绿盲蝽体内寄主植物DNA提取方法的优化

An improved method for extracting host plant DNA from Apolygus lucorum(Hemiptera:Miridae)

【目的】优化绿盲蝽Apolygus lucorum体内植物DNA提取方法,为绿盲蝽寄主利用习性研究发展DNA分子追踪技术。【方法】首先分别利用CTAB法、两种试剂盒法提取绿盲蝽中肠内DNA样本,比较分析其中的棉花DNA检出率。其次,利用1%~1.5%的次氯酸钠溶液漂洗绿盲蝽成虫,比较不同漂洗处理对绿盲蝽体表植物DNA的清除效率以及对体内棉花DNA检出率。【结果】不同提取方法对绿盲蝽中肠内棉花DNA检出率存在显著影响,其中CTAB法提取的中肠样本中棉花DNA的检出率最高。将绿盲蝽成虫在1%~1.5%次氯酸钠中漂洗5 s,可清除成虫体表的植物DNA污染,且不破坏体内的棉花DNA;漂洗30 s后,体内棉花DNA检出率明显降低。【结论】本研究证实了利用整头绿盲蝽成虫代替其中肠提取的绿盲蝽DNA样本的可行性,并明确了成虫体内植物DNA高效提取的方法,为进一步利用DNA分子追踪技术研究绿盲蝽田间寄主选择与取食行为奠定了重要基础。

[Objectives] To develop an improved method of extracting host plant DNA from Apolygus lucorum （Hemiptera：Miridae） in order to facilitate the use of DNA-based technology to explore patterns of host plant utilization by this mirid bug in the field. [Methods] We used a CTAB-based method and two commercial DNA extraction kits to extract cotton DNA from the gut of A. lucorum and compared the detection rates of cotton DNA among these three extraction methods. Secondly, we compared the effect of washing A. lucorum adults in a 1%-1.5% sodium hypochlorite solution for different periods of time on removing plant DNA contamination from the body surface, and on the detection rates of cotton plant DNA within the body. [Results] The CTAB-based DNA extraction protocol achieved significantly higher detection rates of cotton plant DNA than the two commercial kits. Washing in 1%-1.5% sodium hypochlorite for 5s successfully removed plant DNA contamination from the body surface of A. lucorum but did not destroy internal cotton plant DNA. The detection rate of cotton plant DNA did, however, decrease significantly after washing for 30 s. [Conclusion] This study established a feasible method to remove external plant DNA contamination from the body surface of A. lucorum and confirmed that DNA can be extracted from the entire body of adult A. lucorum instead of just the gut. It provides an important foundation for the use of DNA-based technology to track the feeding behavior of A. lucorum.