Screening and application of reference genes for qRT-PCR in bamboo wireworm

YE Bihuan; CHEN Youwu; SHU Jinping; ZHANG Wei; ZHANG Yabo; LI Haibo; SONG Qiyan

doi:10.11833/j.issn.2095-0756.20200492

Volume 38 Issue 3

Jun. 2021

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Article Navigation > Journal of Zhejiang A&F University > 2021 > 38(3): 644-651

YE Bihuan, CHEN Youwu, SHU Jinping, ZHANG Wei, ZHANG Yabo, LI Haibo, SONG Qiyan. Screening and application of reference genes for qRT-PCR in bamboo wireworm[J]. Journal of Zhejiang A&F University, 2021, 38(3): 644-651. doi: 10.11833/j.issn.2095-0756.20200492

Citation:

YE Bihuan, CHEN Youwu, SHU Jinping, ZHANG Wei, ZHANG Yabo, LI Haibo, SONG Qiyan. Screening and application of reference genes for qRT-PCR in bamboo wireworm[J]. Journal of Zhejiang A&F University, 2021, 38(3): 644-651. doi: 10.11833/j.issn.2095-0756.20200492

Screening and application of reference genes for qRT-PCR in bamboo wireworm

doi: 10.11833/j.issn.2095-0756.20200492

1.
Zhejiang Academy of Forestry, Hangzhou 310023, Zhejiang, China
2.
Institute of Subtropical Forestry, Chinese Academy of Forestry, Hangzhou 311400, Zhejiang, China

Received Date: 2020-07-28
Rev Recd Date: 2020-11-26

Available Online: 2020-12-23

Publish Date: 2021-06-09

Abstract

Objective The objective of this study was to screen the stable expression of internal reference genes of Melanotus cribricollis larvae infected by Metarhizium pingshaense, so as to lay a foundation for the research on related gene expression in this bamboo wireworm. Method Based on the transcriptome data of M. cribricollis larvae, the correlation (R²) and amplification efficiency were analyzed by qRT-PCR with specific primers. The 6 candidate reference genes including β-actin, GAPDH, α-tubulin, RPL13α, RPS3 and RPS27a were evaluated by GeNorm, NormFinder and BestKeeper softwares. The stabilities of selected candidate reference genes including PRS27a and RPS3 were further validated by analyzing the expression of 6 target genes. Result GeNorm analysis showed that the expression of PRS27a and RPS3 were the most stable, followed by α-tubulin, RPL13α, β-actin and GAPDH. The most suitable number of internal reference genes was 2. NormFinder analysis showed that the expression of RPL13α was the most stable, followed by α-tubulin, RPS3, RPS27a, β-actin, and GAPDH. BestKeeper analysis showed that the P values of β-actin and GAPDH were ＞0.5, which were not suitable for reference genes under the condition of this experiment. There were some differences in the ranking of candidate internal parameters obtained by different software analysis. Comprehensive analysis and expression stability verification showed that PRS27a or RPS3 were the best internal reference genes, and the expression levels of 6 target genes were basically the same. Conclusion PRS27a and RPS3 were the most appropriate reference genes for qRT-PCR analysis in bamboo wireworm infected by M. pingshaense. [Ch, 3 fig. 3 tab. 27 ref.]
- forest protection,
- bamboo wireworm,
- Metarhizium pingshaense,
- reference gene,
- quantitative real-time PCR(qRT-PCR)

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竹林金针虫是取食竹笋的叩甲幼虫的总称，虫害发生时竹笋产量显著减少，严重时竹种无法成竹，制约了竹林的自然更新^[1-2]。筛胸梳爪叩甲Melanotus cribricollis隶属于鞘翅目Coleoptera叩甲科 Elateridae梳爪叩甲属Melanotus，其幼虫为中国南方早园竹Phyllostachys propinqua竹林的优势种。化学药剂防治竹林金针虫易造成环境污染、抗药性和食品安全等问题，阻碍以“生态、健康、有机”为标签的林产品产业发展，因此近年来逐渐采用生物技术防治该虫害。绿僵菌Metarhizium sp.是常见的昆虫致病真菌^[3-4]，对鞘翅目和鳞翅目Lepidoptera等害虫均有一定防效^[5-6]。研究竹林金针虫抗绿僵菌相关基因的表达有利于深入了解害虫免疫抗性机制和虫菌互作机制，也有利于生防菌菌株改造等研究，提高其对农林害虫防治的效果。实时荧光定量聚合酶链式反应(qRT-PCR)是研究基因表达分析的关键技术，省时、高效、应用广泛；为保证目的基因定量表达结果的准确性，需引入表达稳定的内参基因作为参照^[7]。内参基因又称看家基因，理想的内参基因其表达水平应在各条件下保持稳定，而实际上不同条件下许多内参基因的表达并不稳定^[8-9]。因此，即使是同一物种，不同试验条件下的内参基因也不能一概而用，qRT-PCR试验之前应对所选内参基因的表达稳定性进行评估。常用的qRT-PCR内参基因包括ACT (肌动蛋白)、TUB (微管蛋白)、GAPDH (甘油醛-3-磷酸脱氢酶)、UBC (泛素结合酶)、18S rRNA (核糖体蛋白S18)、28S rRNA (核糖体蛋白S28)、EF1 (延伸因子1)、SYN1 (突触融合蛋白1)、SYN6 (突触融合蛋白6)、RPS3 (核糖体蛋白S3)、PRS20 (泛素-核糖体蛋白S20)、PRS27a (泛素-核糖体蛋白S27a)、RPL10 (核糖体蛋白L10)、RPL13α (核糖体蛋白S13α)、AK (精氨酸激酶)和V-ATPase (液泡型ATP合酶)等。符伟等^[10]分析了小菜蛾Plutella xylostella候选内参基因在Bt毒素诱导时的表达稳定性，最终筛选出由PRS13、RPL32和 EF1α组成的最佳内参基因组合。冯波等^[11] 筛选出最适合校正松墨天牛Monochamus alternatus化学感受组织基因表达的GAPDH和TUB内参基因组合。刘金泊^[12]评价了赤拟谷盗Tribolium castaneum的候选内参基因在磷化氢诱导条件下的表达稳定性，筛选出RPS18和 RPL13α最佳内参基因组合。杨苓等^[13]筛选出了适合桃蛀螟Conogethes punctiferalis不同发育时期和不同组织的基因表达研究的2组最佳内参组合(RP49和GAPDH，RPL13和RP49)。陶蓉等^[14]则分别筛选出了美国白蛾Hyphantria cunea不同发育阶段、不同温度和不同组织的3组最佳内参基因(RPL12和EF1β，EF1α和GAPDH，ACT和RPS16)。目前，关于筛胸梳爪叩甲幼虫内参基因的筛选评估尚未见报道。本研究对平沙绿僵菌Metarhizium pingshaense侵染不同时期的筛胸梳爪叩甲幼虫qRT-PCR内参基因进行了筛选和评估，以期选出最佳的内参基因，为今后开展竹林金针虫等昆虫的基因表达研究提供参考。

3. 讨论

实时荧光定量PCR具有快速、准确和灵敏的特性，在基因表达分析研究中应用广泛，然而表达稳定的内参基因选择是获得目的基因准确表达结果的前提条件。同一内参基因在不同物种、组织、发育阶段和处理条件下的表达稳定性也存在差异^[8-9]。目前尚未见有关筛胸梳爪叩甲幼虫内参基因的研究报道。本研究首次对该虫在平沙绿僵菌侵染条件下的候选内参基因进行了筛选评估。GeNorm、NormFinder和BestKeeper软件对候选内参基因的分析结果表明：PRS27a和RPS3的表达稳定。本研究以PGRP、Prx、SDR(1)、SDR(2)、SDR(3)和 SDR(4)为目的基因对PRS27a和RPS3内参基因进行了验证，结果显示：分别以这2个基因为内参时，6个目的基因的表达趋势一致，证明了这2个基因作为内参的可靠性。PGRP隶属于模式识别受体蛋白(pattern recognition receptor proteins，PRRs)家族，可与革兰氏阳性菌和革兰氏阴性菌的肽聚糖结合，激活Toll和Imd信号转导通路，诱导免疫防御相关的水解级联反应，发生吞噬作用^[22-23]。以PRS27a和RPS3为内参时，目的基因PGRP在筛胸梳爪叩甲幼虫体内被平沙绿僵菌诱导表达，7 d时较对照组显著升高，该现象与赤拟谷盗和油菜花露尾甲Meligethes aeneus PGRP基因被寄生物或病原菌诱导后表达上调相似^[24-25]。Prx基因编码过氧化物还原酶(peroxiredoxin)，是抗氧化家族中的一员，能够通过消除细胞内的活性氧(ROS)以维持氧化还原平衡^[26]。以PRS27a和RPS3为内参时，目的基因Prx整体表达呈上升趋势，推测其可能在病程后期发挥作用。SDR蛋白(short-chain dehydrogenases/reductases，短链脱氢还原酶)是一个超级家族，由多种酶编码基因家族组成，在多种生物合成途径、细胞信号通路和病理途径中都扮演重要角色^[27]。本研究发现：筛胸梳爪叩甲幼虫4个SDR基因[SDR(1)、SDR(2)、SDR(3)和 SDR(4)]对绿僵菌侵染的响应机制不同，可能是因为参与过程不同。由上可见，以PRS27a和RPS3为内参时，筛胸梳爪叩甲幼虫PGRP、Prx和SDR这3类基因的表达模式符合赤拟谷盗和油菜花露尾甲等昆虫对病原菌的响应机制，进一步证实了该内参组合适用于绿僵菌侵染条件下的目的基因表达水平研究。

本研究首次报道了在平沙绿僵菌侵染条件下筛胸梳爪叩甲幼虫可用的内参基因(PRS27a和RPS3)，为后续研究绿僵菌和竹林金针虫互作相关基因的表达奠定基础，也对其他昆虫基因表达以及虫菌互作相关研究提供参考。

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基因	引物序列	产物长度/bp	扩增效率/%	R²
β-actin	F：GGATACCTCTTTTGCTCTGGG，R：ATCAGGGTGTCATGGTTGG	75	112.48	0.993
GAPDH	F：CTACTCATGGTCGTTACAAGGG，R：TTCTACAACGTATTCAGCTCCAG	140	101.79	0.993
α-tubulin	F：GAAGCTCGTGAAGATTTGGC，R：ACCTTCGCCTTCTCCTTCTC	137	100.63	0.996
RPL13α	F：CTGAGGAAGAGCGTAAGGTG，R：TCAGCACGAGCCTTTCTTAAG	145	106.97	0.990
RPS27a	F：CTTGTCCTGAATCTTTGCCTTG，R：GTTCTTTTGGTAGCGTGTCATG	146	96.20	0.998
RPS3	F：CAATAGCGCACAAACCACG，R：TGTATTGGGTGAAAAGGGAAGG	128	111.93	0.992
PGRP	F：TGTCGTACTTCTGGCTATCATTG，R：TGTGATGGAGGGTTTACTTGC	123	88.69	0.989
Prx	F：CTATCCCTTAGACTTCACCTTCG，R：ATTTCTCCCAAACCTCCCTG	171	106.67	0.996
SDR(1)	F：CGGCATTGACGGAAACTTTAC，R：GGTTTCCACAGACTTTTGCG	126	109.16	0.995
SDR(2)	F：AGGTGCTAGTTCGGGAATTG，R：CGTGTAATTTGCCAGGTTTTCC	140	99.49	0.997
SDR(3)	F：GGATTACGAGCATAAGTCCTGG，R：CGGCGATGTCTTCAGATTTTAAC	126	106.58	0.996
SDR(4)	F：TTAGGGTTTCAGTCAAGGCAG，R：GAAGCCGTCCAAGATATGAAAG	148	93.34	0.994

基因	GeNorm		NormFinder		BestKeeper
基因	稳定值	排名	稳定值	排名	标准差	P	排名
β-actin	0.391	4	0.244	5	0.27	0.313	6
GAPDH	0.426	5	0.293	6	0.16	0.261	5
α-tubulin	0.242	2	0.177	2	0.24	0.042	4
RPL13α	0.319	3	0.164	1	0.32	0.024	3
RPS27a	0.197	1	0.206	4	0.35	0.012	2
RPS3	0.197	1	0.202	3	0.39	0.002	1

V_n/n+1	配对变异值	V_n/n+1	配对变异值
V_2/3	0.084	V_4/5	0.092
V_3/4	0.092	V_5/6	0.074
说明：n表示内参基因数目

Screening and application of reference genes for qRT-PCR in bamboo wireworm

doi: 10.11833/j.issn.2095-0756.20200492

Abstract

References

Proportional views

通讯作者: 陈斌, bchen63@163.com

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Proportional views