牧草盲蝽实时定量PCR内参基因的筛选

doi:10.16380/j.kcxb.2019.12.004

昆虫学报 ›› 2019, Vol. 62 ›› Issue (12): 1379-1391.doi: 10.16380/j.kcxb.2019.12.004

牧草盲蝽实时定量PCR内参基因的筛选

贾冰¹, 马亿¹, 庞保平¹, 单艳敏², 鲍青龙³, 韩海斌⁴, 谭瑶^1,*

(1. 内蒙古农业大学草原昆虫研究中心, 呼和浩特 010020; 2. 内蒙古草原工作站, 呼和浩特 010020; 3. 赤峰市草原工作站, 内蒙古赤峰 024000; 4. 中国农业科学院草原研究所, 呼和浩特 010019)

出版日期:2019-12-20 发布日期:2019-12-12

Screening of reference genes for quantitative real-time PCR in Lygus pratensis (Hemiptera: Miridae)

JIA Bing¹, MA Yi¹, PANG Bao-Ping¹, SHAN Yan-Min², BAO Qing-Long³, HAN Hai-Bin⁴, TAN Yao^1,*

(1. Research Center of Grassland Entomology, Inner Mongolia Agricultural University, Hohhot 010020, China; 2. Inner Mongolia Grassland Station, Hohhot 010020, China; 3. Chifeng Grassland Station, Chifeng, Inner Mongolia 024000, China; 4. Institute of Grassland Research, Chinese Academy of Agricultural Sciences, Hohhot 010019, China)

Online:2019-12-20 Published:2019-12-12

摘要/Abstract

摘要： 【目的】筛选出适合牧草盲蝽Lygus pratensis在不同条件下稳定表达的内参基因。【方法】选取编码牧草盲蝽α-微管蛋白、β-微管蛋白、肌动蛋白、延伸因子、琥珀酸脱氢酶复合体A、泛素、转录起始因子TFIID亚基、谷胱甘肽S-转移酶、核糖体蛋白L32及TATA盒结合蛋白的10条基因为候选内参基因，采用qRT-PCR技术测定其在牧草盲蝽不同性别成虫、成虫不同组织、不同龄期、对功夫菊酯不同抗性成虫、不同温度及不同杀虫剂分别处理后的成虫共6类样品中的表达量；采用BestKeeper, geNorm, NormFinder及RefFinder 4种计算程序对各候选基因的表达稳定性进行评价。【结果】依据获得的有效qRT-PCR数据前提，利用不同计算方法综合分析得出：在牧草盲蝽成虫不同组织中和对功夫菊酯不同抗性成虫中最稳定表达的内参基因均为RPL32；不同温度处理、不同性别成虫中、不同杀虫剂处理和不同龄期牧草盲蝽中最稳定表达的内参基因分别为UBQ, SDHA, β-tubulin和TAF。通过geNorm软件判断配对变异数确定的内参基因最佳数目，结合RefFinder对基因表达稳定性综合排序的结果可得：牧草盲蝽不同成虫组织中、不同性别成虫中、不同龄期、对功夫菊酯不同抗性成虫中、不同温度及杀虫剂分别处理的成虫等各组的最优内参基因组合分别为RPL32+TAF, SDHA+GST, TAF+GST+UBQ, RPL32+GST, UBQ+ACT+β-tubulin和β-tubulin+TAF+RPL32。【结论】本研究获得的牧草盲蝽在不同条件下表达最稳定及最适内参基因组合，都可用于后续的基因定量表达研究。

关键词: 牧草盲蝽, qRT-PCR, 内参基因, 基因表达分析, 表达稳定性

Abstract: 【Aim】 To screen out the most stably expressed reference genes in Lygus pratensis under specific conditions. 【Methods】 qRT-PCR was adopted to determine expression levels of 10 candidate reference genes including alpha-tubulin, beta-tubulin, actin, elongation factor, succinate dehydrogenase, ubiquitin, transcription initiation factor TFIID subunit, glutathione S-transferase, ribosomal protein L32, and TATA-box binding protein genes in six categories of samples of L. pratensis including different sex adults, different adult tissues, different developmental stages, adults with different lambdacyhalothrin resistance, and adults respectively exposed to different temperatures and different insecticides. BestKeeper, geNorm, NormFinder and RefFinder were adopted to analyze the mRNA expression stability of the candidate reference genes. 【Results】 According to the results of different analysis methods based on the effective qRT-PCR data, it was concluded that the most stably expressed reference gene was RPL32 for different adult tissues and adults of L. pratensis with different lambda-cyhalothrin resistance. The most stably expressed reference genes for the groups of adults exposed to different temperatures, different sex adults, adults exposed to different insecticides and different developmental stages were UBQ, SDHA, β-tubulin and TAF, respectively. Based on the comprehensive analysis from geNorm and RefFinder calculation, the best combinations of stably expressed reference genes for the groups of different adult tissues, different sex adults, different developmental stages, adults with different lambda-cyhalothrin resistance, and adults exposed to different temperatures and different insecticides were RPL32+TAF, SDHA+GST, TAF+GST+UBQ, RPL32+GST, UBQ+ACT+β-tubulin and β-tubulin+TAF+RPL32, respectively. 【Conclusions】 All the most stably expressed reference genes and their best combinations obtained from this study could be acceptable as reference genes for gene expression analysis in L. pratensis for future studies.

Key words: Lygus pratensis, qRT-PCR, reference gene, gene expression analysis, expression stability

贾冰, 马亿, 庞保平, 单艳敏, 鲍青龙, 韩海斌, 谭瑶. 牧草盲蝽实时定量PCR内参基因的筛选[J]. 昆虫学报, 2019, 62(12): 1379-1391.

JIA Bing, MA Yi, PANG Bao-Ping, SHAN Yan-Min, BAO Qing-Long, HAN Hai-Bin, TAN Yao. Screening of reference genes for quantitative real-time PCR in Lygus pratensis (Hemiptera: Miridae) [J]. Acta Entomologica Sinica, 2019, 62(12): 1379-1391.

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牧草盲蝽实时定量PCR内参基因的筛选

Screening of reference genes for quantitative real-time PCR in Lygus pratensis (Hemiptera: Miridae)

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