The Quantitative Study And Analysis Of Transcription Levels Of Carboxylesterase Genes From Bombyx Mori

Carboxylesterase is a multifunctional superfamily and ubiquitous in all living organisms, including animals, plants, insects, and microbes. Carboxylesterases have a broad range of functions; they can effectively catalyze the hydrolysis of endogenous and exogenous compounds containing carboxylester, amide and thioester bonds. They play important roles in xenobiotic detoxification, and pheromone degradation, neurogenesis and regulating development. In order to explore the roles of carboxylesterase genes from Bombyx mori (BmCarEs), we investigated the transcription levels of three types of tissue-specific expression genes BmCarEs with real-time quantitative PCR (qPCR) method. The main results are as follows.1 The quantitative analysis of head-specific expression genes BmCarEsWe used real-time fluorescent quantitative PCR method to detect the transcription levels of head-specific expression genes BmCarEs BmCarE-6,BmCarE-12,BmCarE-15 and BmCarE-16 in head, antennae and maxilla of the 5th instar larvae feeding on both normal mulberry leaves and 4μg/mL phoxim treated mulberry leaves. The detection results were normalized by using Bombyx mori housekeeping gene Actin3. The results indicated that these head-specific expression genes BmCarEs are mainly expressed in antennae and maxilla which are the olfactory organs of larvae. What's more, the transcription levels of these genes gradually decreased when the mulberry leaves were treated with phoxim. Considering that antennae and maxilla are the important olfactory sensilla of the silkworm larvae. Olfactory sensilla is responsible for the recgonition,combination and degradation of pheromone and odorant molecular. Besides, carboxylesterase has the activity of hydrolase. We supposed that these head-specific expression genes BmCarEs may play important roles in odor and pheromone degradation. 2 Transcription levels of midgut-specific expression genes BmCarEsWe have detected the transcription levels of midgut-specific expression genes BmCarEs BmCarE-8,BmCarE-11,BmCarE-13 and BmCarE-14in the midgut of the 5th instar larvae feeding on both normal mulberry leaves and 4μg/mL phoxim treated mulberry leaves by qPCR. The detection results were normalized by using Bombyx mori housekeeping gene Actin3. The results showed that compared with control group, the transcription levels of midgut-specific expression genes in the poisonous group increased dramatically at 24h or 48h after treated, and then decreased to the normal levels. The midgut of silkworm is the important xenobiotics-degrading tissue. The change in transcription levels of these four carboxylesterase is in accordance with slight toxic symptom. In other words, the transcription levels increased with silk worm larvae appeared to have toxic symptom after 24 h feeding with phomix, then the transcription levels decreased with the toxic symptom disappeared. We speculated that these midgut-specific expression genes BmCarEs might be relevant to the insecticide resistance.3 Identification and expression analysis of carboxylesterase gene BmCarE-9 and BmCarE-7 from Bombyx moriWe cloned a carboxylesterase gene named BmCarE-9 from Bomyx mori by using rapid amplification of cDNA ends (RACE) and reverse transcription polymerase chain reaction (RT-PCR) methods, and this gene was deposited in GenBank under the accession no. EU523534. We have determined the transcription levels of silk gland-specific expression genes BmCarEs BmCarE-7 and BmCarE-9 in different tissues of 3rd day 5th instar larvae and in silk glands of 5th instar each day larvae, and in the fat body and silk glands of the 5th instar larvae feeding on phoxim treated mulberry leaves by qPCR. The detection results were normalized by using Bombyx mori housekeeping gene Actin3. The results suggested that these BmCarEs expressed specifically in silk gland at high levels, and mainly expressed in median and posterior silk glands. Furthermore, the expression of these BmCarEs increased as silk gland developed, and decreased at the end of 5th instar stage. Besides, compared with control group, the transcription levels of silk gland-specific expression genes in the poisonous group increased immediately after treated and decreased at the end. The change in transcription levels of these four carboxylesterase is in accordance with slight toxic symptom It was postulated that these silk gland-specific expression genes BmCarEs might be involved in the development of silk gland and the synthesis of silk proteins or the detoxification of xenobiotics entering into the silk gland.Recently, the research about carboxylesterase is mainly focused on its involvement in insecticide resistance. However, the other functions of Bombyx mori carboxylesterase such as odor or pheromone degradation and development regulation have not been studied enough. Here, based on some researches before, we investigated the transcription levels of three types of tissue-specific expression genes BmCarEs and found that head-specific expression carboxylesterase genes mainly expressed in antennae and maxilla; the transcription levels of midgut-specific expression carboxylesterase genes changed while the silkworm larvae appeard the toxic symptom to disappeard the toxic symptom; silk gland-specific expression carboxylesterase genes mainly expressed in median and posterior silk glands. Furthermore, the expression of these BmCarEs increased as silk gland developed, and decreased at the end of 5th instar stage, the transcription levels of silk gland-specific expression carboxylesterase genes changed while the silkworm larvae appeard the toxic symptom to disappeard the toxic symptom. All of these results provide some new insights into functions of BmCarEs in silkworm.