The Clone And Expression Research Of Ecdysone Receptor And The Ultraspiracle And Hexokinase Gene Of Antheraea Pernyi

Diapause is a predefined resting form of dormancy through stable genetic mode,when insect is stimulated by external environmental conditions.Diapause plays an important role in ensuring population and individual survival,when insect cope with adverse environment.A.pernyi is a complete metamorphosis economic insect with pupae diapause.It has high application value,nutritive value and medicinal value.A.pernyi has two characteristics because of the different geographical environment,which are divided into univoltine and bivoltine.A.pernyi diapause pupa is conducive to preservation of cocoon for egg production,and the release of diapause controls the beginning of A.pernyi production,so the study of the A.pernyi diapause has a great significance for A.pernyi production,and may also provide new ideas for the prevention and treatment of Lepidoptera pest of the same diapause model.In this study,the diapause pupae of A.pernyi was used as the material,and the method of molecular biology was used to study the role in the hormonal regulation of ecdysone receptor(EcR)gene and the ultraspiracle(USP)gene and in sugar metabolism regulation of hexokinase(HK)gene during the process of diapause termination and development.The result is as follows:1.The research of ecdysone receptor and ultraspiracle binding protein gene cloning and expression of A.pernyiThe ecdysone receptor and ultraspiracle binding protein gene primers were designed to clone the 2 genes according to A.pernyi transcriptome database,and named as ApEcRB1(GenBank accession No.KY411159)and ApUSP1(GenBank accession No.KY411160).The open reading frames(ORF)of 2 genes are 1 758 bp and 1 401 bp long,encoding 585 and 466 amino acids respectively.The two genes expressed in the tissues of Spermary/ovary,silk gland,fat body and so on of A.pernyi larvae by semi-quantitative RT-PCR detection,among which silk gland and fat body had higher expression levels over other tissues.The expression of these two genes was substantially up-regulated before larva ecdysis and around pupation as revealed by real-time fluorescent quantitative PCR(qRT-PCR).After injection with 20-hydroxyecdysone(20E),pupae at diapause were induced to develop.The pupae of induced group appeared eclosion after 17 d,which was ahead of the contrast.The expression levels of both genes in pupae at diapause significantly decreased after 20 E injection.ApEcRB1 gene maintained at a low level during 1 d to 12 d,but increased sharply before ecosion,and reached the highest level at 16 d(the day before eclosion).The expression level of ApUSP1 gene started to increase at 8 d,and reached the highest level at 16 d.The above results show that the ecdysone mediated the expression of ecdysone receptor and ultraspiracle genes to control the growth,development and metamorphosis of A.pernyi.2.Study on the cloning and expression of hexokinase gene during A.pernyi diapause and diapause terminationInsects can turn glucose from digestion into trehalose or reserve it as glycogen in fat body.Trehalose can be converted into glucose used for glycoysis when energy is needed.Hexokinse(HK)plays an important role in the process of transformation.In order to study glycometabolism during pupa diapause termination and pupa development in A.pernyi,two hexokinase isozyme genes were cloned,and named as ApHK1(GenBank accession No.KY624592)and ApHK2(GenBank accession No.KY624593).Their full-length open reading frames are 1 455 bp and 1 362 bp,encoding 484 and 453 amino acids respectively.Phylogenetic analysis indicated that ApHK1 and ApHK2 are lacated in two different evolutionary clades,and the same HK isoform from different insects has higher identity.In different tissues of the 5th instar A.pernyi larvae,the two HK gene have high expression level in fat body.ApHK2 was expressed in all tested tissues,showing wider distribution than ApHK1.Its expression in midgut and muscle was higher than in other tissues.Expression levels of ApHK genes were detected in fat body of univoltine diapause pupa by using real-time quantitative(qRT-PCR)under long photoperiod(17 h/d)condition.The results showed that the relative expression level of the two genes was increased obviously at 21 d after treatment,among which the expression of ApHK2 was increased by 3.1 times.At 42 d after treatment,ApHK1 expression was increased by 2.8 times while relative expression of ApHK2 had no significant change.The above results indicate that ApHK1 and ApHK2 play an important role in the process diapause termination and pupa development by light in A.pernyi,and the regulatory functions of the 2 genes are different in those of the tissue different glycometabolic pathways.3.Study on the activity of hexokinase and the change of glucose content during A.pernyi diapause terminationThe changes of glucose content and enzyme activity were determined by ultraviolet spectrophotometer during A.pernyi diapause and diapause termination.The result showed that the hexokinase activity in fat body of pupae under long photoperiod treatment was higher than that of the control group.It increased in early stage,reached peak level at 28 d after treatment,and stabilized after decline at 35 d;Glucose content in pupa haemolymph exhibited a first-decline then-rise trend,which was correlated with hexokinase activity in fat body.These data show that hexokinase acticity has complementary relationship with gluose content during middle stage of A.pernyi pupa development,i.e.glucose content in haemolymph decreases with increase of hexokinase activity.