Study On The Resistance Of Insect-resistant Rice Lines To Brown Planthopper, Nilaparvata Lugens And The Role Of ApterousA In Brown Planthopper

The brown planthopper (BPH), Nilaparvata lugens (Stal) are serious pests in rice fields in many areas of Asia, they are distributed mainly over the Yangtze River, South China and southwestern China. The reproduction of BPH is amazing, they also can migrate to other areas and cause further damage. To illustrate inherent mechanism of disasters caused by BPH, it is necessary to study the molecular mechanism of growth and development of BPH, by molecular biological technology. In this study, the biological effects of insect-resistant rice on BPH was investigated, and that was about to expound insect-resistant mechanism of insect-resistant varieties. Fuhthermore, we cloned N. lugens apterousA(NlapA) gene relating to growth and development, analyzed its function, and providing new genetic resources for BPH controlling.1. Biological effects of rice harbouring Bphl4and Bphl5on BPH①The results of seedling resistance to BPH and honeydew measurement during tillering stageThe severity scores of MH63::14, MH63::15and MH63::14&15were<3.0, which indicated that insect-resistant rice lines have a high resistance to BPH. In addition, honeydew production of BPH on those insect-resistant lines was significantly lower than that on TN1and MH63. This indicated that the resistance level of introgression lines was obviously improved during seedling and tillering stage.②The biological effects of insect-resistant lines on BPHCompared with MH63, the development duration of BPH nymph was significantly retarded, the development duration of female was significantly decreased, and the brachyptery ratio, female ratio, copulation rate and fecundity were significantly decreased by MH63::15and MH63::14&15. This indicated that resistant rice lines were not beneficial to the growth and development of BPH.③The antixenosis of resistant rice lines on BPHThe average number of nymphs and female adults settled on resistant rice lines (MH63::15and MH63::14&15) was significantly lower than that on MH63, and the number of BPH eggs per tiller was significantly lower than MH63. This indicated that the antixenosis of resistant rice lines on BPH was high.④The expression of P450gene was regulated by insect-resistant rice in BPH Among the five P450genes of BPH, the expression of three genes (CYP4C61, CYP4C62and CYP6CW1) was up-regulated, the expression of one gene (CYP6CS1) was down-regulated and one gene (CYP303A1) was unchanged by resistant hosts. This indicated that CYP4C61, CYP4C62and CYP6CW1were important during the insect-resistant course of resistant rice lines.2. Gene clone and the role of NlapA in the wing development and reproduction of BPH①Cloning and sequence analysis of NlapAThe NlapA cDNA is3004bp long and contains a unique open reading frame (ORF) spanning nucleotides58-1326bp. The polypeptide deduced from the ORF comprises422amino acides, with a calculated molecular mass of46.6kDa. The predicted protein contains two conseved LIM domains and one homeodomain.②The expression of NlapA in different tissues and different periods during BPH developmentThe results show that the relative expressions of NlapA was investigated in all stages of BPH, the relative expressions of NlapA during first-fifth instar nymphs and one day after BPH emergence were higher than those during two days-eight days after BPH emergence. And the relative expression of NlapA in the third instar nymphs is the highest. In addition, the relative expressions of NlapA are stable during different tissues of BPH, but the relative expression of NlapA is the highest in the wing buds of BPH. And the relative expression of NlapA in wing buds after NlapA RNAi is2.5-6times of that in other tissues.③The expression of NlapA in BPH after NlapA RNAiIn this study, we design three different dsapA according the sequence of NlapA, he results of NlapA RNAi shows that the relative expressions of NlapA in different periods during BPH development were significantly lower than those in control, the relative expression of NlapA is gradually decreasing over days after injection of dsapA, and the relative expression of NlapA in BPH is the lowest on the tenth day after injection of dsapA. Furthermore, the relative expression of NlapA in eggs after NlapA RNAi is significantly lower than control, and it is only19%of that in control.④NlapA RNAi result in wing abnormalityThe abnormality rates of BPH after injection of dsapAs are above93%, however, the abnormality rates of BPH after injection of dsGFP is zero. NlapA RNAi results in wing morphological defects in BPH, the main wing defects as follows:I:The veins on the wing is crooked, and that results in BPH not to form normal folded wing; Ⅱ:No sensory bristles are existed on the forewing veins, and veins become thinner; Ⅲ:The membranous regions between veins become thinner.⑤NlapA RNAi decreased the reproduction of BPHThe fecundity of BPH after NlapA RNAi is significantly decreased by dsapA than that in control, and it is only15.67-23.13%of that in control. The emergence rate of BPH after NlapA RNAi is zero, but the emergence rate of BPH after GFP RNAi is92.7%. Although dsapA stops the development of eggs, it has no effect on the morphological characteristics of ovary.Taken together, the results indicated that resistant rice lines were not beneficial to the growth and development of BPH, and they slao decreased the reproduction of BPH; knockdown of NlapA expression in vivo RNAi generated phenotypic defects in the wing, decreased the number of offspring and stopped the development of eggs. This technique will provide powerful tools for functional genomics of BPH, so as to lay a solid foundation of theoretical knowledge for the study of BPH development genes.