基因UB和L40与溴氰菊酯抗性关系的初步研究

发布时间：2018-08-26 20:05

【摘要】：小菜蛾(Plutella xylostella L.)属于鳞翅目菜蛾科,其寄主高达四十多种,被认为是十字花科的蔬菜如甘蓝、青菜等的主要害虫之一。如今控制害虫的一个重要手段就是化学防治。然而随着杀虫剂的不断使用,小菜蛾对许多杀虫剂都已经产生了抗药性,并且已经成为病虫害防治中的一大障碍。研究和发现新的与抗药性相关基因将有助于了解抗药性的分子基础,有利于寻找新的治理抗性的靶标。本实验室前期研究证明UBL40与小菜蛾的溴氰菊酯抗性有关,但UBL40是融合基因,UB基因编码泛素蛋白,L40基因编码核糖体蛋白。在抗药性产生的过程中,是UB基因还是L40基因或是二者都发挥作用,需要进一步研究。本文在前期实验的基础上,对UB和L40与抗药性的关系分别进行了研究,主要结果如下：1)在果蝇细胞中,用不同浓度的溴氰菊酯分别刺激细胞48h,然后荧光定量分别检测细胞中UB和L40的表达量,结果表明,UB和L40的表达量均有不同程度的上升。随后选取一个适宜的溴氰菊酯浓度,刺激果蝇细胞不同时间,结果发现,UB和L40的表达量也均上升。这为下一步的实验奠定了基础。2)扩增UB和L40基因全长,然后在果蝇细胞中进行转染,并在基因和蛋白质水平上检测转染效率,在检测转染成功的果蝇细胞中,用不同浓度的溴氰菊酯进行刺激,然后用CCK-8试剂盒检测细胞的死亡率,结果表明,转染了UB或转染了L40的果蝇细胞与对照组相比对溴氰菊酯的抗性均有显著的增加。3)用RNAi试剂盒合成UB和L40的dsRNA,然后在果蝇细胞中进行干扰,同样在基因水平和蛋白质水平进行检测干扰效率,在干扰成功的果蝇细胞中,用不同浓度的溴氰菊酯刺激48h,然后CCK-8检测细胞死亡率。结果发现,干扰了UB或干扰了L40的果蝇细胞与对照组相比对溴氰菊酯的抗性均有不同程度的降低。4)运用RNAi技术在溴氰菊酯抗性品系小菜蛾幼虫中对UB和L40进行干扰,然后运用生测方法检测小菜蛾的死亡率,结果显示干扰了UB或干扰了L40的小菜蛾与对照组相比,对溴氰菊酯的抗性水平显著降低,小菜蛾的成活率下降。由此我们得出结论,在融合基因UBL40中,UB和L40基因均参与了溴氰菊酯的抗性形成,这为溴氰菊酯抗性机理研究提供了新的线索,但其相关机制尚需进一步研究。
[Abstract]:Diamondback moth (Plutella xylostella L.) Lepidoptera belongs to the family Lepidoptera with more than 40 hosts and is considered to be one of the main pests of cruciferous vegetables such as cabbage and green vegetables. Chemical control is an important means of pest control nowadays. However, with the continuous use of pesticides, Plutella xylostella has developed resistance to many insecticides and has become a major obstacle in pest control. The research and discovery of new genes related to drug resistance will be helpful to understand the molecular basis of drug resistance and to find new targets for resistance management. Our previous study proved that UBL40 is related to the deltamethrin resistance of Plutella xylostella, but UBL40 is a fusion gene encoding UB gene encoding ubiquitin protein and L40 gene encoding ribosomal protein. Whether the UB gene or L 40 gene or both play a role in the development of drug resistance needs further study. On the basis of previous experiments, the relationship between UB and L40 and drug resistance were studied respectively. The main results are as follows: 1) in Drosophila cells, The cells were stimulated with different concentrations of deltamethrin for 48 hours, and then the expression of UB and L40 in the cells were detected quantitatively by fluorescence. The results showed that the expression of UB and L40 increased in different degrees. Then a suitable concentration of deltamethrin was selected to stimulate Drosophila cells for different time. The results showed that the expression of UB and L40 also increased. This laid a foundation for the next experiment. 2) amplifying the full length of UB and L40 genes, then transfecting them in Drosophila cells, detecting transfection efficiency at gene and protein levels, and detecting transfection efficiency in successfully transfected Drosophila cells. Different concentrations of deltamethrin were used to stimulate, and then CCK-8 kit was used to detect the cell death rate. The resistance of Drosophila cells transfected with UB or L40 to deltamethrin was significantly increased by 3. 3) UB and L40 dsRNA, were synthesized by RNAi kit and then interfered in Drosophila cells. The interference efficiency was also detected at the gene level and protein level. In Drosophila melanogaster cells with different concentrations of deltamethrin for 48 hours, the cell death rate was measured by CCK-8. The results showed that the resistance to deltamethrin of Drosophila melanogaster cells which interfered with UB or L40 decreased to varying degrees compared with the control group. The RNAi technique was used to interfere with UB and L40 in diamondback moth larvae, a Deltamethrin resistant strain. Then the death rate of Plutella xylostella was detected by bioassay. The results showed that the level of resistance to deltamethrin and survival rate of diamondback moth decreased significantly compared with the control group which interfered with UB or L40. It is concluded that both UB and L40 genes are involved in the formation of deltamethrin resistance in the fusion gene UBL40, which provides a new clue for the study of deltamethrin resistance mechanism, but the related mechanisms need to be further studied.
【学位授予单位】：南京师范大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S433.4

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