Expression Analysis Of BmorCSP9in Larvac And Pupae Of Silkworm, Bombyx Mori

Chemosensory proteins (CSPs) are small soluble proteins andabundantly distributed in the chemosensory organs of insects. They have manydifferent physiological functions. In order to provide useful informaiton for furtherclarify the function and mechanism of insect CSPs. In the present study, Quantitativereal-time RT-PCR(qRT-PCR), western blot and immunofluorescence analysis wereused to investigate the expression pattern of Bmorcsp9gene and BmorCSP9proteinof model insect silkworm Bombyx mori at different developmental stages and indifferent tissues of larvae. Finally, in order to find the best condition of RNAinterference, we tried to use double-stranded RNA to block the expression ofBmorcsp9gene by injection or feeding. The results were shown below:1. Expression pattern of Bmorcsp9gene of Bombyx mori at larval and pupalstagesqRT-PCR was used to investigate the expression pattern of Bmorcsp9gene ofBombyx mor at larvae, pupae and in different tissues of larvae. The results showedthat Bmorcsp9gene is expressed abundantly in the head, thorax, abdomen and less inthe ovary of the larval stage. The Bmorcsp9gene couldn’t be detected in the silkglandand testis. The Bmorcsp9gene showed regular expression patterns at the larval andpupal stages. The expression level was low in all the moulting larvae, increased innewly molted larvae, and decreased in molted larvae after feeding12hours; but itincreased rapidly again at end of the fifth larvae before spinning, decreased at2daysafter pupation, and increased again at the end of pupal stage.2. Expression pattern of BmorCSP9protein of Bombyx mori in the differentlarval instarsWestern blot and immunofluorescence analysis were used to investigate theexpression pattern of BmorCSP9protein of Bombyx mori at the different larval instars.The results of western blot showed that BmorCSP9protein was distributed in themoulting larvae, newly moulted larvae and molted larvae after feeding12hours ofdifferent developemental stages. The results of immunofluorescence indicated thatBmorCSP9were abundantly distributed in the epidermal chemosensory organs of the larval stage. The expression pattern of BmorCSP9protein is consistent with theresults of qRT-PCR. It is speculated that the BmorCSP9protein may play animportant regulatory role in the different larval stages.3. Exploring the condition of RNA interference to block the expression ofBmorcsp9genePrimers with T7promerter were used to clone Bmorcsp9gene by PCR. Thentranscription was performed in vitro with T7RNA polymerase. The products ofdouble-stranded RNA was used to block the expression of Bmorcsp9gene byinjection or feeding. Then qRT-PCR was used to detect the effect of RNAi. Theresults showed that the expression quantity of several treatment group was lower thanthe control group, but the Bmorcsp9gene was not blocked completely. Further more,the conditions of RNAi need to be optimized.