| With the world’s fish consumption rising and diminishing wild fish stocks,transgenic fish might alleviate pressure on wild stocks.However,commercial implementation of transgenic fish technology for food production has remained elusive.Researchers worry that genetically engineered fish,if they escaped,could wreak ecological havoc.The only way to address this issue is to control the fertility of transgenic fish.There is no doubt that the production of sterile triploid transgenic fish will address concerns about ecological risk,however,the method of artificial induction can not satisfy the need for a large scale commercial production of transgenic fish and not offers a number of benefits to commercial growers.The tetraploid produced by distant hybridization(common carp,2n=100♂×red crucian carp,2n=100 ♀),is the first case artificial polyploidy fish with the bisexual fertile and stable inheritance in bony fish in the world.Hybridization between the tetraploid(4n=200)and diploid transgenic yellow river carp(2n=100)species produce all triploid transgenic progeny,transgenic triploid carp have maintain the sterile characteristic of triploid hybrid fish but have a faster growth rate,the use of sterile transgenic triploid carp resolve the risk of ecology safety,whereas,hybridization can satisfy the need for a large scale commercial production,there is no doubt that the production will address concerns about industrialization.The growth development of fish is an integral biological process.A variety of endocrine hormones interact each other between the gonadotropic axis and somatotropic axis and regulate the growth and development of fish.Growth hormone(GH)is known to be key factor in the control of growth in teleosts,Gonadotropin hormone(GTH)plays a crucial role in regulating gonadal development and stimulating the gametes mature in teleosts,GH and GTH are both primarily controlled by the neutoendocrine factors of hypothalamus-pituitary-gonad axis(HPG axis)and interaction between themselves.The pituitary is an important endocrine organ,which plays a significant role in regulating the synthesis and secretion of hormones related to the growth development.In this study we firstly analyzed the expression and feedback regulation of GH and GTH in different ploidy fishes.The major results in this paper were presented as follows:(1)By crossing the females of tetraploid red crucian carp with the males of diploid transgenic yellow river carp(homozygote),we obtained the progeny of transgenic triploids.By PCR testing,the present study indicated that the progeny are all transgenic triploids(positive triploid fish,100%).In this way,we can obtain a number of transgenic triploids to offer insight into the expectation for successfully industrialization,and make up the instability and inefficiency through manual work.(2)By RT-PCR,the cDNAs encoding FSHβ,i and LHβ subunits were obtained from tetraploids and cloned into PMD18-T vector.Confirmed by sequencing,two right cDNAs were cloned into PET-32(a)vector,and then transformed into E.coli BL21 to construct two expression strains.After confirmed by sequencing again,the recombinant FSHβ and LHfi subunits were efficiently expressed in the form of fusion protein after induction with IPTG.After roughly purified,the fusion proteins were injected into New Zealand White rabbits to produce polyclonal antibodies.(3)The tissue-specific expression of GH and GTH were studied by RT-PCR in different ploidy fishes.GH mRNA was virtually restricted to the pituitaries with very strong signal,interestingly we found.that GH mRNA was also detected in the muscle of transgenic triploids with strong signal,the direct expression of GH in the muscle perhaps induced the faster growth rate in the transgenic triploids.whereas,expression for two gonadotropin subunits,Fshβ and Lhβ could only be detected in the pituitary of different ploidy.(4)The expression of GTH in different season were investigated in different ploidy fishes at the mRNA and protein level respectively.In the spawning season(May),GTH transcripts in the pituitaries were significantly lower in transgenic triploids than in diploids or tetraploids.In contrast,expression of GTH in the prespawning season(November)was significantly higher in transgenic triploids than in diploids and tetraploids.In both prespawning(November)and spawning seasons(May),GTH protein in the pituitaries were significantly lower’ in transgenic triploids than in diploids and tetraploids,while no significant dirrerences were observed between diploids and tetraploids.The possible reason for this difference is that GH can exert a direct negative feedback control on GTH secretion at the pituitary level.Finally,these results suggest the abnormal expression of GTH in transgenic triploids is related to their abnormal gonadal’ development and probably even to their sterility.(5)The expression of GH was investigated in different ploidy fished at the mRNA and protein level respectively.Sterile common triploids showed a significantly higher expression of GH mRNA in pituitary compared with sterile transgenic triploids and other fertile fish,meanwhile GH mRNA was exclusively expressed in the muscle of transgenic triploids,at the protein level,the result of Western-blot was consistent with the former.These results imply that exogenous GH can impart its feedback on GH release and synthesis and pituitary GH expression is regulated by negative feed-back controls as occurs in other vertebrate systems。We suspected that the direct expression of GH in the muscle induced the faster growth rate in the transgenic triploids.In conclusion,the expression of GH was detected in the pituitary and muscles of transgenic triploids,the direct expression of GH in the muscle may induce the faster growth rate in the transgenic triploids;Overexpression of exogenous GH can exert a direct negative feedback control on GTH secretion at the pituitary level in different season,the phenomenon explain the sterility of triploids.The present research offered insight into the expectation for successfully industrialization by hybridization between the tetraploids and transgenic diploids obtaining a number of transgenic triploids;comparatively analyzed the expression characterizations of GH and GTH in different ploidy and provided the data for the sterility of gonad and fast growth of endocrine mechanism in triploids. |
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