Studies On Chloride Ion Channel Protein Genes And Coenzyme In Diapause Termination Of Silkworm,Bombyx Mori

Bombyx mori is one of the important insects in the research of egg diapause. It is easy to be control and convenient to be operated and has been achieved a lot of research results. In this study, 932 strain of silkworm eggs was used as experimental material. Through different treatment to select the most suitable way of diapauses breaking, using bioinformatics, molecular biology and genetic engineering technology methods, the chloride ion channel protein genes were screened in the hydrochloric acid treatment. The genes were cloned and expression analyzed. Four kinds of coenzyme changes were also studied which involved in the process of embryonic development. The main results were described as follows:(1) Hatchability was varied according to silkworm eggs in hydrochloric acid and DMSO dipping time. Postpartum 20 h of real-time pickling in 0.5~1.5 minutes, hatchability gradually rising when stimulus increases with time; pickle lasting for 1.5~6 minutes, the hatching rate can be maintained between 95%~97% in the picking. Hatchability increased when postpartum 20 h eggs were stimulated by DMSO and lasted for 15~40 minutes. Lasting for 40~50 minutes’ stimulation, the hatching rate could be maintained between 88%~90%; when stimulation lasting for 60 minutes, the hatching rate reduced to 86%. Egg postpartum 10 days, it completely enter diapause under 5℃. Cold storage preservation after 90 days, with hydrochloric acid to stimulate and lasted for 4~6 minutes, hatching rate remained relatively stable which located between 94%~95%;part of eggs going to die when stimulus last for 7min, the hatching rate also reduced to 93%。(2) Three genes of chlorine ion channels(Bm CLIC), silkworm glutamic acid chloride ion channels(Bm Glu Cl) and silkworm γ-aminobutyric acid chloride ion channel receptors(Bmγ-Cl) were cloned. According to the sequence of CDS design primers, through PCR, agarose electrophoresis, plastic recycling, connecting carrier, enzyme digestion and sequencing method, Bm CLIC, Bm Glu Cl and Bmγ-Cl were successfully cloned.(3) By real-time fluorescent PCR technology, m RNA expression amounts Bm CLIC, Bm Glu Cl and Bmγ-Cl candidate genes in different processing eggs and the process of embryonic development. Bm CLIC, Bm Glu Cl and Bmγ-Cl gene expression quantity increased in postpartum after 20 h eggs and refrigerated eggs. It suggested that HCl treatment and the amounts of target gene transcriptional expression correspondingly responded. In diapause eggs, the amount of Bm CLIC and Bm Glu Cl gene expression reached the highest level on the first day, but Bm CLIC gradually decreased with development; however, Bm Glu Cl gene expression increased firstly and then decreased in the process of development; on the other hand, Bmγ-Cl gene expression reached the highest amount of value 2.04 on the second day and gradually reduced along with the development of embryo. Three candidate genes have the same change trend in the process of pickled eggs processing and DMSO treated eggs.(4) Some enzymes activity changes were detected such as acetyl coenzyme A, flavin adenine dinucleotide, NAD and NADP in embryonic development of diapause eggs and instant pickled eggs 1~9d. The results showed that acetyl coenzyme A in diapause egg trend to decrease; but it decreased after acid treatment during development of eggs in 5 days, then increased slowly. FAD activity showed upward trend in the process of diapause egg from 1~5d after being laid, and then presents downward trend; Instant acid treatment process resulted FAD quantity per gram protein increased from 0.00024 pg of first day to 0.00102 pg of 9th day. NAD in the process of egg diapause dropped from 41.549523 mol/g of first day to 13.45342 mol/g of 4th day. It remained stable at relatively low after 4th day; In the process of pickled egg, it presents a trend of increasing to the peak of 122.04747mol/g in 9th day. NADP in the process of egg diapause maintained a stable level, while in the process of pickled egg, it presents a trend of increasing to the peak of 59.45774 mol/g in 9th day.