Study On The Detection Method Of Nosema Bombycis And Cloning And Analysis Of The RPB1Gene Of Nosema Sp. PA

Microsporidia are a group of obligate intracellular parasites that can parasitize a widevariety of other eukaryotes ranging from protists to invertebrates and vertebrates. More than1,300microsporidian species belonging to160genera have been reported. Nosemabombycis is a typical species of Nosema genus. Silkworm pebrine disease is a devastatingdisease in sericulture. Nosema bombycis can not only parasitize in the silkworm larva, pupa,and moth but also transmit to the next generation through the silkworm eggs, which leads toa decline in the yield and quality of cocoons, and causes great losses in sericulture.Detection methods for silkworm pebrine disease have been researched and explored fromthe earliest visual identification to microscopic examination and to serological andmolecular biology techniques. Microscopic examination method is still applied to inspectionand quarantine of silkworm pebrine disease because there are limitations in practicality ofthe new methods developed by laboratory. It is possible that the sample （moths） and theactual product （silkworm eggs） do not correspond due to human factors of the mothexamination method. So inspection of silkworm eggs will inevitably replace the mothexamination method. It’s necessary to establish a simple and efficient method for thedetection of Nosema bombycis in sericulture.In this study, we successfully established a detection method which exhibited amarkedly higher sensitivity than previously developed detection methods for Nosemabombycis by combining glass beads, FTA card, and LAMP. Spores of N. bombycis werefirst broken by acid-washed glass beads, the DNA was subsequently extracted and purifiedwith the FTA card, and LAMP was performed using primers （LSU296） designed based onthe sequence of the LSU rRNA of N. bombycis. The minimum detection concentration was10spores/mL. The LAMP method established here will not provide false positive resultsbecause the LAMP primers cannot amplify the genome of the bacteria and fungi that infectsilkworm. Furthermore, the LAMP method established in our study could detect N.bombycis infection in silkworm24h earlier than microscopy. When this method was used todetect pebrine disease in silkworm egg, the detection rate for500silkworm eggs, in whichonly one egg was infected with N. bombycis, was100%under our optimized conditions.The detection rate decreased as the total number of silkworm eggs increased. If the numberof eggs in the sample increased to800or1000, the sample was divided into two equalportions, and the eggs were smashed with glass beads after the addition of1mL of TE buffer. The liquid in two tubes was later mixed and applied to the FTA card, and thedetection rates were100%. Our experiment showed that glass beads reaching1/2of thevolume of the tube and1mL of TE buffer for a total of500eggs can result in a satisfactorybreaking of the spores and DNA extraction.RPB1gene is the main functional subunit of RNA polymerase II. In this paper, theRPB1gene of Nosema sp. PA was cloned and characterized using bioinformatics methods.First six pairs of homologous primers were designed for the RPB1gene of Nosema sp. PAbased on the RPB1gene of Nosema bombycis. Partial sequence of the RPB1gene ofNosema sp. PA was cloned by PCR amplification （GenBank Accession No. KJ728831）. Theresult showed that the partial sequence of the RPB1gene of Nosema sp. PA had2933nucleotides which contained a ORF with2922bp encoding a polypeptide of974aminoacids with a molecular weight of109.38277kD and an isoelectric point of7.087. Thestructure of the partial sequence of the RPB1gene was a single exon. The encoded proteincontained four domains: RPOLA_N, RNA_pol_Rpb1₄, RNA_pol_Rpb1₅andRNA_pol_Rpb1₆. The protein contained four main secondary structures: α-helix, randomcoil, β-turn and extended strand. The proportion of extended strand and random coil wasquite high. Extended strand were mainly located between the α-helix and random coil.Sequece comparison showed that the encoded protein was99.6%identical to Nosemabombycis and had a close relationship with Nosema bombycis. The resultes confirmed thatNosema sp. PA was a member of Nosema clade at the aspect of molecular biology.In this study, we successfully established one method that was convenient and efficient,exhibited high performance, and could be widely used. This article provides a new approachfor the inspection and quarantine of silkworm pebrine disease in sericulture and may behelpful for microsporidian detection in other fields.The RPB1gene of Nosema sp. PA wascloned and characterized using bioinformatics methods. This article also provides a newfoundation for further study of it’s concrete biological function and mechanism ofexpression and regulation of Nosema sp. PA.