Cloning And Expression Analysis Of BmNADK And Its Research Of High Temperature Stress Reaction In Bombyx Mori

NAD kinaseis the sole NADP-biosynthetic enzyme that catalyzes phosphorylationofNAD+/NADH to yield NADP+/NADPH using ATP as a phosphoryl donor.We useRT-PCR, qRT-PCR, RNAi and the test of enzyme activity to study regulatorymechanism of the silkworm’s oxidation-reduction system.We also find the changes ofBmNADK gene expression level in the same variety and differentvarieties ofBombyx mori by high temperature.The results are as follows:1We clone the BmNADK gene and its down-regulated made theconcentration of NADPH and GSH decreasedUsing RACE technique，we extend the5’and3’ end of the gene sequence andobtain the cDNA sequence of NADK gene located in chromosome3and its geneticevolution is nearest to Papilio polytes. We investigate the NADK gene expressionlevel of5instars silkworm,and find its expression in swing period is higher, wechoose the swing period to do the RNAi experiment.As the BmNADK genedown-regulated， the time of swing period will be shortened,and theNADPH,GSHcontent are decreased,the content of H2O2and NAD+are rised.Theresults show BmNADK gene involves in the silkworm oxidation-reduction system,mainly through influencing the concentration of NADPH and GSH.2BmNADK gene are related to high temperatureWe do treatment with34℃high temperature continuously to Dazao andinvestigate female male silkworms’ fat body, midgut, silk gland.The results show theBmNADK gene will be up-regulated when the time of treatment increases. Malesilkworm’NADK genes increase more obvious than female silkworm, and raise the most significant in the fat body tissue.In order to furtherly study the functionofBmNADK gene,using42℃to treat Dazao with3h and investigate female malesilkworms’ fat body, midgut, silk gland，we find the BmNADK gene will beup-regulated afte the treatment. This phenomenon is most obvious in the malesilkworm and fat body. When the temperature is normal，the BmNADK geneexpression level will be down.These indicate BmNADK gene are related to hightemperature.3The influenceof BmNADK gene expression indifferent silkworm varieties byhigh temperatureWe treat7532（high temperature resistance） and Haoyue （weakly hightemperature resistance）with34℃high temperature and investigate female malesilkworms’ fat body, midgut, silk gland.The result shows the BmNADK geneexpression is difference between different varieties，and high expressions in resistantvarieties.This shows that NADK gene can influence the antioxidant capacity of thesilkworm and it is related to tolerate high temperature among different varieties ofsilkworms.