利用末龄幼虫蜕和蛹壳对苹果蠹蛾基因的无创伤检测方法  

A non-destructive method for the detection of codling moth genotypes using the exuviate of the final instar larvae or puparia

作　　者：魏子涵 王雅琪 赵诗词 杨雪清[1,2,3] WEI Zi-Han;WANG Ya-Qi;ZHAO Shi-Ci;YANG Xue-Qing(College of Plant Protection,Shenyang Agricultural University,Shenyang 110866,China;Key Laboratory of Economical and Applied Entomology of Liaoning Province,Shenyang 110866,China;Key Laboratory of Major Agricultural Invasion Biological Monitoring and Control of Shenyang,Shenyang 110866,China)

机构地区：[1]沈阳农业大学植物保护学院,沈阳110866 [2]辽宁省经济与应用昆虫教育厅重点实验室,沈阳110866 [3]沈阳市重大农业入侵生物监测与防控重点实验室,沈阳110866

出　　处：《环境昆虫学报》2024年第4期1018-1024,共7页Journal of Environmental Entomology

基　　金：国家重点研发计划(2021YFD1400200);兴辽英才计划(XLYC1907097)。

摘　　要：苹果蠹蛾Cydia pomonella(L.)是我国重大农业入侵害虫,对我国果业健康发展造成严重威胁。在利用基因编辑等技术对相关基因进行功能研究时,通常采用单对交配策略对纯合突变体进行筛选。因此,在配对前明确个体基因型,避免对虫体造成损伤尤为重要。本研究分别收集末龄幼虫蜕(10个蜕)和蛹壳(1个、5个、10个、15个蛹壳),通过基因组DNA提取、目的基因PCR扩增、琼脂糖凝胶电泳检测和PCR产物测序,以评估该方法作为苹果蠹蛾基因检测的可行性。结果显示,以苹果蠹蛾末龄幼虫10个蜕及5个以上蛹壳提取的基因组DNA作为模板,扩增精巢特异性丝氨酸/苏氨酸蛋白激酶(Testis specific serine/threonine protein kinase,TSSK1)基因,PCR产物经琼脂糖凝胶电泳检测得到单一、明亮的条带,经测序证实该产物是目的基因序列。基于高效及节约成本的原则,拟推荐利用10个末龄幼虫蜕或10个蛹壳提取基因组DNA、通过PCR扩增和测序进行苹果蠹蛾的无损伤基因型检测。本研究建立了一种利用幼虫蜕和蛹壳进行苹果蠹蛾无损伤、高效的基因检测方法,为提高苹果蠹蛾基因功能研究的效率奠定了基础。The codling moth,Cydia pomonella,is a quarantine pest that threatens the health of the fruit farming industry in China.In gene editing and other functional study techniques,homozygous mutant lines are usually screened by single pair mating strategy.Consequently,it is of paramount importance to ascertain the individual genotype prior to pairing,while avoiding any potential damage to the body.In this study,the genomic DNA(gDNA)was extracted from exuviate of the final instar larvae(10 individuals)and puparium(1,5,10,and 15 individuals,respectively)of C.pomonella.Then PCR amplification,agarose gel electrophoresis,and PCR product sequencing was performed to evaluate the feasibility of this method for gene detection in C.pomonella.Results showed that using the Testis specific serine/threonine protein kinase 1(TSSKI)gene as a target gene and the gDNA extracted from 10 exuviates and 5 or more pupariums as templates,a single and bright band was amplified by PCR.The PCR products were verified as target sequence by DNA sequencing.Based on the principle of efficiency and cost saving,it is recommended to use gDNA extracted from 10 final-instar of larval exuviates or 10 pupariums to non-destructive detect the genotypes of C.pomonella by sequencing.In this study,we established a non-destructive and efficient gene detection method of C.pomonella by using the exuviates of final-instar larvae and pupariums,which laid a foundation for improving the efficiency of gene function research.

关 键 词：苹果蠹蛾 基因型 无创伤检测 CRISPR/Cas9 突变品系 

分 类 号：Q963[生物学—昆虫学] S433[农业科学—农业昆虫与害虫防治]