Screening The Differentially Expressed Genes In Brown Planthopper Biotype Ⅰ And Ⅱ By Fluorescent Differential Display-PCR

Brown Planthopper (Nilaparvata lugens Stal) has long been one of the most devastating pests to rice crop in Asia. Many comprehensive study has been launched in the insect's develop regular and prevention .In order to clear some confused problems in BPH biotype we conducted a series of experiments including 'The purification of BPH through obtaining a BPH offspring from one female adult BPH ' 'Screening the differentially expressed genes in brown planthopper biotype I and II by Fluorescent Differential Display-PCR' etc. The following results were obtained:1) We have purified BPH through obtaining a BPH offspring from one female adult BPH. And we has identified two BPH biotypes through shoot phase colony identification.2) We acquired the high purity RNA is extracted by chloroform repeatedly and the solid impurity and pigment is eliminated by centrifuge after many experiments, which can size up constructing cDNA library . The reseach will contribute to the reseach of Brown Planthopper molecular biology, but also will provide favorable reference for the allied research about some other insect.3) The technique known as Fluorescential display polymerase chain reaction(FDD-PCR) was used to explore the difference of gene expression between BPH biotype I and II. The results showed that there is a remarkable difference in gene expression between the two planthopper biotypes. There were more than 40 differential cDNA fragments being screened by both FHTn-M(A,C,G) and 8 oligonucletide arbitrary primers.4) Some of cDNA fragments are biotype I specific and the others biotype II specific. Two differential cDNA fragments named g!2-1(487 bp, biotype II specific) and 37-2 (314 bp, biotype I specific) were cloned and sequenced, and no homologous sequences were searched in GenBank. It can be concluded that the damage differentiation of brown planthoppers results from their genes' differential expression. Moreover, those cloned specific cDNA fragments are very useful in the study of brown planthopper's damage differentiation-related genes.5) We have constructed two BPH biotypes' cDNA libraries . And we have tittered the unamplified libraries. BPH biotype I 's library have 4.2 x 106 independent clones,and BPH biotype II 's library have 3.1 X 106. These cDNA libraries may provide basis for obtaining integral genes associated with Biotype and studying the molecular mechanism of Brown planthopper Biotype.6} A pair of primers based on conservative sequences of Drosophila melanogaster was designed to amplify the p53 gene. The fragment with 1156 base pair was obtained and sequenced.