昆虫学报, 2020, 63(10): 1159-1170.
	GONG Xuefang, XIE Shouan, YANG Ping, et al. Analysis of the antennal transcriptome and olfaction-related genes of Agrilus zanthoxylumi (Coleoptera: Buprestidae)[J]. Acta Entomologica Sinica, 2020, 63(10): 1159-1170.
[38]	胡佳萌, 徐丹萍, 卓志航, 等. 云斑天牛成虫触角转录组及嗅觉相关基因分析[J]. 应用昆虫学报, 2019, 56(5): 1037-1047.
	HU Jiameng, XU Danping, ZHUO Zhihang, et al. Analysis of antennal transcriptome and olfaction-related genes of adult Batocera horsfieldi (Hope)[J]. Chinese Journal of Applied Entomology, 2019, 56(5): 1037-1047.
[39]	宋月芹, 董钧锋, 陈庆霄, 等. 点蜂缘蝽触角转录组及化学感受相关基因的分析[J]. 昆虫学报, 2017, 60(10): 1120-1128. DOI
	SONG Yueqin, DONG Junfeng, CHEN Qingxiao, et al. Analysis of the antennal transcriptome and chemoreception-related genes of the bean bug, Riptortus pedestris (Hemiptera: Alydidae)[J]. Acta Entomologica Sinica, 2017, 60(10): 1120-1128. DOI
[40]	张健, 谢婉莹, 董曼羽, 等. 青杨天牛嗅觉相关基因的鉴定及表达谱分析[J]. 江苏农业科学, 2022, 50(4): 23-28.
	ZHANG Jian, XIE Wanying, DONG Manyu, et al. Identification and expression profile analysis of olfactory related genes of Anoplophora populnea[J]. Jiangsu Agricultural Sciences, 2022, 50(4): 23-28.
[41]	龙亚芹, 罗梓文, 王雪松, 等. 茶谷蛾成虫触角转录组及嗅觉相关基因分析[J]. 茶叶科学, 2021, 41(4): 553-563.
	LONG Yaqin, LUO Ziwen, WANG Xuesong, et al. Analysis of the antennal transcriptome and olfactory-related genes in the Agriophara rhombata[J]. Journal of Tea Science, 2021, 41(4): 553-563.
[42]	杨爽, 赵慧婷, 徐凯, 等. 大蜡螟触角转录组及嗅觉相关基因分析[J]. 应用昆虫学报, 2019, 56(6): 1279-1291.
	YANG Shuang, ZHAO Huiting, XU Kai, et al. Analysis of the antennal transcriptome and olfaction-related genes of the greater wax moth Galleria mellonella(Lepidoptera: Pyralidae)[J]. Chinese Journal of Applied Entomology, 2019, 56(6): 1279-1291.
[43]	Wu G X, Su R R, Ouyang H L, et al. Antennal transcriptome analysis and identification of olfactory genes in Glenea cantor Fabricius (Cerambycidae: Lamiinae)[J]. Insects, 2022, 13(6): 553.
[44]	巩雪芳, 谢寿安, 杨平, 等. 花椒窄吉丁雌、雄成虫触角差异表达基因的初步筛选[J]. 农业生物技术学报, 2019, 27(7): 1233-1245.
	GONG Xuefang, XIE Shouan, YANG Ping, et al. Preliminary screening of differentially expressed genes of female and male antenna of Agrilus zanthoxylumi[J]. Journal of Agricultural Biotechnology, 2019, 27(7): 1233-1245.
[45]	Tanaka K, Shimomura K, Hosoi A, et al. Antennal transcriptome analysis of chemosensory genes in the cowpea beetle, Callosobruchus maculatus (F.)[J]. PLoS One, 2022, 17(1): e0262817.

基因ID Gene ID	基因名称 Gene name	上游引物(5’-3’) Forward	下游引物(3’-5’) Reverse	扩增片段长度 Product length
Cluster-12689.30761	MsigOBP2	TGGTTTCAGCGAACGCAATC	TCCTTCATGACTGTCTGGTTCC	152
Cluster-12689.31568	MsigOBP3	TAGAGGCAAATGCACCACCAT	TAGAGGCAAATGCACCACCAT	194
Cluster-12689.26420	MsigOBP5	AAATGCGGATGGCTCCATGA	TCCCAGTGCTCGTCCTTCTA	199
Cluster-12689.31318	MsigOBP6	GCAACCCATTTGTTGCCTCA	TGTACGTGTGTCGGACAGTG	165
Cluster-12689.8427	MsigOBP8	CATCCATGCGCCTTTCAAGA	GCCAAAGAAGAGTGTCAAGCTG	199
Cluster-12689.26435	MsigOBP15	TGGGAATCTTAGCTGGTGGC	CACACCAATTTCATCGAAAACGC	191
Cluster-12689.34706	MsigOBP18	AGGACTCCAAAATGAAGACGGT	ACAAGGACACGTTTGGGGTT	189
Cluster-12689.31354	MsigOBP20	ACGGTTGCGTCGTCTATTCC	AAGATCCAGAAACACGAGCGG	181
Cluster-12689.30900	MsigOBP21	TGGACACCAAGGCATCTCAC	TCTGAGGCGCCTGAATAACA	196

基因ID Gene ID	基因名称 Gene name	上游引物(5’-3’) Forward	下游引物(3’-5’) Reverse	扩增片段长度 Product length
Cluster-12689.30761	MsigOBP2	TGGTTTCAGCGAACGCAATC	TCCTTCATGACTGTCTGGTTCC	152
Cluster-12689.31568	MsigOBP3	TAGAGGCAAATGCACCACCAT	TAGAGGCAAATGCACCACCAT	194
Cluster-12689.26420	MsigOBP5	AAATGCGGATGGCTCCATGA	TCCCAGTGCTCGTCCTTCTA	199
Cluster-12689.31318	MsigOBP6	GCAACCCATTTGTTGCCTCA	TGTACGTGTGTCGGACAGTG	165
Cluster-12689.8427	MsigOBP8	CATCCATGCGCCTTTCAAGA	GCCAAAGAAGAGTGTCAAGCTG	199
Cluster-12689.26435	MsigOBP15	TGGGAATCTTAGCTGGTGGC	CACACCAATTTCATCGAAAACGC	191
Cluster-12689.34706	MsigOBP18	AGGACTCCAAAATGAAGACGGT	ACAAGGACACGTTTGGGGTT	189
Cluster-12689.31354	MsigOBP20	ACGGTTGCGTCGTCTATTCC	AAGATCCAGAAACACGAGCGG	181
Cluster-12689.30900	MsigOBP21	TGGACACCAAGGCATCTCAC	TCTGAGGCGCCTGAATAACA	196

样品 Sample	原始读数 Raw reads	筛选后读数 Clean reads	总核酸数(G) Clean bases	错误率 Error rate (%)	Q₂₀ (%)	Q₃₀ (%)	GC比值 GC content (%)
FA₁	21 774 061	20 648 522	6.2	0.03	97.79	93.3	34.16
FA₂	20 757 300	20 132 668	6.0	0.03	97.88	93.58	36.30
FA₃	22 134 770	21 179 365	6.4	0.03	98.07	93.99	35.45
MA₁	21 323 904	20 130 023	6.0	0.03	97.82	93.43	35.49
MA₂	20 885 385	20 454 004	6.1	0.03	97.75	93.23	33.98
MA₃	21 288 624	20 829 381	6.2	0.03	97.83	93.42	34.95

样品 Sample	原始读数 Raw reads	筛选后读数 Clean reads	总核酸数(G) Clean bases	错误率 Error rate (%)	Q₂₀ (%)	Q₃₀ (%)	GC比值 GC content (%)
FA₁	21 774 061	20 648 522	6.2	0.03	97.79	93.3	34.16
FA₂	20 757 300	20 132 668	6.0	0.03	97.88	93.58	36.30
FA₃	22 134 770	21 179 365	6.4	0.03	98.07	93.99	35.45
MA₁	21 323 904	20 130 023	6.0	0.03	97.82	93.43	35.49
MA₂	20 885 385	20 454 004	6.1	0.03	97.75	93.23	33.98
MA₃	21 288 624	20 829 381	6.2	0.03	97.83	93.42	34.95

长度范围 Length range (bp)	Transcripts		Unigenes
长度范围 Length range (bp)	数量 Number (个)	百分比 Percentage (%)	数量 Number (个)	百分比 Percentage (%)
300~500	53 300	33.39	29 009	39.71
500~1k	42 661	26.72	21 009	28.76
1k~2k	31 535	19.75	12 325	16.87
>2k	32 162	20.14	10 707	14.66
总数Total number	159 658		73 050
总长度Total length	216 704 872		83 267 839
平均长度Mean length	1 357		1 140
N₅₀长度N₅₀ length	2 339		1 896
N₉₀长度N₉₀ length	516		440