陈锐^,1, 高贺¹, 张国军¹, 朱克岩², 成卫宁^,11西北农林科技大学植物保护学院/农业农村部西北黄土高原作物有害生物综合治理重点实验室,中国陕西杨凌 712100
²Department of Entomology，Texas A&M University，Texas 77843，USA

Effects of Secondary Metabolites in Wheat Kernels on Activities of Three Detoxifying Enzymes and Related Gene Expression in Sitodiplosis mosellena

CHEN Rui^,1, GAO He¹, ZHANG GuoJun¹, ZHU KeYan², CHENG WeiNing^{,1 1}College of Plant Protection, Northwest A&F University/Key Laboratory of Integrated Pest Management on Crops in Northwestern Loess Plateau, Ministry of Agriculture and Rural Affairs, Yangling 712100, Shaanxi, China
²Department of Entomology，Texas A&M University，Texas 77843，USA

通讯作者: 成卫宁,E-mail: cwning@126.com

责任编辑: 岳梅
收稿日期:2020-03-7接受日期:2020-04-22网络出版日期:2020-10-16

基金资助:

国家重点研发计划.2018YFD020040302 陕西省重点研发计划.2020NY-059 国家自然科学基金.31371933 杨凌示范区科技计划项目.2018NY-07

Received:2020-03-7Accepted:2020-04-22Online:2020-10-16
作者简介 About authors
陈锐,E-mail: ruichen0828@qq.com。







摘要
【目的】明确小麦灌浆期籽粒次生物质含量与麦红吸浆虫（Sitodiplosis mosellena）幼虫谷胱甘肽转移酶（glutathione S-transferase,GST）、羧酸酯酶（carboxylesterase,CarE）和细胞色素P450 酶系（cytochrome P450,CYP450）活性及相关基因表达的关系,探讨次生物质在小麦对麦红吸浆虫抗性中的作用及麦红吸浆虫的解毒代谢机制。【方法】2016年5月采用剥穗调查法对陕西省周至县试验田种植的4个抗虫和4个感虫小麦品种（系）进行麦红吸浆虫幼虫危害调查,并收集各品种（系）灌浆期籽粒及取食各品种（系）灌浆期籽粒的麦红吸浆虫幼虫;采用比色皿法、香草醛法、Folin-Ciocalteu法和亚硝酸钠-硝酸铝显色法分别测定收集的小麦籽粒阿魏酸、单宁、总酚和总黄酮含量,酶联免疫吸附法和qPCR方法分别检测收集的麦红吸浆虫幼虫GST、CarE和CYP450活性及其编码基因GST1、CarE2和CYP6A1 mRNA表达水平,分析次生物质含量与小麦对麦红吸浆虫抗性以及麦红吸浆虫解毒酶活性和基因表达的相关性。【结果】不同小麦品种（系）灌浆期籽粒阿魏酸、单宁、总酚和总黄酮含量差异明显,其中阿魏酸和单宁含量以抗虫品种科农1006最高,总酚含量以抗虫品种晋麦47最高,总黄酮含量以感虫品种西农88最高;取食不同品种（系）小麦的吸浆虫幼虫GST、CarE和CYP450活性及GST1、CarE2和CYP6A1表达水平差异亦明显,GST和CarE活性分别以取食抗虫品种科农1006和陕麦139的最高,CYP450活性以取食感虫品种小偃6号的最高;GST1和CarE2表达水平在取食抗虫品种的幼虫中均高于取食感虫品种,CYP6A1表达水平在取食抗、感性品种的幼虫中表现无规律。相关分析表明,小麦籽粒阿魏酸含量与穗被害率、粒被害率、单穗虫口和估计损失4个小麦对吸浆虫的抗性指标均呈显著负相关（P<0.05）,单宁、总酚和总黄酮含量与4个指标相关性不显著。取食不同品种（系）小麦的吸浆虫幼虫GST和CarE活性与小麦籽粒阿魏酸含量呈显著正相关,CYP450活性与单宁含量呈显著负相关（P<0.05）,总酚和总黄酮含量与3种解毒酶活性相关性不显著;GST1和CarE2表达水平分别与其GST和CarE活性呈显著正相关（P<0.05）,但CYP6A1表达水平与CYP450活性相关性不显著。【结论】阿魏酸是小麦抗麦红吸浆虫的主要次生物质,能够诱导麦红吸浆虫幼虫GST、CarE活性和相关基因的表达,它们共同作用参与麦红吸浆虫对小麦次生物质的代谢和适应。
关键词： 小麦籽粒;次生物质;麦红吸浆虫;解毒酶活性;基因表达

Abstract
【Objective】The objective of this study is to clarify the relationship between secondary metabolite contents at grain filling stage of wheat and detoxification enzyme activities of glutathione S-transferase (GST), carboxylesterase (CarE) and cytochrome P450 (CYP450) and related gene expression in Sitodiplosis mosellena, and to explore the role of secondary metabolite in wheat resistance to S. mosellena and the metabolic mechanism.【Method】The numbers of S. mosellana larvae in wheat kernels of four resistant and four susceptible wheat varieties (lines) planted in experimental fields in Zhouzhi County, Shaanxi Province were investigated by dissecting wheat ears in May 2016. Wheat kernels at filling stage from eight varieties (lines) and S. mosellena larvae feeding wheat kernels of eight varieties (lines) were simultaneously collected. The contents of ferulic acid, tannin, total phenol and total flavonoid were determined by colorimetry, vanillin assay, Folin-Ciocalteu assay and sodium nitrite-aluminium nitrite assay, respectively. The activities of GST, CarE and CYP450 and expression levels of GST1, CarE2 and CYP6A1 in collected S. mosellena larvae were determined using enzyme linked immunosorbent assay (ELISA) and qPCR methods, respectively. And then, the relationship between secondary metabolite contents in various wheat varieties (lines) and resistance indictors of wheat to S. mosellena, as well as the interaction of secondary metabolites with detoxification enzyme activity and gene expression in S. mosellena larvae were studied by correlation analysis.【Result】The contents of ferulic acid, tannin, total phenol and total flavonoid of wheat kernel at filling stage of eight wheat varieties (lines) were significantly different. The highest contents were in the resistant variety Kenong 1006 for ferulic acid and tannin, in the resistant variety Jinmai 47 for total phenol, and in the susceptible variety Xinong 88 for total flavonoid. The activities of GST, CarE and CYP450 and expression levels of GST1, CarE2 and CYP6A1 in S. mosellena larvae from eight wheat varieties (lines) were also significantly different. The highest enzyme activities were in larvae from resistant varieties Kenong 1006 and Shaanmai 139 for GST and CarE, respectively, and from the susceptible variety Xiaoyan 6 for CYP450. The expression levels of GST1 and CarE2 were higher in larvae feeding four resistant varieties than those feeding four susceptible varieties, but CYP6A1 expression level had not obvious regularity between larvae feeding resistant and susceptible varieties. Correlation analysis results showed that the ferulic acid content of wheat kernel had significant negative correlation (P<0.05) with the four resistance indicators of wheat to S. mosellena including percentage of infested ears, percentage of infested grains, insect number per ear and estimated loss rate. There was no significant correlation between the contents of tannin, total phenol and total flavonoid and the four resistance indicators. The GST and CarE activities of S. mosellena feeding wheat kernels were significantly positively correlated to the ferulid acid content of wheat kernels, and the CYP450 activity of S. mosellena was significantly negatively correlated to the tannin content (P<0.05). No significant correlation was observed between the total phenol and total flavonoid contents of wheat kernels and the activity of any of the three detoxification enzymes. The expression levels of S. mosellena GST1 and CarE2 were significantly positively correlated with the activities of GST and CarE (P<0.05), respectively, while S. mosellena CYP6A1 expression level was not significantly related with the activity of CYP450.【Conclusion】Ferulic acid is the main secondary metabolite of wheat anti-S. mosellena, and can induce GST and CarE activities of S. mosellena larvae and related gene expression. GST and CarE jointly participate in the detoxification metabolism and adaptation of S. mosellena to wheat secondary metabolites.
Keywords：wheat kernel;secondary metabolite;Sitodiplosis mosellena;detoxification enzyme activity;gene expression


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本文引用格式
陈锐, 高贺, 张国军, 朱克岩, 成卫宁. 小麦籽粒次生物质对麦红吸浆虫幼虫解毒酶活性及基因表达的影响[J]. 中国农业科学, 2020, 53(20): 4204-4214 doi:10.3864/j.issn.0578-1752.2020.20.009
CHEN Rui, GAO He, ZHANG GuoJun, ZHU KeYan, CHENG WeiNing. Effects of Secondary Metabolites in Wheat Kernels on Activities of Three Detoxifying Enzymes and Related Gene Expression in Sitodiplosis mosellena[J]. Scientia Acricultura Sinica, 2020, 53(20): 4204-4214 doi:10.3864/j.issn.0578-1752.2020.20.009

0 引言

【研究意义】麦红吸浆虫（Sitodiplosis mosellana）是小麦生产上间隙性猖獗成灾的重要害虫^[1,2],以幼虫潜伏在颖壳内吸食正在灌浆的籽粒汁液,造成麦粒瘪疮、空壳或霉烂,一般使小麦减产10%—20%,重者30%—50%,甚至颗粒无收,严重影响小麦的产量和品质,甚至根本不能食用^[3]。实践证明,选育和推广种植抗虫品种是降低吸浆虫危害最安全和经济有效的途径^[4,5]。探究小麦灌浆期籽粒次生物质含量与小麦抗吸浆虫的关系,以及不同抗性品种次生物质含量对麦红吸浆虫幼虫解毒酶活性及其编码基因表达的影响,对深入研究小麦对吸浆虫的抗性机制及抗虫品种的选育具有重要意义。【前人研究进展】为了避免或减轻植食性昆虫的危害,植物与昆虫在长期的协同进化过程中形成了一系列防御机制,抗生性是植物抗虫的主要机制之一,植物的抗生性与次生物质含量关系密切,如高粱对西南玉米杆螟（Diatraea grandiosella）、小麦籽粒对玉米象（Sitophilus zeamais）、棉花对棉铃虫（Helicoverpa armigera）和绿盲蝽（Apolygus lucorum）的抗性分别与生腈糖苷、阿魏酸、单宁和棉酚等含量有密切关系^[6,7,8,9]。为抵御次生物质的毒害,植食性昆虫也演化出了多种反防御机制。当取食不同寄主或不同类型次生物时,昆虫体内的解毒酶会变化,从而对摄入的有毒物质进行解毒和排毒^[10]。谷胱甘肽-S-转移酶（glutathione S-transferase,GST）、羧酸酯酶（carboxylesterase,CarE）和细胞色素P450 酶系（cytochrome P450,CYP450）是昆虫体内最重要的解毒酶,其活性和基因表达水平能够被多种次生物质诱导,以此增强对次生物质的代谢能力和对寄主的适应性^{[11,12,13,14]}。研究表明,儿茶素和芦竹碱可以诱导麦长管蚜（Sitobion avenae）CarE和GST活性升高^[15],单宁可以诱导杨小舟蛾（Micromelalopha troglodyte）GSTd1的表达^[16];亚洲小车蝗（Oedaleous asiaticus）取食含单宁、黄酮、苯酚等次生物质含量高的抗性寄主冷蒿后,体内GST、CarE和P450活性及相关基因表达量显著升高^[17]。【本研究切入点】多年来,国内外对小麦抗吸浆虫性进行了大量研究,发现不同品种（系）小麦对吸浆虫的抗性不同^[18,19,20],抗性不同品种间次生物质含量亦存在差异^[21,22],但小麦籽粒次生物质是否对吸浆虫解毒酶产生影响,解毒酶在吸浆虫对植物次生物质代谢及寄主适应性中所起作用尚不清楚。【拟解决的关键问题】分析不同抗性品种（系）小麦次生物质（单宁、总黄酮、阿魏酸、总酚）含量与小麦抗吸浆虫指标,以及与取食不同品种（系）的麦红吸浆虫幼虫3种解毒酶（GST、CarE和CYP450）活性及相关基因表达的关系,明确影响小麦抗麦红吸浆虫的关键次生物质及麦红吸浆虫对次生物质的解毒机制,为麦红吸浆虫综合治理和抗虫品种的培育提供新思路。

1 材料与方法

试验于2015—2017年分别在陕西省西安市周至县和西北农林科技大学农业农村部西北黄土高原作物有害生物综合治理重点实验室完成。

1.1 供试小麦品种（系）

选取生育期相同或相近,抽穗期（敏感期）与麦红吸浆虫羽化高峰期吻合,前期鉴定连续多年对麦红吸浆虫抗性表现稳定的抗虫小麦品种（系）晋麦47、科农1006、陕麦139和陕农33,及感虫品种（系）小偃22、西农822、西农88和小偃6号^[20]。所有品种（系）均来源于西北农林科技大学农学院。

1.2 田间试验设计

将供试材料于2015年小麦适播期播种于麦红吸浆虫重发区陕西省西安市周至县试验地（N34°9′,E109°10′）,每品种3个重复,每重复10行,行长1.5 m。翌年春季淘土调查虫口密度为390万头/667 m²,试验地常规管理,不施用任何农药,各品种（系）在田间自然感虫。

1.3 小麦品种（系）被害调查与试验样品收集

小麦灌浆期,每个品种（系）小麦各重复随机取50穗,带回室内逐穗、逐粒剥查并记载麦粒中的麦红吸浆虫幼虫数;同时收集各品种（系）健康籽粒,保存于-20℃,用于次生物质含量测定;收集足够多危害各品种（系）小麦籽粒的虫龄一致的幼虫,龄期依据虫体大小、颜色和Y型剑骨片有无区分^[18],收集的幼虫每30头分装于2.0 mL冻存管,液氮速冻后-80℃保存,用于解毒酶活性和基因表达分析。调查结束后,计算每个品种（系）的平均穗被害率、粒被害率、单穗幼虫数和估计损失率。计算估计损失率时,麦粒中的幼虫数分为5级:0级:无虫/粒;1级:1头/粒;2级:2头/粒;3级:3头/粒;4级:≥4头/粒^[20]。

$估计损失率\text{ }\!\!%\!\!\text{ }=\frac{\sum _{1}^{4}各级粒数\times 相应级别值}{4\times 总粒数}\times 100$。

1.4 小麦灌浆期籽粒次生物质含量测定

将收集的小麦籽粒80℃烘干至恒重,研磨成粉,过100目筛子后用于次生物质含量的测定,其中阿魏酸含量以阿魏酸为标品,采用比色皿法测定^[23],单宁含量以儿茶素为标准品,采用香草醛法测定^[6],总酚含量以没食子酸为标准品,采用Folin-Ciocalteu法测定^[24],总黄酮含量以芦丁为标准品,采用亚硝酸钠-硝酸铝显色法测定^[25]。每处理重复3次。

1.5 麦红吸浆虫幼虫GST、CarE和CYP450活性分析

1.5.1 酶液制备 从冰箱中取出各样品,称重后放入1.5 mL离心管,分别加入500 μL 0.04 mol·L^-1 pH 7.0、0.1 mol·L^-1 pH 6.5和0.1 mol·L^-1 pH 7.8的磷酸缓冲液（含1 mmol·L^-1 EDTA、1 mmol·L^-1 PMSF和1 mmol·L^-1 DTT）,充分研磨后,匀浆液于4℃ 12 000×g下离心10 min,上清液分别为CarE、GST和CYP450酶液^[26]。测试时,酶液再用缓冲液稀释成最佳取样浓度。每处理30头试虫,重复3次。

1.5.2 酶活力测定 3种解毒酶的活力均采用上海泛柯实业有限公司生产的酶联免疫分析试剂盒测定,具体操作步骤按试剂盒说明进行。

GST活力测定原理:用纯化的昆虫GST抗体包被微孔板,制成固相抗体,往包被单抗的微孔中依次加入GST标准品和待测酶液,再与辣根过氧化物酶（HRP）标记的GST抗体结合,形成抗体-抗原-酶标抗体复合物,经彻底洗涤后加底物四甲基联苯胺（TMB）显色。用酶标仪在450 nm波长下测定OD值,通过标准曲线计算样品中GST活性。

CarE和CYP450活力测定原理与GST相同,区别仅在于以相应的酶标抗体进行包被。

以37℃下每毫克组织蛋白每分钟转化1 μmol底物（TMB）的酶量为一个活力单位（U/mg protein）。

1.5.3 酶蛋白浓度测定 以牛血清蛋白（BSA）为标准蛋白,采用考马斯亮蓝G-250染色法测定^[27]。

1.6 麦红吸浆虫GST、CarE和CYP450表达水平分析

1.6.1 总RNA提取和cDNA合成 分别取危害各品种（系）小麦的麦红吸浆虫幼虫30头,按照TRNzol总RNA提取试剂盒（TIANGEN,北京）操作说明提取总RNA,PrimeScript^TM RT reagent Kit with gDNA Eraser（Perfect Real Time）反转录试剂盒（TaKaRa,大连）说明合成单链cDNA。重复3次。

1.6.2 荧光定量PCR检测 在麦红吸浆虫幼虫转录组数据库搜索GST、CarE和CYP450酶编码基因,从中挑选表达量高的GST1、CarE2和CYP6A1 Unigene序列设计定量引物（表1）。以麦红吸浆虫GAPDH（GenBank登录号:KR733066）为内参,合成的cDNA为模板,按照SuperReal PreMix Plus（SYBR Green）试剂盒说明书（TIANGEN,北京）,在iQ5 Real-Time PCR Detection System（Bio-Rad,Hercules,CA）上进行qPCR分析。反应体系（20 μL）:2×SuperReal PreMix Plus 10 μL,上下游引物（10 μmol·L^-1）各0.8 μL,cDNA模板1.0 μL,RNase Free H₂O 7.4 μL。扩增程序:95℃预变性15 min;95℃变性30 s,55℃退火30 s,72℃延伸30 s,40个循环;最后95℃ 30 s,55℃ 1 min,95℃ 15 s,进行熔解曲线分析,反应结束后读取Ct值。每样品进行3次技术重复。

Table 1
表1
表1qPCR中应用的引物序列
Table 1Primer sequences for qPCR

引物名称 Primer name	上游引物 Forward primer (5′-3′)	下游引物 Reverse primer (5′-3′)
GST1	ATAACGGCGTGCTAATAG	AACGAGAATCCACTTGAG
CarE2	AAATGGCTCGGACTCTTTACG	ATGGCTCGGATGCTGACT
CYP6A1	TTACGAAGCGATGATGGA	GCAACTGGTGGATACTTTC
GAPDH	CCATCAAAGCAAGCAAGA	CAGCACGGAGCACAAGAC

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1.7 数据分析

以取食陕麦139的麦红吸浆虫幼虫为基准,采用2^-ΔΔCT法^[28]计算危害各品种（系）小麦的麦红吸浆虫幼虫解毒酶基因相对表达量。小麦品种（系）间受害程度、次生物质含量、解毒酶活性和基因表达量进行单因素方差分析（P<0.05）,Duncan氏新复极差法进行多重比较;Pearson法分析次生物质含量与小麦抗虫指标、解毒酶活性,以及基因表达量与酶活性间的相关性,所有分析在SPSS 19.0（Chicago,IL,美国）统计软件包上进行。

2 结果

2.1 不同品种（系）小麦受麦红吸浆虫危害程度

由表2可知,不同品种（系）小麦受害程度差异明显,其中前期鉴定为抗虫的4个品种晋麦47、科农1006、陕麦139和陕农33穗被害率、粒被害率、单穗虫口数和估计损失率分别为30.67%—69.67%、3.20%—7.11%、1.10—5.07头和0.42%—2.43%,均显著低于4个感虫品种小偃22、西农822、西农88和小偃6号对应的89.33%—95.33%、11.51%—15.15%、7.57—14.24头和4.25%—6.12%（P<0.05）。抗虫品种和感虫品种内部受害程度也存在一定的差异,在抗虫品种中以陕麦139受害最轻,科农1006和晋麦47次之,陕农33受害较重;感虫品种中以小偃22受害最重,除穗被害率外,其他几个指标均显著高于西农822、西农88和小偃6号。

Table 2
表2
表2麦红吸浆虫幼虫对8个品种（系）小麦的危害程度
Table 2Damage degree of eight wheat varieties (lines) caused by S. mosellana larvae

小麦品种（系） Wheat variety (line)	穗被害率 Percentage of infested ears (%)	粒被害率 Percentage of infested grains (%)	单穗虫口数 Insect number per ear	估计损失率 Estimated loss rate (%)
陕麦139 Shaanmai 139	30.67±0.88f	3.20±0.35e	1.10±0.14f	0.42±0.05f
科农1006 Kenong 1006	50.33±1.76e	3.75±0.41e	2.30±0.27ef	1.11±0.13e
晋麦47 Jinmai 47	59.67±1.76d	5.44±0.41d	3.61±0.32de	1.80±0.15d
陕农33 Shaannong 33	69.67±2.60c	7.11±0.49c	5.07±0.39d	2.43±0.19d
小偃6号Xiaoyan 6	89.33±0.88b	11.51± 0.75b	7.57±0.53c	4.25±0.30c
西农88 Xinong 88	95.33±1.20a	12.13±0.62b	9.94±0.63b	4.97±0.29b
西农822 Xinong 822	92.00±1.53ab	12.90±0.61b	10.91±0.69b	5.02±0.29b
小偃22 Xiaoyan 22	91.33±1.86ab	15.15±0.73a	14.24±0.88a	6.12±0.36a

表中数据为3次重复的平均值±标准误,同列数据后标有不同字母分别表示Duncan氏多重比较差异显著（P<0.05）。表3、表5同
The data in the table are mean±SE of three repetitions, and data followed by different letters in the same column indicate significant difference at 0.05 level by Duncan’s multiple range test. The same as Table 3, Table 5

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2.2 不同品种（系）小麦籽粒次生物质含量比较

由表3可知,不同小麦品种（系）灌浆期籽粒阿魏酸、单宁、总酚和总黄酮含量差异明显,其中阿魏酸和单宁含量在抗虫品种科农1006中最高,分别为6.57和2.17 mg·g^-1,显著高于所有感虫品种,在感虫品种西农822和小偃6号中最低,分别为4.61和1.02 mg·g^-1,显著低于其他品种（系）。总酚含量在抗虫品种晋麦47中最高（24.86 mg·g^-1）,低抗品种陕农33中最低（12.56 mg·g^-1）。总黄酮含量在感虫品种西农88中最高（3.93 mg·g^-1）,显著高于其他品种（系）,小偃6号中最低（1.91 mg·g^-1）。

Table 3
表3
表38个品种（系）小麦籽粒次生物质含量
Table 3Contents of secondary metabolites in wheat kernels of eight varieties (lines)

小麦品种（系） Wheat variety (line)	阿魏酸含量 Ferulic acid content (mg·g-1)	单宁含量 Tannin content (mg·g-1)	总酚含量 Total phenol content (mg·g-1)	总黄酮含量 Total flavonoid content (mg·g-1)
陕麦139 Shaanmai 139	6.23±0.09b	1.69±0.05b	24.64±0.85a	2.19±0.08ef
科农1006 Kenong 1006	6.57±0.10a	2.17±0.04a	23.74± 0.15a	2.94±0.06c
晋麦47 Jinmai 47	6.44±0.02ab	1.66±0.03bc	24.86±1.15a	2.67±0.05cd
陕农33 Shaannong 33	6.33±0.11ab	1.28±0.01d	12.56±0.30c	3.29±0.12b
小偃6号 Xiaoyan 6	5.51±0.16c	1.02±0.06e	23.99±1.34a	1.91±0.05f
西农88 Xinong 88	4.77±0.10d	1.55±0.02c	24.09±1.04a	3.93±0.02a
西农822 Xinong 822	4.61±0.04d	1.12±0.01e	14.55±0.61bc	2.39±0.15de
小偃22 Xiaoyan 22	5.72±0.15c	1.55±0.05c	15.83±1.25b	3.44±0.06b

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2.3 小麦籽粒次生物质含量与抗虫性的关系

以抗、感程度不同的各品种（系）小麦穗被害率、粒被害率、单穗虫口数和估计损失率为抗虫指标,与小麦籽粒4种次生物质含量进行相关性分析,结果表明,阿魏酸含量与4个抗虫指标均呈显著负相关（P<0.05）,单宁、总酚和总黄酮含量与4个抗虫指标的相关性均未达显著水平（P>0.05）（表4）。

Table 4
表4
表4小麦籽粒次生物质含量与小麦抗吸浆虫指标的相关性
Table 4Correlation between resistance indicators of wheat to S. mosellena and contents of secondary metabolites in wheat kernels

次生物质 Secondary metabolite	穗被害率 Percentage of infested ears		粒被害率 Percentage of infested grains		单穗虫口数 Insect number per ear		估计损失率 Estimated loss rate
	r	P	r	P	r	P	r	P
阿魏酸 Ferulic acid	-0.774	0.024*	-0.778	0. 023*	-0.726	0.041*	-0.774	0.024*
单宁 Tannin	-0.620	0.101	-0.597	0.118	-0.477	0.232	-0.550	0.158
总黄酮Total flavonoid	0.332	0.422	0.275	0.509	0.378	0.356	0.337	0.415
总酚 Total phenol	-0.392	0.337	-0.438	0.278	-0.495	0.212	-0.426	0.292

*表示在P<0.05水平显著相关,表6同
* indicates significant correlation at P<0.05 level. The same as Table 6

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2.4 小麦品种（系）对麦红吸浆虫GST、CarE和CYP450活性的影响

由表5可知,取食抗、感程度不同品种（系）小麦的麦红吸浆虫幼虫体内GST、CarE和CYP450活性差异明显。除小偃6号外,取食感虫品种小偃22、西农822和西农88的幼虫GST和CarE活性均低于4个抗虫品种,其中取食西农88的GST和CarE活性最低,分别为0.0963和0.1016 U/mg protein,取食科农1006和陕麦139的最高,分别为0.2144和0.2284 U/mg protein。对于CYP450,以取食小偃6号的活性最高,其次为西农822,分别为0.1885和0.1791 U/mg protein,取食其他品种的幼虫间差异不显著。

Table 5
表5
表5取食8个品种（系）小麦的麦红吸浆虫幼虫解毒酶活性
Table 5Detoxification enzyme activities of S. mosellana larvae feeding on eight wheat varieties (lines)

小麦品种（系） Wheat variety (line)	GST活性 GST activity (U/mg protein)	CarE 活性 CarE activity (U/mg protein)	CYP450活性 CYP450 activity (U/mg protein)
陕麦139 Shaanmai 139	0.1633±0.0185bcd	0.2284±0.0054a	0.1446±0.0173bc
科农1006 Kenong 1006	0.2144±0.012a	0.1823±0.0103b	0.1124±0.0046c
晋麦47 Jinmai 47	0.1707±0.0242abc	0.1414±0.0087c	0.1248±0.0107c
陕农33 Shaannong 33	0.2038±0.0177ab	0.2116±0.0135a	0.1163±0.0065c
小偃6号 Xiaoyan 6	0.1913±0.0022ab	0.1784±0.0079b	0.1885±0.0038a
西农88 Xinong 88	0.0963±0.0055e	0.1016±0.0071d	0.1304±0.0143c
西农822 Xinong 822	0.1206 ±0.0101de	0.1074±0.0037d	0.1791±0.0217ab
小偃22 Xiaoyan 22	0.1333±0.0064cde	0.1242±0.0038cd	0.1306±0.0001c

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2.5 麦红吸浆虫幼虫体内解毒酶活性与小麦籽粒次生物质含量的关系

对取食不同小麦品种（系）的麦红吸浆虫幼虫3种解毒酶活性与小麦籽粒次生物质含量进行相关性分析,结果表明,GST和CarE活性与阿魏酸含量呈显著正相关（P<0.05）,与单宁、总酚和总黄酮含量相关性不显著（P>0.05）;CYP450活性与单宁含量呈显著负相关（P<0.05）,与阿魏酸、总酚和总黄酮含量相关性不显著（P>0.05）（表6）。

Table 6
表6
表6麦红吸浆虫幼虫体内解毒酶活性与小麦籽粒次生物质含量的相关性
Table 6Correlation between detoxification enzyme activities of S. mosellana larvae and contents of secondary metabolites in wheat kernels

解毒酶 Detoxification enzyme	阿魏酸Ferulic acid		单宁Tannin		总酚 Total phenol		总黄酮 Total flavonoid
	r	P	r	P	r	P	r	P
GST	0.805	0.016*	0.229	0.586	0.083	0.845	-0.375	0.361
CarE	0.717	0.045*	0.167	0.693	0.114	0.788	-0.412	0.311
CYP450	-0.608	0.110	-0.742	0.035*	-0.003	0.995	-0.682	0.063

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2.6 小麦品种（系）对麦红吸浆虫幼虫GST1、CarE2和CYP6A1表达的影响

取食不同小麦品种（系）的麦红吸浆虫幼虫GST1和CarE2表达量存在明显差异,其中取食抗虫品种晋麦47、科农1006、陕麦139和陕农33的表达水平显著高于取食感虫品种小偃22、西农822、西农88和小偃6号（P<0.05）。GST1表达量在取食陕农33的幼虫中最高,为陕麦139的1.42倍,显著高于其他品种;在取食小偃22的幼虫中最低,为陕麦139的0.15倍。CarE2表达量在取食陕麦139的幼虫中最高,显著高于取食其他品种,在取食小偃22的幼虫中最低,仅为取食陕麦139幼虫的0.07倍（图1-A、1-B）。

图1

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图1取食8个小麦品种（系）的麦红吸浆虫幼虫GST1、CarE2和CYP6A1相对表达量

SM139:陕麦139 Shaanmai139;KN1006:科农1006 Kenong1006;JM47:晋麦47 Jinmai47;SN33:陕农33 Shaannong 33;XY6:小偃6号Xiaoyan 6;XN88:西农88 Xinong 88;XN822:西农822 Xinong 822;XY22:小偃22 Xiaoyan 22。图中数据为3次重复的平均值±标准误,柱上不同字母表示经Duncan氏多重比较后差异显著（P<0.05）The data in the figure are mean±SE of three repetitions, different letters above bars indicate significant difference at 0.05 level by Duncan’s multiple range test
Fig. 1Relative expression level of GST1, CarE2 and CYP6A1 in S. mosellana larvae feeding on eight wheat varieties (lines)



取食不同抗虫性小麦品种（系）的麦红吸浆虫幼虫CYP6A1表达量没有表现出一定的规律,取食抗虫品种陕麦139、晋麦47和感虫品种小偃22、西农88的表达量较高且无显著差异,取食抗虫品种科农1006和感虫品种西农822的表达量较低且无显著差异（图1-C）。

2.7 取食不同小麦品种（系）的麦红吸浆虫解毒酶活性与基因表达水平的关系

相关性分析结果表明,取食不同小麦品种（系）的麦红吸浆虫幼虫GST1表达水平与GST活性（r=0.772,P=0.043）、CarE2表达水平与CarE活性均呈显著正相关（r=0.756,P=0.030）,CYP6A1表达水平与CYP450活性相关性不显著（r=0.558,P=0.150）。

3 讨论

植物次生物质是化学防御的物质基础,能影响或帮助植物抵御昆虫的侵害,植物品种间抗生性差异与体内各类型次生物质组成和含量密切相关^[6,7,8,9]。本研究发现小麦对麦红吸浆虫的抗性与小麦灌浆期籽粒单宁、总酚和总黄酮含量相关性不显著,但与阿魏酸含量呈显著正相关,即阿魏酸对吸浆虫抗性的贡献较单宁、总酚和总黄酮大,其含量越高,对吸浆虫抗性越强。这与小麦对麦长管蚜、玉米象及水稻对褐飞虱（Nilaparvata lugens）抗性与阿魏酸含量显著正相关的结果一致^[9,29-30]。研究表明,阿魏酸不仅是构成植物细胞壁的重要成分,而且可显著抑制麦二叉蚜（Schizaphis graminum）和斜纹夜蛾（Spodoptera litura）的取食和生存^[31,32],并能降低棉铃虫幼虫、蛹的体重和影响幼虫对食物的吸收与利用^[7]。据此推测,阿魏酸作为小麦抗麦红吸浆虫的关键次生物质,可能通过降低麦红吸浆虫对食物的吸收效率,抑制其取食和生长发育,以此达到抵御侵害的目的。

解毒酶及解毒机制在昆虫对植物次生物质的代谢和寄主适应中起着重要作用。作为昆虫重要的解毒酶,谷胱甘肽-S-转移酶（GST）可催化还原型谷胱甘肽与各种亲电有毒次生物进行加成反应,形成亲水性更强的无毒或低毒物而排出体外^[33];羧酸酯酶（CarE）则能催化含酯键、酰胺键的外源与内源次生代谢物质水解^[15]。许多研究证明寄主抗虫性和植物次生物质可影响解毒酶活性及相关基因表达,以此增加对次生物质的防御适应性。绿豆象（Callosobruchus chinensis）取食抗虫绿豆品种、麦长管蚜取食抗虫品种小麦后体内CarE和GST活性显著高于取食感虫品种^[34,35];饲喂阿魏酸可显著提高褐飞虱若虫GST和CarE活性及其编码基因NlGSTe1、NlGSTd1和NlCarE的表达^[30];用含0.5%单宁、0.2%芦丁或1%没食子酸的饲料饲喂斜纹夜蛾后,5龄幼虫中肠和脂肪体GST和CarE活性及SlGSTe1和SlCarE表达量显著增加^[12]。本研究表明,麦红吸浆虫幼虫取食阿魏酸含量高的抗虫品种小麦后,GST和CarE活性及GST1和CarE2表达量均显著高于取食感虫品种,GST和CarE活性与阿魏酸含量及对应基因表达水平显著正相关,与以上结果相符,说明GST和CarE及其基因GST1和CarE2在麦红吸浆虫对寄主小麦有毒次生物质的代谢和防御中起着重要作用。

细胞色素P450酶系（CYP450）在昆虫对植物次生物质的解毒代谢中也发挥了重要作用,许多研究报道了植物次生物质对昆虫CYP450活性的影响。萜类植物次生代谢物棉酚可诱导棉铃虫CYP450的活性,棉铃虫取食棉酚含量高的寄主植物后,CYP450活性显著升高^[36];吲哚-3-甲醇、花椒毒素、2-十三玩酮等多种植物次生物质可诱导草地贪夜蛾（Spodoptera frugiperda）幼虫CYP450的活性^[37]。与之不同,2-十三烷酮却通过降低棉铃虫6 龄幼虫CYP450活性,使其在含量较低的情况下具有较高的毒性^[38]。本研究发现,麦红吸浆虫幼虫CYP450活性与小麦籽粒单宁含量显著负相关,即单宁可降低CYP450活性。单宁具有靴化蛋白的特性,是多种酶促反应的抑制剂^[39,40],据此推测单宁可能与吸浆虫CYP450相关蛋白结合,通过抑制其活性和作用过程,阻止或降低其解毒代谢,使虫体生命力“弱化”而发挥毒杀作用。CYP6亚家族基因代谢解毒作用是昆虫寄主适应的重要机制,不同次生物质所诱导的CYP6不同。棉酚能够诱导棉铃虫CYP6AE11和CYP6B7过量表达,而槲皮素和单宁酸仅能诱导CYP6B6的表达^[36,41]。本研究发现单宁虽然能够抑制麦红吸浆虫CYP450的活性,但是对CYP6A1表达无显著影响,这可能是由于CYP450活性由多基因控制,特定的植物次生物质对不同CYP6诱导或抑制作用不同^[11,41-42],而CYP6A1可能不是麦红吸浆虫幼虫响应单宁胁迫的关键基因。

4 结论

小麦灌浆期籽粒阿魏酸是影响小麦对麦红吸浆虫抗性的主要次生物质,阿魏酸含量高的寄主能够诱导麦红吸浆虫幼虫GST和CarE活性及编码基因GST1和CarE2的表达,这些酶及基因在麦红吸浆虫对阿魏酸的解毒代谢和防御反应中发挥着重要作用。单宁却通过降低吸浆虫幼虫CYP450的活性使其在含量较低的情况下具有相对高的毒杀作用。

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[9] 韩宪琪, 李雪娇, 冯淑娟, 成卫宁, 朱克岩. 小麦籽粒营养物质和次生物质含量与其对玉米象抗性的关系
植物保护学报, 2017,44(5):721-728.
[本文引用: 3]

HAN X Q, LI X J, FENG S J, CHENG W N, ZHU K Y. Relationships between the contents of nutrients and secondary metabolites in wheat seeds and their resistance to Sitophilus zeamais (Coleoptera: Curculionidae)
Journal of Plant Protection, 2017,44(5):721-728. (in Chinese)
[本文引用: 3]

[10] MAINGUET A M, LOUVEAUX A, EL SAYED G, ROLLIN P. Ability of a generalist insect, Schistocerca gregaria, to overcome thioglucoside defense in desert plants: tolerance or adaptation?
Entomologia Experimentalis et Applicata, 2000,94(3):309-317.
[本文引用: 1]

[11] 陈澄宇, 康志娇, 史雪岩, 高希武. 昆虫对植物次生物质的代谢适应机制及其对昆虫抗药性的意义
昆虫学报, 2015,58(10):1126-1139.
[本文引用: 2]

CHEN C Y, KANG Z J, SHI X Y, GAO X W. Metabolic adaptation mechanisms of insects to plant secondary metabolites and their implications for insecticide resistance of insects
Acta Entomologica Sinica, 2015,58(10):1126-1139. (in Chinese)
[本文引用: 2]

[12] 黄敏燕, 李雪峰. 植物次生物质对斜纹夜蛾解毒酶活性的影响
基因组学与应用生物学, 2018,37(8):3495-3502.
[本文引用: 2]

HUANG M Y, LI X F. Effects of plant secondary metabolite on detoxification enzyme activity of Spodoptera litura
Genomics and Applied Biology, 2018,37(8):3495-3502. (in Chinese)
[本文引用: 2]

[13] VOGEL H, MUSSER R O, DE LA PAZ CELORIO-MANCERA M. Transcriptome responses in herbivorous insects towards host plant and toxin feeding
Annual Plant Reviews, 2014,47:197-233.
[本文引用: 1]

[14] ZHONG H, LI F, CHEN J, ZHANG J, LI F. Comparative transcriptome analysis reveals host-associated differentiation in Chilo suppressalis (Lepidoptera: Crambidae)
Scientific Reports, 2017,7:13778.
DOI:10.1038/s41598-017-14137-xURLPMID:29062034 [本文引用: 1]
The striped stem borer, Chilo suppressalis Walker (Lepidoptera: Crambidae), is one of the most serious rice pests. Besides attacking rice, it also feeds on an economically important vegetable crop, water-oat Zizania latifolia. The species feeding on water-oat has higher growth and survival rate than those on rice, suggesting their success in adaptation to the new host plant. However, little is known about the molecular mechanisms of host plant adaptation. Here we investigated the midgut transcriptome responses of C. suppressalis larvae reared on rice and water-oat. A total of 1,633 differentially expressed genes were identified, with a greater number up-regulated on the more delicious new host. The up-regulation of most digestive and detoxification-related genes may be the result of adaptation to the changes in nutritional requirements and toxic chemicals during host shift. In contrast, down-regulation of ribosomal genes may be related to their better development performance when feeding on the new host. In conclusion, our results suggest that transcriptional regulation of genes related to digestion, detoxification and ribosome may play an important role in adaptation of C. suppressalis to a new host plant.

[15] ZHANG M, FANG T, PU G, SUN X, ZHOU X, CAI Q. Xenobiotic metabolism of plant secondary compounds in the English grain aphid,
Sitobion avenae (F.), 2013,107(1):44-49.
[本文引用: 2]

[16] CHENG H, TANG F, LI W, XU M. Tannic acid induction of a glutathione S-transferase in Micromelalopha troglodyta (Lepidoptera: Notodontidae) larvae
Journal of Entomological Science, 2015,50(4):350-362.
[本文引用: 1]

[17] HUANG X B, MA J C, QIN X H, TU X B, CAO G C, WANG G J, NONG X Q, ZHANG Z H. Biology, physiology and gene expression of grasshopper Oedaleus asiaticus exposed to diet stress from plant secondary compounds
Scientific Reports, 2017,7:8655.
[本文引用: 1]

[18] LAMB R J, MCKENZIE R I H, WISE I L, BARKER P S, SMITH M A H, OLFERT O O. Resistance to Sitodiplosis mosellana ( Diptera: Cecidomyiidae) in spring wheat (Gramineae)
The Canadian Entomologist, 2000,132(5):591-605.
[本文引用: 2]

[19] 屈振刚, 温树敏, 屈赟, 刘桂茹. 小麦品种抗麦红吸浆虫鉴定与抗性分析
植物遗传资源学报, 2011,12(1):121-124.
[本文引用: 1]

QU Z G, WEN S M, QU Y, LIU G R. Evaluation and identification of wheat varieties resistant to Sitodiplosis mosellana
Journal of Plant Genetic Resources, 2011,12(1):121-124. (in Chinese)
[本文引用: 1]

[20] 郝亚楠, 张箭, 龙治任, 王越, 成卫宁. 小麦品种(系)对麦红吸浆虫抗性指标筛选与抗性评价
昆虫学报, 2014,57(11):1321-1327.
[本文引用: 3]

HAO Y N, ZHANG J, LONG Z R, WANG Y, CHENG W N. Screening of resistance indicators and evaluation of the resistance of wheat varieties to orange wheat blossom midge, Sitodiplosis mosellana (Diptera: Cecidomyiidae)
Acta Entomologica Sinica, 2014,57(11):1321-1327. (in Chinese)
[本文引用: 3]

[21] ABDEL-AAL E S M, HUCL P, SOSULSKI F W, GRAF R, GILLOTT C, PIETRZAK L. Screening spring wheat for midge resistance in relation to ferulic acid content
Journal of Agricultural and Food Chemistry, 2001,49(8):3559-3566.
DOI:10.1021/jf010027hURLPMID:11513628 [本文引用: 1]
The concentration of ferulic acid (FA), the major phenolic acid in the wheat kernel, was found to differ significantly in the mature grain of six wheat cultivars known to have a range of tolerance to the orange wheat blossom midge (Sitodiplosis mosellana). Differences in FA content were correlated with floret infestation level of the cultivars. The wheat cultivars ranked similarly in FA content at the four locations where they were tested, despite a significant effect of environment. Ferulic acid was synthesized mainly during the early stages of grain filling but at different rates among cultivars. Ferulic acid was concentrated primarily in the shorts and bran fractions in an insoluble-bound form. A high correlation was obtained between FA contents as determined by GLC, fluorometry, UV, and colorimetry. The colorimetric procedure was modified as a qualitative, simple, and rapid test for identifying midge-resistant wheat and evaluated in several field trials. The method should provide a rapid tool in the preliminary screening of experimental lines in the development of midge-resistant wheat cultivars.

[22] 史忠良, 仇松英, 马爱萍, 许钢垣, 武计平, 逯腊虎. 冬小麦对麦红吸浆虫抗性机制研究初报
华北农学报, 2003,18(1):100-102.
[本文引用: 1]

SHI Z L, QIU S Y, MA A P, XU G Y, WU J P, LU L H. Studies on the resistance mechanism of wheat to blossom midge
Acta Agriculturae Boreali-Sinica, 2003,18(1):100-102. (in Chinese)
[本文引用: 1]

[23] 王丹, 杜金华. 比色法快速测定小麦及其麦芽中的阿魏酸
食品与发酵工业, 2011,37(1):146-151.
[本文引用: 1]

WANG D, DU J H. Rapid determination of ferulic aciol in wheats and malts by colorimetry
Food and Fermentation Industries, 2011,37(1):146-151. (in Chinese)
[本文引用: 1]

[24] MAHBOUBI A, ASGARPANAH J, SADAGHIYANI P N, FAIZI M. Total phenolic and flavonoid content and antibacterial activity of Punica granatum L. var. pleniflora flowers (Golnar) against bacterial strains causing foodborne diseases
BMC Complementary and Alternative Medicine, 2015,15:366.
URLPMID:26470879 [本文引用: 1]

[25] SHENG Z L, WAN P F, DONG C L, LI Y H. Optimization of total flavonoids content extracted from Flos populi using response surface methodology
Industrial Crops and Products, 2013,43(1):778-786.
[本文引用: 1]

[26] 申君, 鲁艳辉, 张淑真, 游红, 李建洪. 亚致死浓度氰氟虫腙对小菜蛾三种解毒酶及P450 mRNA表达量的影响
应用昆虫学报, 2016,53(2):256-263.
[本文引用: 1]

SHEN J, LU Y H, ZHANG S Z, YOU H, LI J H. Effects of sublethal concentrations of metaflumizone on detoxifying enzyme activity, and expression of cytochrome P450 genes, in Plutella xylostella (L.)
Chinese Journal of Applied Entomology, 2016,53(2):256-263. (in Chinese)
[本文引用: 1]

[27] BRADFORD M M. A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye binding
Analytical Biochemistry, 1976,72(1/2):248-254.
[本文引用: 1]

[28] LIVAK K J, SCHMITTGEN T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2^-ΔΔCT method
Methods, 2001,25(4):402-408.
DOI:10.1006/meth.2001.1262URLPMID:11846609 [本文引用: 1]
The two most commonly used methods to analyze data from real-time, quantitative PCR experiments are absolute quantification and relative quantification. Absolute quantification determines the input copy number, usually by relating the PCR signal to a standard curve. Relative quantification relates the PCR signal of the target transcript in a treatment group to that of another sample such as an untreated control. The 2(-Delta Delta C(T)) method is a convenient way to analyze the relative changes in gene expression from real-time quantitative PCR experiments. The purpose of this report is to present the derivation, assumptions, and applications of the 2(-Delta Delta C(T)) method. In addition, we present the derivation and applications of two variations of the 2(-Delta Delta C(T)) method that may be useful in the analysis of real-time, quantitative PCR data.

[29] 胡远, 韩颖, 赵欣, 杨晓琴, 黄永, 罗盘, 蔡青年. 小麦不同抗蚜品种中3种酚酸类化合物的含量变化及其作用评价
应用与环境生物学报, 2008,14(6):753-756.
[本文引用: 1]

HU Y, HAN Y, ZHAO X, YANG X Q, HUANG Y, LUO P, CAI Q N. Dynamics and effect evaluation of three phenolic compound contents in wheat varieties with different resistances to Sitobion avenae
Chinese Journal of Applied and Environmental Biology, 2008,14(6):753-756. (in Chinese)
[本文引用: 1]

[30] YANG J, SUN X Q, YAN S Y, PAN W J, ZHANG M X, CAI Q N. Interaction of ferulic acid with glutathione S-transferase and carboxylesterase genes in the brown planthopper, Nilaparvata lugens
Journal of Chemical Ecology, 2017,43(7):693-702.
URLPMID:28647840 [本文引用: 2]

[31] CABRERA H M, MU?OZ O, Zú?IGA G E, CORCUERA L J, ARGANDO?A V H. Changes in ferulic acid and lipid content in aphid-infested barley
Phytochemistry, 1995,39(5):1023-1026.
DOI:10.1016/0031-9422(95)00065-FURL [本文引用: 1]

[32] SU Q, ZHOU Z, ZHANG J, SHI C, ZHANG G, JIN Z, WANG W, LI C. Effect of plant secondary metabolites on common cutworm, Spodoptera litura (Lepidoptera: Noctuidae)
Entomological Research, 2018,48(1):18-26.
DOI:10.1111/enr.2018.48.issue-1URL [本文引用: 1]

[33] SHEEHAN D, MEADE G, FOLEY V M, DOWD C A. Structure, function and evolution of glutathione transferases: Implications for classification of non-mammalian members of an ancient enzyme superfamily
Biochemical Journal, 2001,360(1):1-16.
DOI:10.1042/bj3600001URL [本文引用: 1]

[34] 蔡青年, 张青文, 高希武, 王宇, 周明牂. 小麦体内次生物质对麦蚜的抗性作用研究
中国农业科学, 2003,36(8):910-915.
URL [本文引用: 1]
采用蚜量比值法评价了 7个小麦品种 (系 )对麦长管蚜的抗性 ,并用紫外分光光度法测定了各品种 (系 )旗叶和穗部总酚和吲哚生物碱含量 ,并通过酶标仪法测定了来自不同抗级小麦穗部麦长管蚜体内的羧酸酯酶活性的差异 ,以此研究小麦体内次生物质含量的差异及其抗麦长管蚜的关系和抗性生化机制。结果表明 ,旗叶和穗部总酚和吲哚生物碱含量不同 ,表现出对麦长管蚜种群影响的差异。穗部吲哚生物碱含量与麦长管蚜蚜量比值之间呈极显著的负相关 ,相关指数R =- 0 .9896。而旗叶吲哚生物碱、旗叶总酚和穗部总酚含量与麦长管蚜蚜量比值的相关系数分别为 - 0 .6 82 6、- 0 .4 2 0 8和 - 0 .5 6 2 3,相关性均不显著。取食不同抗蚜品种 (系 )穗部的蚜虫羧酸酯酶活性与穗部吲哚生物碱含量呈显著相关性 (R =0 .96 4 6 ) ,而与穗部总酚含量相关性不显著 (R =0 .4 95 3)。与总酚含量相比 ,小麦穗部吲哚生物碱含量高低与小麦对麦长管蚜抗性关系更密切。
CAI Q N, ZHANG Q W, GAO X W, WANG Y, ZHOU M Z. Effects of the secondary substances on wheat resistance to Sitobion avenae (F.)
. Scientia Agricultura Sinica, 2003,36(8):910-915. (in Chinese)
URL [本文引用: 1]
采用蚜量比值法评价了 7个小麦品种 (系 )对麦长管蚜的抗性 ,并用紫外分光光度法测定了各品种 (系 )旗叶和穗部总酚和吲哚生物碱含量 ,并通过酶标仪法测定了来自不同抗级小麦穗部麦长管蚜体内的羧酸酯酶活性的差异 ,以此研究小麦体内次生物质含量的差异及其抗麦长管蚜的关系和抗性生化机制。结果表明 ,旗叶和穗部总酚和吲哚生物碱含量不同 ,表现出对麦长管蚜种群影响的差异。穗部吲哚生物碱含量与麦长管蚜蚜量比值之间呈极显著的负相关 ,相关指数R =- 0 .9896。而旗叶吲哚生物碱、旗叶总酚和穗部总酚含量与麦长管蚜蚜量比值的相关系数分别为 - 0 .6 82 6、- 0 .4 2 0 8和 - 0 .5 6 2 3,相关性均不显著。取食不同抗蚜品种 (系 )穗部的蚜虫羧酸酯酶活性与穗部吲哚生物碱含量呈显著相关性 (R =0 .96 4 6 ) ,而与穗部总酚含量相关性不显著 (R =0 .4 95 3)。与总酚含量相比 ,小麦穗部吲哚生物碱含量高低与小麦对麦长管蚜抗性关系更密切。

[35] 成小芳, 张耀文, 张仙红. 抗虫绿豆对绿豆象生长发育及体内几种酶活性的影响
植物保护学报, 2017,44(3):420-426.
[本文引用: 1]

CHENG X F, ZHANG Y W, ZHANG X H. Effects of insect-resistant mung bean on development and activities of several enzymes of Chinese bean weevil Callosobruchus chinensis L. (Coleoptera: Bruchidae)
Journal of Plant Protection, 2017,44(3):420-426. (in Chinese)
[本文引用: 1]

[36] TAO X Y, XUE Y X, HUANG Y P, CHEN X Y, MAO Y B. Gossypol-enhanced P450 gene pool contributes to cotton bollworm tolerance to a pyrethroid insecticide
Molecular Ecology, 2012,21(17):4371-4385.
DOI:10.1111/j.1365-294X.2012.05548.xURL [本文引用: 2]
Cotton plants accumulate phytotoxins, including gossypol and related sesquiterpene aldehydes, to resist insect herbivores and pathogens. To counteract these defensive plant secondary metabolites, cotton bollworms (Helicoverpa armigera) elevate their production of detoxification enzymes, including cytochrome P450 monooxygenases (P450s). Besides their tolerance to phytotoxin, cotton bollworms have quickly developed resistance to deltamethrin, a widely used pyrethroid insecticide in cotton field. However, the relationship between host plant secondary metabolites and bollworm insecticide resistance is poorly understood. Here, we show that exogenously expressed CYP6AE14, a gossypol-inducible P450 of cotton bollworm, has epoxidation activity towards aldrin, an organochlorine insecticide, indicating that gossypol-induced P450s participate in insecticide metabolism. Gossypol-ingested cotton bollworm larvae showed higher midgut P450 enzyme activities and exhibited enhanced tolerance to deltamethrin. The midgut transcripts of bollworm larvae administrated with different phytochemicals and deltamethrin were then compared by microarray analysis, which showed that gossypol and deltamethrin induced the most similar P450 expression profiles. Gossypol-induced P450s exhibited high divergence and at least five of them (CYP321A1, CYP9A12, CYP9A14, CYP6AE11 and CYP6B7) contributed to cotton bollworm tolerance to deltamethrin. Knocking down one of them, CYP9A14, by plant-mediated RNA interference (RNAi) rendered the larvae more sensitive to the insecticide. These data demonstrate that generalist insects can take advantage of secondary metabolites from their major host plants to elaborate defence systems against other toxic chemicals, and impairing this defence pathway by RNAi holds a potential for reducing the required dosages of agrochemicals in pest control.

[37] GIRAUDO M, HILLIOU F, FRICAUX T, AUDANT P, FEYEREISEN R, LE GOFF G. Cytochrome P450s from the fall armyworm (Spodoptera frugiperda): Responses to plant allelochemicals and pesticides
Insect Molecular Biology, 2015,24(1):115-128.
[本文引用: 1]

[38] 于彩虹, 高希武, 郑炳宗. 2-十三烷酮对棉铃虫细胞色素P450的诱导作用
昆虫学报, 2002,45(1):1-7.
URL [本文引用: 1]
将2-十三烷酮按0.005%～0.01%（重量比）的浓度加到棉铃虫人工饲料中，连续诱导3代，测定棉铃虫中肠和脂肪体中细胞色素P450(cyt-P450)含量以及与标准配基（正丁醇、吡啶、苯胺、环己烷）形成的氧化型结合光谱。2-十三烷酮诱导品系的中肠cyt-P450与CO结合光谱的最大吸收峰在449 nm处，脂肪体cyt-P450与CO结合光谱的最大吸收峰在450.7 nm处。中肠cyt-P450除了在450 nm附近存在一个吸收峰外，在通入CO后依次在414、415、418 nm附近出现吸收峰，随后该峰消失，随着时间的推移（第31次扫描）在420 nm处又开始出现一个弱吸收峰。2-十三烷酮诱导品系的中肠、脂肪体cyt-P450与4种标准配基形成的差光谱与对照相比在峰型上存在着不同程度的差异。中肠cyt-P450与正丁醇形成双峰双谷的光谱；脂肪体cyt-P450与正丁醇形成的光谱最大吸收峰在416.61 nm处，波谷在424.91 nm处；中肠cyt-P450和脂肪体cyt-P450与吡啶形成的光谱为典型的Ⅱ型光谱，而与环己烷形成的光谱为不典型Ⅰ型光谱；中肠和脂肪体的cyt-P450与苯胺形成典型的Ⅱ型光谱，最大吸收峰分别在443.30和428.92 nm处，最小吸收分别在402.30和401.00 nm处。
YU C H, GAO X W, ZHENG B Z. Induction of the cytochrome P450 by 2-tridecanone in Helicoverpa armigera
Acta Entomologica Sinica, 2002,45(1):1-7. (in Chinese)
URL [本文引用: 1]
将2-十三烷酮按0.005%～0.01%（重量比）的浓度加到棉铃虫人工饲料中，连续诱导3代，测定棉铃虫中肠和脂肪体中细胞色素P450(cyt-P450)含量以及与标准配基（正丁醇、吡啶、苯胺、环己烷）形成的氧化型结合光谱。2-十三烷酮诱导品系的中肠cyt-P450与CO结合光谱的最大吸收峰在449 nm处，脂肪体cyt-P450与CO结合光谱的最大吸收峰在450.7 nm处。中肠cyt-P450除了在450 nm附近存在一个吸收峰外，在通入CO后依次在414、415、418 nm附近出现吸收峰，随后该峰消失，随着时间的推移（第31次扫描）在420 nm处又开始出现一个弱吸收峰。2-十三烷酮诱导品系的中肠、脂肪体cyt-P450与4种标准配基形成的差光谱与对照相比在峰型上存在着不同程度的差异。中肠cyt-P450与正丁醇形成双峰双谷的光谱；脂肪体cyt-P450与正丁醇形成的光谱最大吸收峰在416.61 nm处，波谷在424.91 nm处；中肠cyt-P450和脂肪体cyt-P450与吡啶形成的光谱为典型的Ⅱ型光谱，而与环己烷形成的光谱为不典型Ⅰ型光谱；中肠和脂肪体的cyt-P450与苯胺形成典型的Ⅱ型光谱，最大吸收峰分别在443.30和428.92 nm处，最小吸收分别在402.30和401.00 nm处。

[39] 陈巨莲, 倪汉祥, 孙京瑞, 程登发. 小麦几种主要次生物质对麦长管蚜几种酶活力的影响
昆虫学报, 2003,46(2):144-149.
URL [本文引用: 1]
借助麦蚜人工饲料研究明确了小麦几种主要次生物质单宁酸、总酚（没食子酸）和香豆素对麦长管蚜Sitobion avenae的存活、生长和发育有明显的抑制作用，其抗蚜阈值浓度分别为0.06%、0.08%和0.065%。用昆虫酶系体外抑制法，研究上述三种次生物质的抗蚜阈值浓度对麦长管蚜的糖转化酶和解毒酶活力的影响，结果表明: 0.06%单宁酸强烈抑制麦长管蚜蔗糖酶、海藻糖酶、羧酸酯酶和谷胱甘肽S-转移酶的活力；0.08%没食子酸显著抑制蔗糖酶、羧酸酯酶和谷胱甘肽S-转移酶的活力；0.065%香豆素极显著地抑制谷胱甘肽S-转移酶活力。
CHEN J L, NI H X, SUN J R, CHENG D F. Effects of major secondary chemicals of wheat plants on enzyme activity in Sitobion avenae
Acta Entomologica Sinica, 2003,46(2):144-149. (in Chinese)
URL [本文引用: 1]
借助麦蚜人工饲料研究明确了小麦几种主要次生物质单宁酸、总酚（没食子酸）和香豆素对麦长管蚜Sitobion avenae的存活、生长和发育有明显的抑制作用，其抗蚜阈值浓度分别为0.06%、0.08%和0.065%。用昆虫酶系体外抑制法，研究上述三种次生物质的抗蚜阈值浓度对麦长管蚜的糖转化酶和解毒酶活力的影响，结果表明: 0.06%单宁酸强烈抑制麦长管蚜蔗糖酶、海藻糖酶、羧酸酯酶和谷胱甘肽S-转移酶的活力；0.08%没食子酸显著抑制蔗糖酶、羧酸酯酶和谷胱甘肽S-转移酶的活力；0.065%香豆素极显著地抑制谷胱甘肽S-转移酶活力。

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