| Chinese mitten crab,Eriocheir sinensis,commonly known as crab.It is a unique freshwater crabs in China and has become one of the major global economic species because of its delicious and nutritious.In recent years,the aquaculture was develop with the direction of scale intensive cultivation,improving the growth performance and reducing premature rate had always been the focus of people.As a kind of essential amino acid tryptophan,its metabolism will produce many kinds of active substancessuch as 5-HT,they were closely related to many aspects of animal body,such as growth,development,feeding and behavior.Studies have shown that tryptophan that added to the feed,can raise growth performance,improve the survival rate,promote food intake and reduce the attack behavior of some animal,etc.And the previous immunohistochemical analysis showed that 5-HT receptor in Eriocheir sinensis bowel and widely distributed in the organization,suggests the tryptophan and5-HT in Eriocheir sinensis has many physiological functions in the body.Immersion and injection experiments suggest that 5-HT can promote molt reduce duration in the intestines of Eriocheir sinensis.Feed the different tryptophan content of diet for 30 d,found that tryptophan feed can improve the survival rate and decrease the rateof Eriocheir sinensis.On the other hand,it can improve the food intake and increased protease,lipaseactivity of hepatopancreas,subsequent experiment used high-definition camera camera,it shows that tryptophan increase feeding rate and moving time in the process of feeding.This shows tryptophan can promote the feeding of Eriocheir sinensis,which is similar to other animal studies.This study also found that tryptophan can promote molt of Eriocheir sinensis.in order to further research,the third chapter selected organ what was major regulate molt(Y-organ),and we analysed two kinds of circumstances in normal and precocious puberty transcriptome.5-HT is a kind of important neurotransmitters.Its role is mediated through the implementation of the corresponding receptors.5-HT gene of Eriocheir sinensis clone progress was slow,we can not analysis function of tryptophan and 5-HT.So in the fourth chapter,we using the transcriptome data from the third chapter to clone 5-HT1 B receptor gene.Now wegot the 3' and 5' fragment,and through qPCR technology to study expression of different tissues and when feeding different TRP content in Eriocheir sinensis.Provided data for futher study in the future.The concrete results were present as follows:1)In this experiment,different L-tryptophan supplemented in diets were fed to Eriocheir sinensis,in order to study the effects on growth performance and digestive physiology of juvenile Eriocheir sinensis.Four experimental diets were formulated to supplement with graded levels of tryptophan,resulting in different dietary tryptophan levels at 0.36%(A,control),0.47%(B),0.73%(C),? 1.05%(D).A total of 360carbs(about 2.45g)were divided into four treatments with three replicates and fed with the respective tryptophan diets in an indoor recirculating system.Before and after the experiment,recorded body weight,body length,body width,body height,situation of limb and molting,measuring food intake every seven days.Gathering hepatopancreas form each group for measuring the activity of trypsin,amylase and lipase.The results showed that no significant differences were found in the rate of length gain,rate of width gain,rate of height gain,percent weight gain rate and hepatopancreas index among all the treatments(P>0.05).Survival rate were obviously increased by dietary tryptophan levels,and the maximum was obtained at D group(P < 0.05).On the contrary,the rate of leg amputated crab were obviously reduced by dietary tryptophan levels,and the minimum was obtained at C group(P<0.05).C group on molting rate and the rate of carb during premolt was significantly better than A group and B group.Food intake of B group and D group were significantly higher than A group when feed7 d,while D group significantly higher than B group when feed 21 d.2)Then we measured enzyme activity of each group,the result show that no significant differences were found in activity of amylase among four groups.Activity of lipase was significantly higher in C groupn than group A.B group had significantly higher activity of trypin than other groups.In conclusion,L-tryptophan supplemented in diets could enhance survival rate,molting rate,food intake,activity of lipase and trypsin,as well as improve the rate of leg amputated composition.3)Activity while taking feed and feeding rate.Tryptophan is precursor of 5-HT,melatonin and several important physiological activators,it could increase food intake in animal,decrease the use of high quality protein of feed and the aggressive behavior,and improve the survival rate.To examine effects of L-tryptophan supplemented inactivity while taking feed and feeding rate in Eriocheir sinensis,explore appropriate levels of tryptophan of feed,and lay the foundation for further researches.Four experimental diets were formulated to supplement with graded levels of tryptophan,resulting in different dietary tryptophan levels at 0.36%(A,control),0.47%(B),0.73%(C)? 1.05%(D).60 carbs with the average body weight of(14.27±1.29 g)that were randomly allocated into 4 groups with 15 Polyethylene tank(35×25×10cm)for 7d.We provided 1g of feed at 8:00 during the test for each carb,then used high-definition cameras to record total duration of activity within 1h,and collected residue by 200-mesh sieveafter1 h,analysed data by SPSS software.The results showed that total duration of activity within 1h of B group(30.36±13.27%)and C group(32.26±14.50%)were significantly increased than A group(17.13±8.48%).Feeding rate were obviously increased by dietary tryptophan levels,and feedingrate of C group(5.7±1.16%)and D group(5.52±1.03%)significantly higher than A group(4.46±0.68%).In conclusion,L-tryptophan could improve activity while taking feed as well as increase eedingrate.Comprehensive consideration,0.75% in the feed was optimum content.4)There were a lot of 5-HT receptor sequences that based on the analysis of the transcriptome.Precocity is a common problem in aquaculture of Eriocheir sinensis.To discover and clone of important functional genes,the transcriptome of Y-organ of precocious and normal female crab was sequenced by Illumina Hiseq 2000high-throughput sequencing technology,and comparative transcriptome study was conducted.The results showed that precocious and normal crabs had 44 619 538 and43 052 958 clean reads respectively.Comparison between the sequencing data from precocious and normal female crabs revealed 2655 differentially expressed genes.The differentially expressed genes GO functions in the transcriptome library were broadly divided into three major ontology biological processes,cellularcomponent and molecular function categories of 42 branches.Data in the transcriptome from 134 differentially expressed genes could be divided into 4 classes taking the KEGG database as a reference,according to the metabolic pathway,including synthesis and degradation of ketone bodies,butanoate metabolism,neurotrophin signaling pathway,base excision repair.By RNA-seq,we obtained abundant transcriptome information that could contribute to novel gene identification,causes and prevention of precocity,and research of Y-organ in Eriocheir sinensis.The sequences were 1A?1B?1D?2A?2B?2C?7 types,and they can be used for RACE.5)We got the main sequence of 5-HT1 B receptor,and through qPCR technology to study expression of different tissues and when feeding different TRP content in Eriocheir sinensis.According to the transcriptome of Y-organ of precocious and normal female crab,5HT1 F and 5HT1 R was designed to obtain nuclear cDNA fragments of 5-HT1 B receptors.And this paper used the reverse transcription PCR(RT-PCR)technology and the rapid amplification cDNA end(RACE)technology,cloning partial cDNA fragments of 5-HT1 B receptors.The sequencing results revealed that 5-HT1 B receptors fragments contain 1732 base pairs,whose open reading frame(ORF)is 1080 bp,it encode 359 amino acids.BLAST analysis indicated that the amino acid homology of 5-HT1 B receptor was 96% and 86% similar to Cancer borealis and Homarus americanus respectively.Through real-time fluorescent quantitative PCR(qPCR)technology to study the gene expression of different organizations in Eriocheir sinensis.Results showed that gene was expressed in tissues such as hepatopancreas,muscle,ovary,blood,intestine,gill,thoracic ganglia,Y-organ,cephalic ganglion and eye stalk,meanwhile it was expressed highest inthoracic ganglia,Y-organ?cephalic ganglion and eye stalk which were significantly higher than other tissues(P<0.05).Results in different feeding group showed that it was detected in four tissues,the expression level appeared significantly decreased in thoracic ganglia,intestine and eye stalk.The results showed that theexpression of 5-HT1 B was influenced by feed different TRP levels. |
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