QTL Mapping Of Virulence In Biotype Y BPH And Metabolism Study Of Interaction Between BPH And Rice

Rice (Oryza sativa L.) is a primary staple food crop for more than 50% of the world's population, providing most human nutrition and more than 20% of the calories consumed by humans worldwide. Meanwhile, rice has been one of the most important model plants widely to be researched. Among many rice insect pest, the brown planthopper (BPH; Nilaparvata lugens Stal) is the most damaging insect pest to cause rice production loss in Asia. To develop a sustainable pest management system, it is important to find the right balance between breeding and management strategies to reduce the ecological fitness of BPH and to keep the pest under economic threshold levels. Since the first rice germplasm was shown to exhibit resistance to BPH, there have been detected 27 resistance genes in cultivated and wild species of rice so far. With the selection pressure, BPH evolved new'biotypes' to overcome the resistance of these resistant rice varieties in the field. Our study is helpful to understand evolution of BPH adaption to high resistance rice variety in high selection pressure and it is useful to apply to agriculture to prevent the BPH adaptation for controlling BPH population.Biotype Y is virulent to resistant rice line YHY15 derived from biotype 1 BPHs, by forcing them to feed on the resistant line YHY15 from January 2007. We used highly inbred strains of biotype Y and biotype 1 to construct segregation population due to their pure genetic background. To construct F2 population for QTL mapping survival rate and growth rate on YHY15, we put a virulent biotype Y virgin female or male crossed with an avirulent biotype 1 male or virgin female in a tube to generate F1 populations, F2 populations was obtained from F1 randomly male and female crossing. The survival rate and growth rate didn't fit the Mendelian inheritance in the crossed and reciprocal crossed F1 and F2 populations suggested they were controlled by major genes. Through genome wide QTL scanning by MapQTL6, three QTLs were associated with BPH survival rate on YHY15, two QTLs were on Chr4 and one QTL was on Chr6. CIM analysis of growth rate identified three QTLs, two QTLs were on Chr2 and one QTL was on Chr11. The approximate locations of the virulence QTLs of high virulence lab biotype Y is a starting point for fine mapping or for studying candidate genes close to the identified QTLs.By metabonomics approach, we investigated the differences of response mechanism between YHY15 and TN1. We collected the TN1 and YHY15 leaf sheaths which were treated with BPH for 24hr,48hr and the samples didn't treat with BPH were kept as control. The fresh leaf sheaths were used to extract metabolites, and then the derivatived extracts were analyzed by GC-MS. The results showed that metabolites change depend on feeding time; meanwhile the extent of metabolites variation such as amino acids, sugars, organic acids was greater in susceptible TNI than in resistant YHY15. BPH infestation caused TN1 enhancing fatty acid oxidation, glyoxylate cycle, gluconeogenesis, GABA shunt, while BPH infestation caused YHY15 enhancing glycolysis and shikimate pathway. The results indicated that susceptible and resistant rice plants have different metabolic pathways to respond BPH infestation. We also analyzed the metabolites in honeydew of BPH biotype 1 and biotype Y insects fed on TNI andYHY15 rice plants, respectively. The analysis of metabolites in honeydew indicated that BPHs feeding on YHY15 would enhance amino acid absorption. At the same time, BPH increased ammonia metabolism to relieve stress of toxin from plant secondary metabolites.We studied the function of P450 of brown planthopper (BPH) for their adaptation to BPH resistant rice. We obtained the results as the flowing:1. we detected some secondary metabolism compounds, and these compounds may be toxic to BPH.2. Biotype Y BPH survival rate more than biotype 1 when fed on artificial diets of adding ethanol extracts.3. We had used double-stranded RNA-mediated interference (RNAi) to silence NICPR expression of biotype Y BPHs caused inhibit biotype Y feeding on YHY15.4. Screening 25 BPH P450 genes by using quantitative real time PCR, the result suggested 3 P450 genes induced by YHY15 and expression levels increased with feeding time. Knocking-down of these 3 P450 candidate genes, the results showed the RNA interference of CYP4C61 could inhibit biotype Y feeding on YHY155. The knocking-down of biotype Y BPH CYP4C61 after rearing on artificial diets of adding ethanol extracts should reduce their survival rate when compared to controls. Smi-quantitative PCR result showed CYP4C61 expression was highest in fat body, followed by midgut, the least in salivary gland.The coding region of CYP4C61 gene fragment was cloned into plasmid pET-28a (+) and PGEX-6p-1 respectively, and the recombinant plasmids were transformed into E.coli. Two clones were efficiently expressed; in addition we obtained small amounts of soluble recombinant proteins. The NICPR with N-terminal 15 animo acids truncated was cloned into plasmid PGEX-6p-1, and we also obtained small amounts of soluble recombinant proteins.