Brain Structure Of Octopus Minor And Cloning And Expression Of Oct-GnRH Gene

Five aspects of research were carried out on Octopus minor: The anesthetic effects of 12 reagents on O.minor were studied experimentally.The microstructure of each nerve leaf in central nervous system of O.minor was observed by paraffin section technology.The microscopic and ultramicroscopic observation of the endocrine organ optic gland of O.minor were carried out using paraffin section and transmission electron microscopy technology.Cloning and analysis of oct-GnRH gene cDNA full length,a key neuropeptide in reproductive regulation of O.minor,were carried out by RACE technology.The expression and analysis of oct-GnRH gene in 24 tissues of O.minor were detected by qRT-PCR.The results were as follows:1.The results showed that magnesium sulfate,eugenol,MS-222,manganese(II)chloride,lidocaine,procaine hydrochloride,ethylene glycol phenyl ether,l-menthol,benzocaine and 2-trichloromethyl-2-propanol had no anesthetic effect on O.minor.The degree of anesthesia of O.minor was divided into five periods,the recovery process was divided into four periods.Samples stayed in 15～35 g/L magnesium chloride and 10～40m L/L ethanol achieved the phase 4 of anesthesia,this stage is most suitable for observation and measurement of O.minor.In a certain concentration,with the increase of the concentration,the anesthesia time gradually shortened,and the recovery time gradually increased.10 m L/L ethanol and 20g/L magnesium chloride showed the shortest anesthesia and recovery time,which were 26 minutes and 40 minutes,respectively.The present study demonstrated that ethanol solution and magnesium chloride solution were effective anesthetic agents for O.minor.2.The brain was divided into three parts according to the location: the supraoesophageal mass,the suboesophageal mass and the optic lobe area on both sides of the supraoesophageal mass and the suboesophageal mass.The supraoesophageal mass includes the vertical lobe,the superior frontal lobe,the inferior frontal lobe,the anterior basal lobes and the posterior basal lobes.The suboesophageal mass includes the brachial lobe,the pedal lobe,the magnocellular lobe,the chromatophore lobe,the visceral lobe,the palliovisceral lobe and the vasomotor lobe.The optic lobe area includes the optic nerve,the optic lobe,the optic gland,the olfactory lobe,the peduncle lobe and the optic tract.Paraffin section,optical microscope and transmission electron microscope were used to observe the microstructure and ultra microstructure of the optic gland.The results showed that there was a layer of connective tissue wrapped outside the optic gland,which was located in the optic tract area,adjacent to the olfactory lobe and the peduncle lobe.A large number of secretory cells were observed in the inner part,with a large nucleus and a diameter range of 4～8μm.The secretory cells were widespread in rough endoplasmic reticulum,Golgi apparatus,secretory vesicles and vacuole secreted by Golgi apparatus.The results showed that the structural characteristics of the optic glands of O.minor were similar to those of Sepiella maindroni and Octopus vulgaris.3.The full length of GnRH cDNA consisted of 853 bp,which contained 153 bp5’UTR,430 bp 3’ UTR,and an open reading frame(ORF)of 270 bp(including termination codon).Oct-GnRH encoded a protein with 89 amino acids,which included a BEN domain and a Pox-A3 L domain.We analyzed the amino acid sequence homology between oct-GnRH of O.minor and the GnRH of other molluscs.The amino acid sequence of oct-GnRH gene of O.minor has the highest similarity with Octopus vulgaris in Octopus,followed by Sepiella japonica and Uroteuthis edulis of Cephalopoda.The phylogenetic tree showed that oct-GnRH gene was most closely related to Octopus vulgaris in Octopus,followed by Sepiella japonica and Uroteuthis edulis of Cephalopoda,but far from Gastropoda and Lamellibranchia in Mollusca.The expression level of oct-GnRH in different tissues of adult O.minor was detected by real-time fluorescence quantitative PCR(qRT-PCR).The results showed that oct-GnRH gene was expressed in 18 tissues(supraoesophageal mass,suboesophageal mass,optic lobe area,wrist muscle,axonal nerve cord,kidney,hepatopancreas,posterior salivary gland,ovary,nidamental gland,testicle,fine buccal pouch,prostate,esophagus,stomach,gastric cecum,intestine,carcass muscle)of 24 tissues(supraoesophageal mass,suboesophageal mass,optic lobe area,sucker,wrist muscle,axonal nerve cord,retina,heart,kidney,hepatopancreas,gill,anterior salivary gland,posterior salivary gland,ovary,nidamental gland,testicle,fine buccal pouch,prostate,esophagus,stomach,gastric cecum,intestine,carcass muscle,skin)of male and female sexually mature O.minor,but not in 6 tissues(sucker,skin,gill,heart,retina,anterior salivary gland).The expression level was higher in the upper and lower esophageal nerve groups,among which the lower esophageal nerve group was the highest.In this study,oct-GnRH gene was cloned in the brain of O.minor for the first time.The expression level and distribution of oct-GnRH gene indicated that oct-GnRH may be the key neuropeptide in the regulation of reproductive development of O.minor.The results provided valuable information for the studies regarding regulation mechanism of reproductive development and industrial breeding technology of O.minor.