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抗氟苯虫酰胺小菜蛾差异表达基因及其通路

本站小编 Free考研考试/2021-12-26
王成花, 孙诗晴, 徐巨龙, 赵小龙, 薛超彬. 抗氟苯虫酰胺小菜蛾差异表达基因及其通路[J]. 中国农业科学, 2018, 51(11): 2106-2115 https://doi.org/10.3864/j.issn.0578-1752.2018.11.008
WANG ChengHua, SUN ShiQing, XU JuLong, ZHAO XiaoLong, XUE ChaoBin. Differential Expressed Genes and Their Pathways of the Resistance to Flubendiamide in Plutella xylostella[J]. Scientia Acricultura Sinica, 2018, 51(11): 2106-2115 https://doi.org/10.3864/j.issn.0578-1752.2018.11.008

0 引言

【研究意义】小菜蛾（Plutella xylostella）属鳞翅目菜蛾科,主要危害甘蓝等十字花科植物,幼虫取食叶片,苗期集中危害心叶,严重影响蔬菜的产量及品质,成为世界性的蔬菜害虫。氟苯虫酰胺（flubendiamide）是由日本农药株式会社和德国拜耳公司联合开发的一种邻苯二甲酰胺类杀虫剂,其主要作用靶标为昆虫鱼尼丁受体（ryanodine receptor,RyR）,该药剂已广泛应用于鳞翅目害虫的防治,效果优良,但近年来小菜蛾等鳞翅目害虫对该药剂的抗性越发突出。因此,明确小菜蛾对氟苯虫酰胺的抗性机理对合理控制小菜蛾及其抗药性发展具有重要意义。【前人研究进展】2008—2011年,泰国Tha Muang地区小菜蛾对氟苯虫酰胺的抗性由1.5倍发展到4 817倍[1];2012年,泰国Sai Noi地区小菜蛾对氟苯虫酰胺的抗性倍数超过750倍;菲律宾Sudlon地区小菜蛾田间种群对氟苯虫酰胺的抗性超过1 300倍[2],2015年该Sudlon种群对氟苯虫酰胺的抗性超过10 000倍[3]。中国广东增城地区的小菜蛾田间种群对氟苯虫酰胺的抗性高达1 779倍[4]。除此之外,其他鳞翅目害虫也对氟苯虫酰胺产生了不同程度的抗性,如茶小卷叶蛾（Adoxophyes honmai）对氟苯虫酰胺的抗性达105倍[5];番茄潜叶蛾（Tuta absoluta）对氟苯虫酰胺的抗性超过1 000倍[6]。抗性机制研究发现,泰国和菲律宾的小菜蛾田间高抗种群中鱼尼丁受体基因（PxRyR）的4 946位均存在一个甘氨酸（G）到谷氨酸（E）的突变[7],其位于受体基因羧基末端的跨膜区附近,且在所有昆虫鱼尼丁受体中高度保守,被认为是导致小菜蛾对氯虫苯甲酰胺产生抗性的主要原因。此外,解毒酶活性的提高,如细胞色素P450单加氧酶活性的提高（P450）、羧酸酯酶（CarE）和谷胱甘肽S-转移酶（GSTs）活性的提高[8,9];PxRyR中G4946E、E1338D、Q4594L和I4790M等4个位点的协同突变[10]和PxRyR mRNA转录表达变化等[4,11-12],也是小菜蛾对氯虫苯甲酰胺产生抗性的重要机制。【本研究切入点】在小菜蛾对双酰胺类杀虫剂抗性机制的研究中,多数报道均以氯虫苯甲酰胺为药剂对象,但小菜蛾对氟苯虫酰胺与氯虫苯甲酰胺的抗性机制是否一致,目前还未有明确的认识。【拟解决的关键问题】以小菜蛾抗氟苯虫酰胺品系为试材,从转录组水平研究小菜蛾对氟苯虫酰胺的抗性机制,进一步揭示其抗性机理。

1 材料与方法

试验于2016年在山东农业大学和深圳华大基因科技服务有限公司完成。

1.1 供试材料

小菜蛾敏感品系（S）于2006年采自山东农业大学南校区实验站园,在室内不接触任何药剂,用甘蓝苗长期饲养且稳定繁殖;抗氟苯虫酰胺品系（Rh）是敏感品系小菜蛾在氟苯虫酰胺药剂的持续选择压力下筛选至28代（Rh28）,其抗性倍数为397.08倍;田间抗性种群（Rz）于2012年10月采集于广州增城市周边蔬菜田,在室内无农药暴露情况下繁殖到第36代（Rz36）,与S品系相比,该种群对氟苯虫酰胺药剂的抗性倍数为98.12倍。

1.2 RNA提取及检测

选取标准一致的小菜蛾3龄幼虫,迅速用液氮冷冻,用RNAsimp Total RNA Kit试剂盒提取RNA,并利用1.0%琼脂糖凝胶电泳检测,将得到的质量较高RNA样品送至深圳华大基因科技服务有限公司,利用Illumina HiSeqTM 2000进行高通量测序。每个样品提供3次重复。并对质检合格的数据进行对比,包括抗氟苯虫酰胺品系与敏感品系对比（Rh28-vs-S）、抗氟苯虫酰胺田间抗性种群与敏感品系对比（Rz36-vs-S）,及抗氟苯虫酰胺品系与抗氟苯虫酰胺田间抗性种群对比（Rh28-vs-Rz36）,并进行相关生物信息分析。

1.3 基因定量方法

将3组样品间两两比较,用RSEM工具[13]进行基因表达定量。表达定量的结果以FPKM为单位,具体计算公式如下：
$FPKM=\frac{{{10}^{6}}C}{NL/{{10}^{3}}}$
式中,FPKM（A）表示为基因A的表达丰度,C为唯一比对到基因A的fragments数,N为唯一比对到参考基因的总fragments数,L为基因A编码区的碱基数。FPKM法能够消除由于基因长度和测序质量差异的影响,使得到的基因量更加准确地比较不同样品间的表达差异。

1.4 明确差异基因

为了挖掘样本间的显著差异基因,基于泊松分布的分析方法,并参照AUDIC等[14]的方法,假设观测到基因A对应的reads数为x,已知在一个大文库中,每个基因的表达量只占所有基因表达量的一小部分,在这种情况下,x的分布服从泊松分布：
$P(x)=\frac{{{e}^{-\lambda }}{{\lambda }^{x}}}{x!}$（λ为基因A的真实转录数）
之后,对差异检验的P值作多重假设检验校正,通过控制FDR（False Discovery Rate）来决定P值的域值[15]。在得到差异检验的FDR值同时,根据基因的表达量（FPKM值）计算该基因在不同样本间的差异表达倍数。FDR值越小,差异倍数越大,则表明表达差异越显著。本研究中,显著差异表达基因定义为FDR≤0.001且倍数差异在2倍以上的基因。

1.5 GO显著性富集分析

基因本体（Gene Ontology,GO）共有3个本体,分别描述基因的分子功能（molecular function）、细胞成分（cellular component）和参与的生物过程（biological process）。GO分析给出与参考基因组背景相比,在差异表达基因中显著富集的GO功能条目,从而筛选出差异表达基因与哪些生物学功能显著相关。本研究首先把所有差异表达基因向Gene Ontology数据库（http://www.geneontology.org/）的各个term映射,计算每个term的基因数目,然后应用超几何检验,找出与整个基因组背景相比,在差异表达基因中显著富集的GO条目。通过GO功能显著性富集分析能确定差异表达基因行使的主要生物学功能。

1.6 KEGG显著性富集分析

京都基因与基因组百科全书数据库（KEGG）是关于通路（Pathway）的主要公共数据库,Pathway显著性富集分析应用超几何检验,该分析的计算公式同GO功能显著性富集分析。在差异表达基因中只有满足Q值≤0.05才属于显著富集的Pathway。通过Pathway显著性富集分析,能够确定差异表达基因参与的最主要的生化代谢途径以及信号转导途径。

2 结果

2.1 差异表达基因

通过将抗氟苯虫酰胺品系（Rh28）与敏感品系（S）的RNA-seq数据对比发现,Rh28中共有540个显著差异表达基因,其中上调表达的基因411个,下调表达的有129个（附表1）;同样的,将田间抗性种群（Rz36）与S品系的RNA-seq数据对比发现,Rz36中共有439个显著差异表达基因,其中上调表达的基因299个,下调表达的有140个;进一步将Rh28品系与Rz36种群数据对比发现,Rh28中共有75个显著差异表达基因,其中上调表达的48个,下调表达的有27个。
Table S1
附表1
附表1Rh28-vs-S显著差异表达基因
Table S1Significantly differentially expressed genes of Rh28-vs-S
编号NumberGene ID基因长度
Gene length (bp)		Means-SMeans- Rh28log2Ratio
(Rh28/S)		Up-Down-
regulation (Rh28/S)		Probability
1XM_011566929.115770.016.5259.349834Up0.883948
2XM_011552794.110850.0111.59510.17929Up0.932143
3XM_011571069.151428.4329.5251.808333Up0.80253
4XM_011552043.1212744.88138.2151.62277Up0.812509
5XM_011551933.1156119.7567.111.764675Up0.815721
6XM_011563919.11775147.755513.51.797157Up0.834865
7XM_011561927.1345917.814510.042.296873Up0.865471
8XM_011562031.1273219.79573.491.892412Up0.825256
9XM_011551180.110780.9414.1853.915562Up0.848007
10XM_011550936.123921.56521.33.766619Up0.861148
11XM_011565830.1107520.10576.2851.923845Up0.827578
12XM_011557685.114570.013.9458.623881Up0.812905
13XM_011570464.115099.8249.5552.335236Up0.843387
14XM_011550942.175821.01247.243.556764Up0.890964
15XM_011566360.114111.911.7052.623054Up0.802875
16XR_960880.11320.015.359.063395Up0.859098
17XM_011554614.13998.0728.191.804543Up0.800578
18XM_011564277.1768355.545983.3051.467607Up0.808285
19XM_011555406.14671.5919.83.638402Up0.855837
20XM_011566251.18527.0528.242.002045Up0.815029
21XM_011565310.1188939.3113.251.52691Up0.801097
22XM_011553389.115403.57537.0453.373264Up0.866558
23XM_011565866.13660.97542.5955.449138Up0.913838
24XM_011556876.139020.013.878.59619Up0.809545
25XM_011565019.11191100.17436.3152.12292Up0.854182
26XM_011552504.115615.6447.423.071729Up0.864878
27XM_011551411.1183552.605255.6352.280814Up0.856899
28XM_011564542.1592187.461138.352.60229Up0.876167
29XM_011570144.152519.135116.252.602945Up0.863767
30XM_011554891.112462.3713.1852.475939Up0.804308
31XM_011569224.134335.7157.843.340505Up0.874339
32XM_011564727.117380.014.3958.779719Up0.830394
33XM_011562845.113828.90545.982.368319Up0.843264
34XM_011552041.1191610.9761.832.494743Up0.852082
35XM_011554540.112808.7634.861.99257Up0.817845
36XM_011552880.116562.21515.3552.79333Up0.822662
37XM_011564837.1128613.76569.162.328933Up0.848254
38XM_011570607.11156165.975466.971.492364Up0.806531
39XM_011565672.127040.015.098.991522Up0.852305
40XM_011565098.1997220.905621.891.493233Up0.808285
41XM_011558736.124087.68537.3652.28157Up0.83442
42XM_011559676.155739.6114.9751.537748Up0.802332
43XM_011566934.1185441.215130.9451.66772Up0.816239
44XM_011556590.164415.3260.431.979849Up0.828492
45XM_011563393.1355210.64535.9351.755214Up0.803122
46XM_011559241.134651.3211.1353.076492Up0.813917
47XM_011558815.1961328.515892.691.4422Up0.805395
48XM_011562159.16704.8727.3052.487171Up0.834593
49XM_011555687.1415121.74583.5551.942042Up0.830171
50XR_960843.13985.48535.4452.692019Up0.848031
51XM_011552317.168162.365353.5152.502963Up0.867225
52XM_011562064.191510.0936.0851.838473Up0.808705
53XM_011552513.111652.6214.9652.513954Up0.812336
54XM_011559483.115744.41522.432.344944Up0.824416
55XM_011570585.114120.0811.0057.103943Up0.901438
56XM_011569848.154599.165626.7452.659976Up0.876044
57XM_011564765.1952158.36446.471.495355Up0.807149
58XM_011565242.1152818.9283.392.139962Up0.84235
59XM_011552165.115943.5516.5552.221376Up0.805222
60XM_011563058.17440.014.98.936638Up0.846796
61XM_011555680.111390.24510.0355.356115Up0.860209
62XM_011564298.13872.5314.5752.526287Up0.811422
63XM_011559169.135720.014.598.84235Up0.837829
64XM_011554544.115041.13515.013.72516Up0.845932
65XM_011558206.113020.016.979.445015Up0.891285
66XM_011551627.1121313.05555.4652.086975Up0.834494
67XM_011552555.1236320.99576.8251.87153Up0.82449
68XM_011558267.188863.33229.9451.860328Up0.833062
69XM_011553657.126538.54547.5752.477051Up0.847216
70XM_011565941.124710.013.7058.53333Up0.802505
71XM_011561945.1131124.15583.521.7898Up0.820661
72XM_011569365.111114.5322.3452.302369Up0.821871
73XM_011558269.18647.4728.661.939858Up0.809965
74XM_011563885.1168326.225117.752.166712Up0.847241
75XM_011553969.1165816.92111.4152.719142Up0.866237
76XM_011566920.1421218.135701.681.685592Up0.828171
77XM_011550519.1346233.765158.2052.228195Up0.851662
78XM_011554605.1488919.92574.011.89314Up0.82533
79XM_011563677.16583.2630.7953.239752Up0.859419
80XM_011570487.19822.9623.763.004866Up0.848155
81XM_011569769.18878.8635.6552.008726Up0.820686
82XM_011555735.111540.919.2553.346295Up0.807939
83XM_011570867.110194.4420.9852.240727Up0.816956
84XM_011566393.138094.6923.8452.346027Up0.826713
85XM_011565161.131587.0130.022.098438Up0.821303
86XM_011550584.19329.38105.713.49438Up0.88427
87XM_011556850.158373.575676.553.200909Up0.891063
88XM_011554560.18367.9433.952.096201Up0.824638
89XM_011561944.132872.14532.7452.884574Up0.881824
90XM_011553397.115024.3720.4152.223924Up0.814708
91XM_011565855.18105.39522.372.05187Up0.810459
92XM_011559326.1651226.541414.382.642332Up0.877402
93XM_011561886.1755212.3951036.2052.286488Up0.864285
94XM_011550249.16464.50584.194.22405Up0.897461
95XM_011550484.111619.56541.742.125594Up0.830789
96XM_011561556.18662.8419.3652.769489Up0.833136
97XM_011555758.118320.019.8759.947637Up0.921768
98XM_011551170.135156.4226.422.040985Up0.815696
99XM_011549516.1172553.75170.3251.663954Up0.8174
100XM_011570514.128440.014.918.939579Up0.847191
101XM_011554017.1263274.56229.11.619504Up0.81577
102XM_011549539.113556.4837.552.534747Up0.843634
103XM_011558700.114180.7222.854.988053Up0.889605
104XR_960941.12936.2338.382.623051Up0.848303
105XM_011553334.11236202.37846.5852.064659Up0.854454
106XM_011551073.1701159.2251055.542.728842Up0.879181
107XM_011556502.118023.81518.072.243842Up0.81036
108XM_011562403.119563.05520.182.723682Up0.833358
109XM_011564769.123412.02518.883.220865Up0.845462
110XM_011560384.12890949.5752.461616Up0.84729
111XM_011550224.123180.0115.5510.6027Up0.947755
112XM_011551813.130361.46513.0653.156735Up0.824836
113XM_011549699.1118959.14419.922.827908Up0.879403
114XM_011570634.148332.765466.2353.83083Up0.902031
115XM_011562158.16720.016.1659.267957Up0.87802
116XM_011568816.121160.52242.6952.00366Up0.84398
117XM_011564058.115480.018.469.724514Up0.909911
118XM_011566603.110328.8730.9651.803632Up0.802431
119XM_011553388.116676.11539.0552.675083Up0.85023
120XM_011562049.1141030.7399.681.697656Up0.816017
121XM_011564936.1232319.88577.331.959348Up0.830814
122XM_011559323.154530.0293.6451.641278Up0.81041
123XM_011565962.17286.17525.0852.022314Up0.812509
124XM_011570160.166423.9873.381.613555Up0.804382
125XR_960670.1106212.35548.581.975267Up0.82449
126XM_011550258.16902.98515.2452.352533Up0.807692
127XM_011564339.115013.1117.882.52336Up0.821081
128XR_960787.121942.365171.942.02096Up0.842794
129XM_011550066.1194343.8175.9752.006368Up0.842424
130XM_011564915.143124.3482.221.75616Up0.818314
131XM_011554355.118862.515.5152.633664Up0.819179
132XM_011568529.17831.5612.272.975517Up0.816215
133XM_011550944.18323.4419.8952.531925Up0.82612
134XM_011568835.12017104.38324.041.634327Up0.81908
135XM_011553196.112581.63512.7852.96709Up0.818438
136XM_011570689.160500.054.9656.633722Up0.815523
137XM_011567286.127950.10511.4756.771961Up0.897708
138XM_011553563.115480.019.5559.900112Up0.919322
139XM_011569900.15826.5557.9153.14437Up0.869522
140XM_011568275.1173811.30542.831.921661Up0.817746
141XM_011570275.121414.5819.1252.06204Up0.804283
142XM_011563260.118130.0110.7210.06609Up0.927276
143XM_011549794.124754.2620.422.261058Up0.816338
144XM_011563090.1712624.8452855.822.192335Up0.861099
145XM_011559970.112752.1114.5952.790159Up0.820118
146XM_011557916.1205952.73219.1252.055058Up0.846598
147XM_011555743.121874.3520.812.25819Up0.817302
148XM_011558444.1631712.8347.181.878654Up0.817005
149XM_011550535.1830573.5852240.9651.966041Up0.849291
150XM_011556719.176145.03131.9851.551416Up0.804481
151XM_011560030.115678.3132.4451.965076Up0.814955
152XM_011563615.16590.014.2658.736402Up0.826021
153XM_011566699.19090.013.888.599913Up0.809866
154XM_011565016.17353.1929.313.199765Up0.858011
155XM_011553974.110421.28512.483.279778Up0.825602
156XR_960864.12717.7930.251.95724Up0.812583
157XM_011564095.1459188.36828.0452.136217Up0.857838
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460XM_011560476.154624.492.345-3.38453Down0.856282
461XM_011568567.169824.8551.2-4.37243Down0.87886
462XM_011563416.145277.49512.28-2.65779Down0.861049
463XM_011562950.1138050.715.2-3.28569Down0.870461
464XM_011553646.1865189.0454.155-1.80353Down0.828566
465XM_011557255.1196017.6750.455-5.2797Down0.887975
466XM_011557683.164118.5350.01-10.856Down0.95519
467XM_011551134.18231618.89420.255-1.94567Down0.847661
468XM_011553673.11408130.7514.785-3.1446Down0.878316
469XM_011562457.12124544.645187.68-1.53704Down0.81246
470XM_011565576.19385.390.01-9.07414Down0.86016
471XM_011549701.1875123.838.725-1.67667Down0.81656
472XM_011563256.1202315.132.93-2.36844Down0.808211
473XM_011551198.15872294.47691.85-1.72963Down0.833259
474XM_011564085.1115953.1959.515-2.48302Down0.849118
475XM_011566928.15466.90.35-4.30117Down0.803468
476XM_011570525.1240016.722.475-2.75607Down0.82617
477XM_011551112.1850108.2236.78-1.55697Down0.803913
478XM_011561407.113259.290.8-3.53761Down0.81162
479XM_011557378.1138727.2255.135-2.4065Down0.832345
480XM_011555588.18241159.33421.67-1.4591Down0.807742
481XM_011559870.1118955.7213.98-1.99483Down0.827454
482XM_011564823.169095.48514.29-2.74027Down0.865101
483XM_011558524.12371210.02-10.0362Down0.958204
484XM_011553973.19119.0950.01-9.82893Down0.915567
485XM_011549922.1117839.16511.425-1.77737Down0.80668
486XM_011570052.1723127.8935.785-1.83748Down0.828195
487XM_011553473.1290790.01-9.81378Down0.914629
488XM_011561706.1137061.18517.97-1.76759Down0.814856
489XM_011565661.1101633.069.78-1.75718Down0.801171
490XM_011556944.131404.2350.01-8.72622Down0.824712
491XM_011560501.171354.5559.4-2.53698Down0.851341
492XM_011550387.126295.540.01-9.11374Down0.863693
493XM_011563741.19701568.92533.985-1.5549Down0.81698
494XM_011570807.116625.2850.01-9.04576Down0.857369
495XM_011562728.1282261.19560.77-2.1037Down0.849785
496XM_011565017.1210821.044.42-2.25102Down0.817277
497XM_011569598.11590103.7235.31-1.55455Down0.802628
498XM_011563247.1118715.132.56-2.5632Down0.814115
499XM_011553349.1432812.6052.095-2.58897Down0.806754
500XM_011554913.141737.610.01-9.57175Down0.899857
501XM_011556689.1927303.81576.37-1.99212Down0.843486
502XM_011553144.143221.115.41-1.96423Down0.801369
503XR_961179.111655.3750.01-9.07012Down0.859888
504XM_011563934.12634105.35.765-4.19104Down0.899486
505XR_960797.13784.5550.01-8.83131Down0.836668
506XM_011552016.184020.733.28-2.65995Down0.833062
507XM_011549868.13502139.3244.095-1.65972Down0.815992
508XM_011569412.1922218.356.57-1.9482Down0.838916
509XM_011566283.1493155.1132.14-2.27085Down0.852996
510XM_011558175.127587.720.01-9.59246Down0.901191
511XM_011567825.123627.170.01-9.48583Down0.894002
512XM_011570580.144065.240.01-9.03342Down0.856183
513XM_011567320.1622161.74545.445-1.83153Down0.82943
514XM_011558819.156295.6816.515-2.53444Down0.858456
515XM_011562480.1193479.214.71-2.4287Down0.853713
516XM_011557864.11325143.0838.25-1.90329Down0.832889
517XM_011559054.117396.7350.01-9.39553Down0.887481
518XM_011558232.11985101.87526.64-1.93513Down0.832518
519XM_011559008.172823.423.625-2.69169Down0.837953
520XM_011550295.1128247.85510.005-2.25795Down0.838521
521XM_011570002.113456.2850.01-9.29577Down0.880268
522XM_011557936.1827352.4958.05-2.60221Down0.871326
523XM_011566025.119235.010.01-8.96867Down0.850057
524XM_011559804.1612857.7856.595-3.13125Down0.8693
525XM_011564212.1199411.7151.375-3.09085Down0.817376
526XM_011568452.16983.880.01-8.59991Down0.809866
527XM_011566931.14492766.3951008.5-1.4558Down0.808384
528XM_011557450.186814.331.065-3.75011Down0.844054
529XM_011552484.112386.710.01-9.39017Down0.886888
530XM_011566305.128321.065.15-2.03186Down0.806704
531XM_011553079.134418.9450.01-9.80494Down0.914406
532XR_960842.1497127.21517.065-2.89816Down0.872289
533XM_011568296.1276164.563.155-5.70483Down0.933674
534XM_011550349.1109149.5650.01-12.2751Down0.978509
535XM_011564893.1893173.7323.215-2.90372Down0.874364
536XM_011558880.12079470.01-12.1984Down0.977546
537XM_011570152.1562299.9479.615-1.91356Down0.838323
538XM_011557424.185629.328.075-1.86035Down0.805099
539XM_011561232.183317.2552.485-2.7957Down0.828418
540XM_011563346.123385.2150.015-8.44156Down0.850971


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2.2 显著差异表达基因的基因本体（GO）分析

在得到每个差异基因的GO注释后,用WEGO软件[16]对3个品系/种群间的对比（Rh28-vs-S、Rz36-vs-S和Rh28-vs-Rz36）得到的差异基因DEGs从生物过程/细胞组件和分子功能3大类进行分类统计,从宏观上认识差异基因的功能分布特征（附图1）。其中生物黏附（biological adhesion）、生物调节（biological regulation）、代谢进程（metabolic process）、免疫系统进程（immune system process）、对刺激物的反应（response to stimulus）、催化活性（catalytic activity）、酶调节活性（enzyme regulator activity）、结合因子（binding）、受体激活（receptor activity）、转运活力（transporter activity）、核酸绑定的转录因子激活（nucleic acid binding transcription factor activity）等功能都均与小菜蛾的抗性及免疫系统具有相关性。
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附图1差异基因功能分类GO统计
-->Fig. S1GO functional classification of differential genes
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2.3 显著差异表达基因的通路（pathway）分析

利用KEGG数据库,对Rh28-vs-S、Rz36-vs-S和Rh28-vs-Rz36 3组样品的差异表达基因可能参与或涉及的通路（pathway）进行分析,结果发现,Rh28与S品系之间共有14 527个被注释到188条pathway上,其中包含基因数量最多的一条是代谢通路（metabolic pathway）,共有2 591个基因,占总数的17.84%,与抗性相关的pathway还包括药物代谢——细胞色素P450（drug metabolism-cytochrome P450）、酪氨酸代谢（tyrosine metabolism）、谷胱甘肽代谢（glutathione metabolism）、促分裂素原活化蛋白激酶信号通路（MAPK signaling pathway）、外来物质代谢的细胞色素P450（metabolism of xenobiotics by cytochrome P450）、心肌收缩（cardiac muscle contraction）、GABA能突触（GABAergic synapse）、咖啡因代谢（caffeine metabolism）、黑色素生成（melanogenesis）等。
Rh28与S品系之间540个显著差异的基因有289个被注释到通路当中,其中显著性富集（Q值<0.05）的pathway只有1个,即过氧化物酶体增殖物激活受体信号通路（PPAR signaling pathway）。Rz36与S品系之间的差异基因中有237个被注释到了KEGG数据库中,得到172条pathway,其中显著富集的也只有1个,即ubiquitin mediated proteolysis。而Rh28与Rz36品系之间的75个显著差异性基因有56个注释到了通路当中,得到71条pathway,没有显著富集的通路。KEGG富集排名前20的pathway散点图见附图2。
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附图2差异基因富集的前20条pathway
-->Fig. S2Top 20 statistics of pathway enrichment
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2.4 抗性显著上调基因

统计Rh28-vs-S,Rz36-vs-S和Rh28-vs-Rz36 3组间显著上调的基因数据发现,与S品系相比,Rh28品系显著上调的411个基因中,有218个在Rz36种群中同样显著上调表达。Rh28相对于Rz36显著上调的48个基因中,有25个是Rh28品系特有的上调表达基因,而在Rz36种群中表现不显著（图1）。针对上述218及25个上调基因展开进一步研究。
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图1上调表达的基因在小菜蛾不同种群中的分布
-->Fig. 1Distribution of up-regulated express genes in different populations of P. xylostella
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2.5 抗性显著上调基因的GO分析

Rh28-vs-S和Rz36-vs-S显著上调的218个交集基因,在GO 3大本体所占的比例及数目分布情况如图2-a 所示;Rh28-vs-S和Rh28-vs-Rz36显著上调的25个交集基因如图2-b所示。结果表明,这部分交集差异基因在GO本体中的分布情况与前述的整体的分布情况基本一致,集中在代谢进程、对刺激物的反应、生物调节、催化活性、结合因子等方面。数据分析表明,RNA-Seq获得的小菜蛾上调的DEGs中,参与生物过程的基因占比较高,达50%以上。
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图2上调交集基因在GO分析中的分布
横坐标为GO 3个大类的下一层级的GO term,纵坐标为注释到该term下（包括该term的子term）的基因个数。红色表示生物过程、蓝色表示细胞组分、绿色表示分子功能 The abscissa is the GO term of the next level belongs to three major categories of GO and the ordinate is the number of genes annotated to the term (including the sub-term of the term). Red indicates biological process, blue indicates cellular component, green indicates molecular function. 1：行为Behavior;2：生物黏附Biological adhesion;3：生物调节Biological regulation;4：细胞组分组织或生物发生Cellular component organization or biogenesis;5：细胞过程Cellular process;6：发育过程Developmental process;7：局部化Localization;8：运动Locomotion;9：代谢过程 Metabolic process;10：多有机体过程Multi-organism process;11：多细胞有机体过程 Multicellular organismal process;12：生物过程调节Regulation of biological process;13：繁殖Reproduction;14：生殖过程Reproductive process ;15：应激反应Response to stimulus;16：信号Signaling;17：单有机体过程Single-organism process;18：细胞Cell;19：细胞组成Cell part;20：高分子配合物Macromolecular complex;21：膜Membrane;22：膜组成Membrane part;23：细胞器Organelle;24：细胞器组成Organelle part;25：抗氧化活性Antioxidant activity;26：结合Binding;27：催化活性Catalytic activity;28：分子传感器活性Molecular transducer activity;29：核苷酸结合转录因子活性Nucleic acid binding transcription factor activity;30：结构分子活性Structural molecule activity;31：转运活性Transporter activity。A：行为Behavior;B：生物调节Biological regulation;C：细胞过程Cellular process;D：局部化Localization;E：运动Locomotion;F：代谢过程 Metabolic process;G：多细胞有机体过程 Multicellular organismal process;H：生物调节过程 Regulation of biological process;I：应激反应Response to stimulus;J：信号Signaling;K：单有机体过程Single-organism process;L：膜Membrane;M：膜组成Membrane part;N：结合Binding;O：催化活性Catalytic activity
-->Fig. 2Distribution of up-regulated intersection genes in GO analysis
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对Rh28-vs-S与Rz36-vs-S交集的218个上调基因,注释到GO数据库BP中,将具有相同生物学功能的基因整合到一起（附表2）。部分差异基因在BP中所占的比例及数目见图3,其中主要涉及脂质代谢、蛋白质代谢、氨基酸转运和代谢、能量代谢、对环境压力的反应、对刺激物的反应、防御反应机制及转运等过程,附表3列举了Rh28-vs-S与Rh28-vs-Rz36交集上调的25个基因所参与的BP。
Table S2
附表2
附表2Rh28-vs-S和Rz36-vs-S显著上调的218个交集差异表达上调的基因注释整合（针对注释到GO-BP中共50个）
Table S2Up-regulated gene annotation integration in 218 intersection differential expression up-regulated by Rh28-vs-S and Rz36-vs-S (annotation to GO-BP for 50)
编号NumberGene ID描述DescriptionNCBI accessionE-value
Lipid metabolic process GO:0006629
1XM_011554560.1PREDICTED: Plutella xylostella uncharacterized LOC105384340 (LOC105384340), mRNAgi|3576272765.08917e-44
2XM_011552811.1PREDICTED: Plutella xylostella acyl-CoA Delta(11) desaturase-like (LOC105382844), mRNAgi|5142563661.12867e-98
3XM_011560272.1PREDICTED: Plutella xylostella sphingomyelin phosphodiesterase (LOC105389189), mRNA——
4XM_011555221.1PREDICTED: Plutella xylostella acyl-CoA desaturase-like (LOC105384925), mRNAgi|5142563945.12548e-149
5XM_011566673.1PREDICTED: Plutella xylostella acyl-CoA Delta (11) desaturase-like (LOC105394761), mRNA——
Protein metabolic process GO:0019538
6XM_011566393.1PREDICTED: Plutella xylostella mucin-17 (LOC105394481), mRNA——
7XM_011549516.1PREDICTED: Plutella xylostella carboxypeptidase B-like (LOC105380035), mRNAgi|3576242310
8XM_011551415.1PREDICTED: Plutella xylostella mannan-binding lectin serine protease 2-like (LOC105381636), partial mRNAgi|45300524.55351e-94
9XM_011554597.1PREDICTED: Plutella xylostella methionine aminopeptidase 2 (LOC105384366), transcript variant X4, mRNAgi|3576258582.41836e-157
10XM_011554639.1PREDICTED: Plutella xylostella serine proteinase stubble (LOC105384407), mRNAgi|3896094933.82422e-109
Metabolic process GO:0008152
11XM_011561204.1PREDICTED: Plutella xylostella putative fatty acyl-CoA reductase CG5065 (LOC105389995), mRNA——
12XM_011554543.1PREDICTED: Plutella xylostella senecionine N-oxygenase (LOC105384325), transcript variant X1, mRNAgi|2964278450
13XM_011557274.1PREDICTED: Plutella xylostella laccase (LOC105386665), mRNA——
14XM_011555197.1PREDICTED: Plutella xylostella peroxidase (LOC105384906), mRNAgi|3576276260
Cellular process GO:0009987
15XM_011567287.1PREDICTED: Plutella xylostella zinc finger RNA-binding protein-like (LOC105395333), transcript variant X6, mRNA——
16XM_011557500.1PREDICTED: Plutella xylostella delta-like protein 1 (LOC105386853), mRNA——
Response to stress GO:0006950
17XM_011550942.1PREDICTED: Plutella xylostella protein lethal(2)essential for life-like (LOC105381254), mRNAgi|709054701.80866e-71
18XM_011555197.1PREDICTED: Plutella xylostella peroxidase (LOC105384906), mRNAgi|3576276260
19XM_011550941.1PREDICTED: Plutella xylostella protein lethal(2)essential for life-like (LOC105381253), mRNAgi|3010701482.40659e-70
20XM_011550975.1PREDICTED: Plutella xylostella uncharacterized LOC105381283 (LOC105381283), mRNAgi|3576131021.79116e-13
Cellular protein metabolic process GO:0044267
21XM_011552622.1PREDICTED: Plutella xylostella FK506-binding protein 2 (LOC105382696), mRNAgi|4318233455.32079e-121
22XM_011569415.1PREDICTED: Plutella xylostella dnaJ protein homolog 1 (LOC105397398), mRNA——
Cell-substrate adhesion GO:0031589
23XM_011569224.1PREDICTED: Plutella xylostella protein mesh-like (LOC105397210), mRNA——
24XM_011553525.1PREDICTED: Plutella xylostella protein mesh-like (LOC105383478), mRNAgi|5129026080
Transcription, DNA-templated GO:0006351
25XM_011550384.1PREDICTED: Plutella xylostella CCAAT/enhancer-binding protein-like (LOC105380784), mRNAgi|1129837303.79764e-106
26XM_011566339.1PREDICTED: Plutella xylostella probable nuclear hormone receptor HR3 (LOC105394379), transcript variant X10, mRNA——
Response to stimulus GO:0050896
Cellular protein metabolic process GO:0044267
27XM_011559625.1PREDICTED: Plutella xylostella heat shock protein 83 (LOC105388667), mRNA——
Response to stimulus GO:0050896
28XM_011560293.1PREDICTED: Plutella xylostella heat shock protein 68-like (LOC105389211), mRNA——
Transition metal ion transport GO:0000041
Response to inorganic substance GO:0010035
29XM_011558700.1PREDICTED: Plutella xylostella high affinity copper uptake protein 1 (LOC105387898), transcript variant X3, mRNA——
30XM_011558698.1PREDICTED: Plutella xylostella high affinity copper uptake protein 1 (LOC105387898), transcript variant X1, mRNA——
Gene expression GO:0010467
31XM_011565866.1PREDICTED: Plutella xylostella 60S ribosomal protein L10-like (LOC105394030), partial mRNA——
32XM_011563058.1PREDICTED: Plutella xylostella DNA-directed RNA polymerase III subunit RPC10-like (LOC105391567), transcript variant X3, mRNA——
Phosphate-containing compound metabolic process GO:0006796
Sulfur compound metabolic process GO:0006790
33XM_011567496.1PREDICTED: Plutella xylostella bifunctional 3'-phosphoadenosine 5'-phosphosulfate synthase (LOC105395544), mRNA——
Transport GO:0006810
Cellular process GO:0009987
34XR_960822.1PREDICTED: Plutella xylostella ATP-binding cassette sub-family D member 2 (LOC105388660), transcript variant X2, misc_RNA——
Dicarboxylic acid metabolic process GO:0043648
35XM_011552336.1PREDICTED: Plutella xylostella NADP-dependent malic enzyme (LOC105382451), transcript variant X2, mRNAgi|1537922700
Carbohydrate metabolic process GO:0005975
36XM_011556990.1PREDICTED: Plutella xylostella heparan sulfate 2-O-sulfotransferase pipe (LOC105386430), mRNA——
Organelle organization GO:0006996
37XM_011561297.1PREDICTED: Plutella xylostella vacuole membrane protein 1-like (LOC105390063), transcript variant X1, mRNA——
Fatty acid metabolic process GO:0006631
Hormone biosynthetic process GO:0033057;GO:0042446
38XM_011563652.1PREDICTED: Plutella xylostella acyl-CoA Delta(11) desaturase-like (LOC105392062), mRNA——
Cell adhesion GO:0007155
39XM_011551813.1PREDICTED: Plutella xylostella integrin beta-PS-like (LOC105381998), transcript variant X2, mRNAgi|2411408570
Cellular component organization GO:0016043
Cellular process GO:0009987
40XM_011560384.1PREDICTED: Plutella xylostella adenylyl cyclase-associated protein 1 (LOC105389289), transcript variant X1, mRNA——
Actin cytoskeleton organization GO:0030036
regulation of cell shape GO:0008360
Cell-substrate adhesion GO:0031589
41XM_011563885.1PREDICTED: Plutella xylostella uncharacterized LOC105392279 (LOC105392279), mRNA——
Coenzyme biosynthetic process GO:0009108
42XM_011561945.1PREDICTED: Plutella xylostella molybdenum cofactor sulfurase (LOC105390609), partial mRNA——
Defense response GO:0006952
Response to protozoan GO:0001562
43XM_011552317.1PREDICTED: Plutella xylostella putative defense protein Hdd11 (LOC105382438), transcript variant X1, mRNAgi|5419055752.47216e-55
Carboxylic acid metabolic process GO:0019752
Catecholamine metabolic process GO:0006584
44XM_011552504.1PREDICTED: Plutella xylostella aromatic-L-amino-acid decarboxylase (LOC105382593), transcript variant X2, mRNAgi|158240410
Alpha-glucoside transport GO:0015766;GO:0000017
45XM_011562031.1PREDICTED: Plutella xylostella facilitated trehalose transporter Tret1-like (LOC105390692), transcript variant X1, mRNA——
GO:0006022
46XM_011557252.1PREDICTED: Plutella xylostella pheromone receptor transcription factor-like (LOC105386640), mRNA——
GO:0006026
47XM_011555687.1PREDICTED: Plutella xylostella probable chitinase 3 (LOC105385326), mRNAgi|3852584750
GO:0006026
48XM_011560083.1PREDICTED: Plutella xylostella endochitinase (LOC105389028), mRNA——
Axon guidance GO:0007411
Regulation of apoptotic process GO:0042981
Regulation of neuronal synaptic plasticity GO:0048168
Anatomical structure formation involved in morphogenesis GO:0048646
49XM_011551940.1PREDICTED: Plutella xylostella uncharacterized LOC105382111 (LOC105382111), mRNAgi|5128979450
GO:0006022
50XM_011557916.1PREDICTED: Plutella xylostella uncharacterized LOC105387220 (LOC105387220), transcript variant X2, mRNA——


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图3上调交集基因部分BP注释
A：运输Transport;B：细胞形态重排Regulation of cell shape;C：防御反应Defense response;D：转录Transcription;E：肌动蛋白细胞骨架组织Actin cytoskeleton organization;F：脂肪酸代谢进程Fatty acid metabolic process;G：基因表达Gene expression;H：对无机物的反应Response to inorganic substance;I：细胞蛋白质代谢过程Cellular protein metabolic process;J：代谢进程Metabolic process;K：脂质代谢进程Lipid metabolic process;L：含磷化合物代谢进程Phosphate-containing compound metabolic process;M：细胞基质黏附Cell-substrate adhesion;N：细胞进程Cellular process;O：蛋白代谢进程Protein metabolic process;P：辅酶生物合成进程Coenzyme biosynthetic process;Q：应压反应Response to stress;R：过渡金属离子运输Transition metal ion transport;S：羧酸代谢进程Carboxylic acid metabolic process;T：二羧酸代谢进程Dicarboxylic acid metabolic process;U：α-葡萄糖苷转运Alpha-glucoside transport;V：儿荼酚胺代谢进程Catecholamine metabolic process;W：应激反应Response to stimulus;X：糖代谢进程Carbohydrate metabolic process;Y：激素生物合成Hormone biosynthetic process;Z：硫化物代谢进程Sulfur compound metabolic process
-->Fig. 3Up-regulated intersection genes annotated to the corresponding BP
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Table S3
附表3
附表3Rh28-vs-S和Rh28-vs-Rz36显著上调的25个交集差异表达上调的基因注释整合（针对注释到GO-BP）
Table S3Up-regulated gene annotation integration in 25 intersection differential expression up-regulated by Rh28-vs-S and Rh28-vs-Rz36 (annotation to GO-BP)
编号
Number		Gene ID描述
Description		NCBI accessionE-value
Response to stimulus GO:0050896
1XM_011550936.1PREDICTED: Plutella xylostella heat shock protein 68-like (LOC105381248), mRNAgi|3196551710
Response to stimulus GO:0050896
Cellular protein metabolic process GO:0044267
2XM_011550224.1PREDICTED: Plutella xylostella heat shock 70 kDa protein cognate 5 (LOC105380633), transcript variant X1, mRNAgi|2230368300
Defense response GO:0006952
3XM_011569900.1PREDICTED: Plutella xylostella cecropin-like (LOC105397888), mRNA——
4XM_011564205.1PREDICTED: Plutella xylostella cecropin-like (LOC105392561), mRNA——
5XM_011566780.1PREDICTED: Plutella xylostella cecropin-like (LOC105394860), mRNA——
6XM_011566777.1PREDICTED: Plutella xylostella cecropin-like (LOC105394858), mRNA——
Coenzyme biosynthetic process GO:0009108
7XM_011563615.1PREDICTED: Plutella xylostella phosphopantothenoylcysteine decarboxylase-like (LOC105392028), mRNA——
Response to stress GO:0006950
8XR_960823.1PREDICTED: Plutella xylostella alpha-crystallin A chain pseudogene (LOC105388668), misc_RNA——
Cell migration GO:0016477
Cell surface receptor signaling pathway GO:0007166
Cellular protein modification process GO:0006464
9XM_011562336.1PREDICTED: Plutella xylostella casein kinase I isoform gamma-3 (LOC105390945), transcript variant X14, mRNA——
Metabolic process GO:0008152
Defense response GO:0006952
10XM_011552765.1PREDICTED: Plutella xylostella lysozyme-like (LOC105382813), mRNAgi|3576120476.79281e-50
Without Process annotation
11XM_011555680.1PREDICTED: Plutella xylostella lipase 3-like (LOC105385319), mRNAgi|3576196192.53335e-72
12XM_011561556.1PREDICTED: Plutella xylostella 15-hydroxyprostaglandin dehydrogenase [NAD(+)]-like (LOC105390283), mRNA——
13XM_011560992.1PREDICTED: Plutella xylostella gloverin-like (LOC105389803), mRNA——
14XM_011563995.1PREDICTED: Plutella xylostella elongation of very long chain fatty acids protein 7-like (LOC105392381), transcript variant X1, mRNA——
15XM_011550636.1PREDICTED: Plutella xylostella DDB1- and CUL4-associated factor 12 (LOC105381005), transcript variant X2, mRNAgi|5129011870
16XM_011569709.1PREDICTED: Plutella xylostella chymotrypsin-like protease CTRL-1 (LOC105397690), mRNA——
17XM_011552980.1PREDICTED: Plutella xylostella ovomucoid-like (LOC105382985), mRNA——
18XM_011553647.1PREDICTED: Plutella xylostella uncharacterized LOC105383567 (LOC105383567), transcript variant X2, mRNAgi|3576128958.06492e-46
19XM_011551124.1PREDICTED: Plutella xylostella uncharacterized LOC105381409 (LOC105381409), transcript variant X2, mRNA——
20XM_011565034.1PREDICTED: Plutella xylostella mucolipin-3-like (LOC105393299), mRNA——
21XM_011570215.1PREDICTED: Plutella xylostella putative odorant-binding protein A10 (LOC105398164), mRNA——
22XM_011555108.1PREDICTED: Plutella xylostella maternal protein exuperantia (LOC105384826), transcript variant X4, mRNAgi|1140531412.5075e-138
23XM_011562321.1PREDICTED: Plutella xylostella alpha-tocopherol transfer protein-like (LOC105390942), transcript variant X2, mRNA——
24XM_011555421.1PREDICTED: Plutella xylostella repetitive proline-rich cell wall protein 1-like (LOC105385099), mRNA——
25XM_011561234.1PREDICTED: Plutella xylostella troponin I (LOC105390008), transcript variant X13, mRNA——


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2.6 抗性显著上调基因的KEGG分析

为了较为准确地定位显著差异表达上调基因参与的生物学功能,针对其所参与的pathway,通过查找统计KEGG数据库,发现Rh28-vs-S和Rz36-vs-S显著上调的218个交集基因中,能够注释到数据库的基因仅有108个,主要集中在代谢通路上,有110个（50.45%）基因在KEGG数据库中没有详细的代谢通路图。小菜蛾响应氟苯虫酰胺药物选择压力下差异显著的表达基因富集到的通路有：代谢通路、PPAR信号通路、MAPK信号通路、Wnt信号通路等。同一个基因可能同时注释到多条pathway之中,经统计,列举了部分可能与抗性相关的通路（图4）。
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图4上调交集基因中与小菜蛾抗氟苯虫酰胺相关的部分通路
1：烟碱代谢Nicotine metabolism;2：恶性肿瘤中的通路Pathway in cancer;3：原发性免疫缺陷Primary immunodeficinecy;4：PPAR信号通路PPAR signaling pathway;5：蛋白质消化吸收Protein digestion and absorption;6：GABA能突触GABAergic synapse;7：甘油脂代谢Glycerolipid metabolism;8：甘油磷脂代谢Glycerophospholipid metabolism;9：昆虫激素生物合成 Insect hormone biosynthesis;10：细胞色素P450药物代谢Drug metabolism-cytochrome P450;11：酪氨酸代谢Tyrosine metabolism;12：Jak-STAT信号通路Jak-STAT signaling pathway;13：肥厚型心肌病Hypertrophic cardiíomyopathy (HCM);14：嘌呤代谢Purine metabolism;15：蛋白质在内质网中加工Protein processing in endoplasmic reticulum;16：嘧啶代谢Pyrimidine metabolism;17：代谢进程Metabolic pathways;18：过氧化物酶体Peroxisome;19：胰液分泌Pancreatic secretion;20：神经活性的配体-受体相互作用Neuroactive ligand-receptor interaction;21：扩张性心肌病Dilated cardiomyopathy;22：心肌收缩Cardiac muscle contraction;23：黑色素生成Melanogenesis;24：肌动蛋白细胞骨架的调控Regulation of actin cytoskeleton;25：不饱和脂肪酸的生物合成Biosynthesis of unsaturated fatty acids;26：脂肪消化吸收 Fat digestion and absorption;27：MAPK信号通路MAPK signaling pathway;28：胆汁分泌Bile secretion;29：ECM受体相互作用ECM-receptor interaction;30：Wnt信号通路Wnt signaling pathway;31：抗原处理与表达Antigen processing and presentation;32：氧化磷酸化Oxidative phosphorylation;33：多巴胺能突触Dopaminergic synapse
-->Fig. 4Partial pathway associated with the resistance to flubendiamide in P. xylostella
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2.7 抗性相关基因的聚类分析

利用高通量数据,对与杀虫剂抗性相关的基因进行了动态的监测,并从中筛选出可能与抗药性相关的通路基因,绘制了聚类热图（图5）。通过聚类热图的红色区域,可以看出Rh28品系与Rz36种群显著表达量上调的基因,很多集中在内质网上蛋白质的加工、酪氨酸代谢、咖啡因代谢、Wnt信号通路。
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图5小菜蛾抗氟苯虫酰胺相关基因表达分析
图中红色代表表达上调基因;绿色代表表达下调基因;黑色代表表达无变化基因Red represents the up-regulated genes; green indicates the down-regulated genes and black represents no change in gene expression。其中图示右侧pathway缩写具体指代如下,MAPK：MAPK信号通路MAPK signaling pathway;PPER：蛋白质在内质网中加工Protein processing in endoplasmic reticulum;TM：酪氨酸代谢Tyrosine metabolism;DM-P450：细胞色素P450药物代谢Drug metabolism-cytochrome P450;GM：谷胱甘肽代谢Glutathione metabolism;MX-P450：外来物质代谢的细胞色素P450 Metabolism of xenobiotics by cytochrome P450;CMC：心肌收缩Cardiac muscle contraction;DC：扩张性心肌病Dilated cardiomyopathy;GS：GABA能突触GABAergic synapse;CM：咖啡因代谢Caffeine metabolism;WSP：Wnt信号通路Wnt signaling pathway;Mel：黑色素生成Melanogenesis;VSMC：血管平滑肌收缩Vascular smooth muscle contraction
-->Fig. 5Expression profiling of genes associated with the resistance to flubendiamide in P. xylostella
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3 讨论

随着高通量测序技术日益成熟,为小菜蛾抗药性研究提供了新方法,通过进行小菜蛾基因的筛选与鉴定,进而分析基因的差异表达,对深入阐述小菜蛾的抗性机制具有重要意义。本文通过基因序列筛选,从小菜蛾S、Rh28和Rz36品系/种群的RNA-Seq序列信息中鉴定出多个与杀虫剂靶标及解毒代谢相关的基因,对这些差异表达基因在GO以及KEGG数据库进行功能预测与分类注释,从获得的2万多条差异表达基因序列中,筛选出满足差异显著性表达条件的只有几百条,这部分显著差异的序列,在GO数据库中差异表达基因显著富集的生物过程均在应激反应（response to stress）和防御反应（defense response）中,导致小菜蛾对氟苯虫酰胺抗性产生的基因主要集中在这部分生物过程中,其生物学功能可能与小菜蛾的抗性之间存在一定的相关性。
通过对差异基因的Pathway统计,信号通路主要集中于PPAR信号通路、蛋白质在内质网上的加工、甘油磷脂代谢、解毒酶代谢等。PPAR是一类配体激活的核转录因子超家族成员,包括PPAR-α、PPAR-β和PPAR-γ 3种表型,其中以PPAR-γ的研究最为深入。现有的研究已证实PPAR-γ主要参与调控脂肪和糖类的代谢、能量平衡等多种生物学功能的调节,小菜蛾的体内脂肪酸LPL、胆固醇、甘油磷脂等各种脂类的代谢相关以及信号受体激活等一系列连锁反应[17]。蛋白质在内质网上的加工,主要参与协助一些蛋白质装配或者折叠,这类蛋白主要是Sec23蛋白和Hsps分子伴侣蛋白,它们与能量代谢、次级代谢途径密切相关。参与各种氨基酸、嘌呤,代谢产物（萜类、半萜类）代谢及生物合成、糖酵解;能量代谢过程中K/Na离子通道ATPase表达上调。研究表明,氯虫苯甲酰胺能显著影响甜菜夜蛾幼虫中枢神经元的兴奋性,使电压门控钠通道电流密度和通道功能发生改变,试验结果说明钠离子通道亦是氯虫苯甲酰胺的作用靶标之一[18]。本文通过聚类热图分析发现,上调表达的基因很多集中在内质网上蛋白质的加工、酪氨酸代谢和Wnt信号通路中,与前人研究结果基本一致。
谷胱甘肽-S-转移酶（GSTs）作为一种超基因家族酶类,可催化昆虫因各种化学药剂产生的有害物质的代谢。依据GSTs所处的细胞位置和功能,主要分为微粒体、线粒体和胞质型,而在昆虫中这种酶主要属于胞质型,其家族中的Delta家族（昆虫中特有的GSTs家族之一）[19],在生物体内起到的作用主要包括催化活性、谷胱甘肽过氧化物酶活性、谷胱甘肽转移酶活性及裂解酶活性等,这些均与昆虫的代谢活性相关。研究发现,抗氯虫苯甲酰胺品系小菜蛾的GSTs 活性是敏感品系的3.34倍,因此GSTs可能在小菜蛾对氯虫苯甲酰胺的代谢抗性中发挥着主要作用[9],且本文的研究也表明主要富集于谷胱甘肽代谢的2个基因（XM_011569702.1、XM_011557174.1）表现为显著表达上调。
细胞色素P450在昆虫生长发育及繁殖等方面起着重要作用,并且在次生物质和杀虫剂代谢方面也有重要作用,在COG数据库中,细胞色素P450被注释到了次生代谢产物的生物合成（second-metabolites biosynthesis）、运输和代谢（transport and catabolism）功能中,这也充分说明了细胞色素P450是昆虫体内主要的代谢解毒酶之一[20]。其中编码CYP2的基因（XM_011556598.1）表达量上调显著,而且已有研究报道CYP家族的细胞色素P450解毒酶在一些昆虫体内与抗药性密切相关[21]。
经过部分筛选分析,从小菜蛾中鉴定出多个热激蛋白（Hsp）,发现该抗性小菜蛾体内的Hsp的多个家族（如Hsp40、Hsp70、Hsp90）表达量显著上调,并且参与了多个生物工程,笔者认为Hsp很可能与昆虫的抗性以及对环境的应激性有关,而且在GO数据库中,这些Hsp家族差异基因均注释进入了“对刺激物的反应”和“细胞蛋白质代谢过程（cellular protein metabolic process）”功能中,同时“对刺激物的反应”的功能注释更加表明了Hsp可能与小菜蛾抗药性的产生有着密切的联系。Hsp基因家族可被压力诱导,在环境压力因素下,如重金属离子、极端温度、营养缺失、氧自由基及细菌或者病毒感染都有可能诱导热激蛋白的过量表达[22]。在粉虱类的抗逆基因研究中已经有过类似的报道,可能由于昆虫的这种蛋白可以应对环境中存在的极端胁迫压力[23,24],导致一些入侵性的有害生物在分布地域方面不断扩大[25]。已有研究证实在高温、低温、辐射、干旱和农药等的环境压力下,都能够诱导昆虫体内不同Hsp的表达,这证实了Hsp可以提高昆虫对不良环境的耐受能力,从而保护昆虫免受和少受胁迫的伤害。如温度驯化的南美斑潜蝇（Lirionyza huidobrebsis）,其体内Hsp70基因大量表达,在性状方面耐热能力也显著提高,表明Hsp表达和昆虫耐热性的获得有着密切关系,而且耐热性的诱导和消退动力学与Hsp的诱导和降解存在着平行关系[26]。抗有机磷类杀虫剂的摇蚊（Chiromomus yoshimatsui）品系Hsp的表达水平与敏感品系相比,是敏感品系的2—3倍,表明Hsp上调增强了昆虫的抗药能力[27]。本研究发现,小菜蛾抗氟苯虫酰胺品系的谷胱甘肽-S-转移酶、细胞色素P450解毒酶及参与昆虫多种生理反应的热激蛋白超基因家族等基因的表达量均显著上调,与前人研究基本一致。因此,今后需要对这些基因及其所在的通路进行深入研究,进一步明确其抗性机制。
昆虫靶标基因的mRNA表达量变化也是昆虫抗药性产生的原因之一。本研究中小菜蛾田间抗性种群鱼尼丁受体相关8个基因表达量均下调,而且有6个显著下调。LIN等[28]通过比较转录组测序发现,对氯虫苯甲酰胺产生不同抗性水平小菜蛾种群的鱼尼丁受体mRNA的表达量均显著下调;WAN等[29]利用RNAi干扰马铃薯甲虫（Leptinotarsa decemlineata）鱼尼丁受体的表达后,再用氯虫苯甲酰胺处理,马铃薯甲虫的死亡率明显降低;YANG等[30]在白背飞虱（Sogatella furcifera）的研究中也发现了类似结果。这些结果均表明鱼尼丁受体基因表达量的下调可能会增强昆虫对双酰胺类药剂的抗性。笔者实验室正在开展鱼尼丁受体表达差异基因的验证工作,以明确该类基因在小菜蛾对双酰胺类杀虫剂抗性中所起的作用。

4 结论

通过转录组测序（RNA-Seq）获得了小菜蛾抗氟苯虫酰胺差异表达基因及抗性显著上调表达基因,其主要富集于代谢过程、应激反应及对刺激的反应等条目中,这些基因的相互协同调控是小菜蛾对氟苯虫酰胺产生抗性的重要机制。
The authors have declared that no competing interests exist.

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