| 5-HT(Serotonin), a monoamine substance, plays an important role in vivo. As a major neurotransmitter,5-HT can control some behaviour, such as depression, anxiety, learning, memory,etc..Moreover,5-HT associates with the development of neurons cell, the release of some hormones, and promotes the vasoconstriction.The function of5-HT must be linked to the specific receptors, and the different5-HT receptors can lead to different physiological responses.5-HT receptors also is closely related to some mental illnesses in human, such as migraine, Alzheimer’s, depression, suicidal tendencies, etc.. Currently, most of the psychiatric disease medicines take5-HT receptors as a therapeutic target. There are14species of5-HT receptors that have been discovered in animals, including1A,1B,1D,1E,1F,2A,2B,2C,3,4,5A,5B,6,7.5-HT3receptor is a ligand-gated ion channel receptor, the others are G protein-coupled receptors. Silkworm is an important model insect, the study of5-HT receptors is important for lepidopteran pest control on forestry, however the knowledge about the5-HT receptors in silkworm is quite limited. So, in this study, we clone the5-HT receptor genes in silkworm by bioinformatics, RT-PCR. In addition, we get the foundamental study on function of5-HTlABm receptor by injection in vivo. The main results of this paper are summarized as follows:1.There are four kinds of5-HT receptors genes in the silkworm,5-HT1ABm、5-HT1BBm、5-HT2Bm and5-HT7Bm.We cloned5-HT1ABm from head, the CDS of5-HT1ABm has1441bp of nucleotides, and has ORF that contained1395nucleotides, encoding464amino acids residues.5-HT1ABm located on the chromosome8contains3exons and2introns in silkworm. The molecular weights of5-HT1ABm is51.752KD, pI is9.13, and5-HT1ABm has been predicted seven transmembrane domains; The results of tissue expression pattern of larvae show that:5-HT1ABm mainly expresses in head, abdominal nerves, testis and ovary, while it has weak expression in other tissues; The results of tissue expression pattern of adults show that:the expressions of5-HT1ABm are similar in male and female, especially in breast, antennae, legs and genitals.We cloned5-HT1BBm from ovary, the CDS of5-HT1BBm has1428bp of nucleotides, and has ORF that contained1341nucleotides, encoding446amino acids.5-HT1BBm located on chromosome10contains3exons and2introns in silkworm. The molecular weights of5-HT1BBm is48.599KD, pI is8.95, and5-HT1BBm has been predicted seven transmembrane domains; The results of tissue expression pattern of larvae show that:5-HT1BBm expresses in many tissues except silk gland; The results of tissue expression pattern of adults show that:5-HT1BBm expresses in breast, abdomen, antennae, wings, legs and genitals in female, and while it expresses in many tissues except breast in male.We cloned5-HT2Bm from testis, the CDS of5-HT2Bm has1881bp of nucleotides, and has ORF that contained1881nucleotides, encoding626amino acids.5-HT2Bm located on chromosome15contains2exons and1introns in silkworm. The molecular weights of5-HT2Bm is69.294KD, pI is9.97, and5-HT2Bm has been predicted seven transmembrane domains; The results of tissue expression pattern of larvae show that:5-HT2Bm only has lower expression in head, abdominal nerves and testis; There is no expression of5-HT2Bm in male and female in adults.We cloned5-HT7Bm from head, the CDS of5-HT7Bm has1510bp of nucleotides, and has ORF that contained1497nucleotides, encoding498amino acids.5-HT7Bm located on chromosome26contains2exons and1introns in silkworm. The molecular weights of5-HT7Bm is56.132KD, pI is9.03, and5-HT7Bm has been predicted seven transmembrane domains; The results of tissue expression pattern of larvae show that:5-HT7Bm has the highest expression in head, while it has lower expression in abdominal nerves, midgut, fat body, testis and ovary; The results of tissue expression pattern of adults show that:5-HT7Bm expresses in breast, abdomen, antennae, wings, legs and genitals in male and female, and the expression of genitals is the highest in male.2. The multiple sequence alignments and phylogenetic analysis of5-HT receptors show that:in insects, the conservative of5-HT1ABm,5-HT1BBm,5-HT2Bm and5-HT7Bm is30.4%. While, the conservation of5-HT1A,5-HT1B,5-HT2and5-HT7are45.4%,61.4%,48.4%,54.1%respectively. In addition, in insects or vertebrates, the amino acids in transmembrane domains(45.2%) of5-HT receptors are more conservative than non-transmembrane domains(12.1%). However, the conserved amino acids in transmembrane domains is possibly indispensable for the stability of structure and the transmission of information. It is likely that the conserved amino acids of non-transmembrane region are necessary for ligand binding, G protein coupled. The evolutionary relationships show that the same type receptor from different species gets together, then the same receptor forms branch as species genetic relationships. The evolutionary relationships of5-HT receptors in silkworm are close to tobacco hornworm. According to the results of the phylogenetic tree, we speculate that:5-HT1,5-HT2,5-HT7receptors maybe have formed before the differentiation of vertebrates and invertebrates,5-HT1receptors has formed two copies in evolution of insects, and the two copies are5-HT1A and 5-HT1B which have different function between them.3. The functional study of5-HT1ABm demonstrates that:no matter larvae or male moth, the inhibitor of5-HT1ABm receptor can reduce the vitality. In other words,5-HT1ABm receptor has a significant relationship with the vitality of silkworm. Therefore, we suggest that5-HT1ABm receptor may be involved in the regulation of nerve signals.5-HT1A receptor agonist have little effect on silkworm, we speculate that the agonist of vertebrate5-HT1A receptor has weak bond strength for5-HT1A receptor of silkworm because of the amino acid sequences of vertebrates and insects are so different.From what has been discussed above, there are four kinds of5-HT receptors genes in silkworm,5-HTlABm,5-HT1BBm,5-HT2Bm,5-HT7Bm. The protein sequences of them have some conserved amino acids, which maybe is important for functions. Furthermore, the study indicates that5-HT1ABm receptor has been significantly associated with the vitality of silkworm. |
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