氯化汞对草地贪夜蛾sf9细胞及核型多角体病毒的损伤与致突变研究

Study on the Damage and Mutation of sf9 Cell and AcMNPV Treated by HgCl_2

目的试验以草地贪夜蛾细胞sf9作为受体,检测氯化汞对其生长发育的影响。方法采用台盼兰染料排斥法测定细胞活力,苏木素-伊红染色法检测细胞中的微核,对经氯化汞处理诱变的病毒AcMNPV的DNA进行PCR扩增,产物经测序后进行分子突变分析。结果sf9细胞在4μg·ml-1的氯化汞浓度作用下,其细胞表面变得粗糙,分裂生长减慢,当剂量增大到7μg·ml-1时,便可观察到一些细胞的细胞膜破裂,在9μg·ml-1时,某些细胞的完整性受到破坏。用苏木素-伊红染色法检测细胞中的微核现象时,9μg·ml-1氯化汞处理区的微核率高达6.8%,有些细胞出现三核甚至多核的核裂现象,反映部分细胞的完整性受到一定程度的损伤。AcMNPV病毒经氯化汞短时间处理后接种于sf9细胞,多角体在sf9细胞中的形成数目较对照区少,异常多角体的比例增加,抽提经氯化汞处理的AcMNPV的DNA,采取PCR技术进行扩增反应,对获得的扩增产物进行测序分析,发现DNA序列上的碱基有2处G→C、T→C的转换和碱基缺失的现象。结论一定剂量的氯化汞将会引起草地贪夜蛾sf9细胞及核型多角体病毒的损伤与致突变。

[Objective] In an experiment, sf9 cell of Autographa californica was used as an acceptor to inspect the effect of HgCI2 on its growth and development. [Method] Evaluated the vigor of cells by repulsion of Trypan Blue dyeing method, detected the microkernel in the ceils by HE dyeing method, amplified the DNA of HgCl2 treated AcMNPV by PCR, and analyzed the molecular mutation of the products after sequencing. [ Result] The results showed that the surface of sf9 cell became rough, and the division became slowly when treated with 4μg·ml^-1 HgCl2. When the dosage increased to 7 μg·ml^-1, the cell membrane broken. The cell integrity was damaged when the concentration increased to 9μg·ml^-1. The microkernel in the cells was detected by using HE dyeing method. When treated with 9μg·ml^-1 HgCl2, the microkernel was as high as 6.8%. Some cells found tri-nucleus and multi-nucleus, reflecting that the integrity of cell was damaged. After shortly treated with HgCl2 AcMNPV was inoculated to sf9 cell. The formation of polyhedra in treated sf9 cells was less than that in CK, and the abnormal polyhedra increased. DNA was extracted from AcMNPV treated with HgCI2, and inspected by PCR. The sequencing of amplification product showed that absent and change （such as G→C, T→C） were found in two bases of DNA. [ Conclusion ] Certain dosage of HgCl2 can cause the damage and mutation of sf9 cell ofAutographa californica and AcMNPV.