| Mytilus coruscus,is one of important aquaculture mussel species in china,and the success of their larvae settlement and metamorphosis directly determines nursery processes and aquaculture seed production of this species.Therefore,it's meaningful to investigate some genes about the processes of larvae settlement and metamorphosis.Result studies have found that the growth and metamorphosis of marine vertebrate larvae could be regulated by the Wnt signaling pathway,which including classical signal pathway and non-classical signal pathway,with different functions in the same species.In present study,we selected four genes including Wnt4,Wnt6,Wnt7 b and ?-catenin,which belong to the Wnt signal pathway,to investigate their effects on the settlement and metamorphosis of larvae of M.coruscus by the techniques of RACE and Real-time Quantitative PCR.The main contents are shown as follows:1.Wnt4 gene expression and functional analysis in the mussel M.coruscusTo understand the potential mechanisms of the Wnt4 gene in different development stages and adult tissues of the mussel M.coruscus,the Wnt4 gene was cloned by RACE technique.The full length of Wnt4 gene was 3342 bp and 1074 bp of open reading frame encoding 357 amino acids.RT-PCR showed that Wnt4 mRNA expression was detected in all tissues and the highest expression was found in mantle,indicating that Wnt4 gene may play an important role in shell formation.Relative gene expression of Wnt4 during larval development stages including trochophore,D-shaped,umbo,pediveliger and the juvenile stage was also detected.The Wnt4 mRNA was highest expressed in umbo stage larvae,suggesting that Wnt4 gene may participate in the process of shell morphological structure change and certain organ formation and development.Taken together,the finding provides new insights on investigating the function of Wnt gene family.2.Wnt7 b gene expression and functional analysis in the mussel M.coruscusTo understand the potential functions of Wnt7 b in different developmental stages and adult tissues of the mussel M.coruscus,the Wnt7 b gene was cloned using the rapid amplification of cDNA ends technique.The full-length Wnt7 b gene was 1910 bp,with a 1059 bp open reading frame encoding 352 amino acids.Wnt7 b mRNA expression was detected by RT-PCR in all tissues studied,with the highest expression found in the gill,and in the male and female gonads.This indicates that Wnt7 b may play an important role in gonadal maturation and in the functions of gills in the mussel M.coruscus.Expression of the Wnt7 b gene during larval development stages,including the trochophore,D-shaped veliger,umbo veliger,pediveliger,and juvenile stages,was also detected.Wnt7 b mRNA was highly expressed in the D-shaped veliger,umbo veliger,and pediveliger larvae stages,suggesting that Wnt7 b may participate in larval development and in the process of metamorphosis in the mussel M.coruscus.Taken together,these findings provide new insights into the functions of the Wnt gene family during mussel larval development and settlement and metamorphosis.3.Wnt6 gene expression and functional analysis in the mussel M.coruscusTo understand the potential functions of Wnt6 in different developmental stages and adult tissues of the mussel M.coruscus,the Wnt6 gene was cloned using the rapid amplification of cDNA ends technique.The full-length Wnt6 gene was 1616 bp,with a 1032 bp open reading frame encoding 343 amino acids.Wnt6 mRNA expression was detected by RT-PCR in all tissues studied,Wnt6 mRNA expression was detected in mantle,gill,foot,male gonads and female gonads,no expression in the digestive gland and adductor muscle.And the highest expression was found in mantle,indicating that Wnt6 gene may play an important role in shell formation.Expression of the Wnt6 gene during larval development stages,was only detected in the umbo veliger and juvenile stages,suggesting that Wnt6 may participate in larval development and in the process of metamorphosis in the mussel M.coruscus,and may participate in the process of shell morphological structure change and certain organ formation and development.Taken together,these findings provide new insights into the functions of the Wnt gene family during mussel larval development and settlement and metamorphosis.4.?-catenin gene expression and functional analysis in the mussel M.coruscusTo understand the potential functions of ?-catenin in different developmental stages and adult tissues of the mussel M.coruscus,and to explore the relationship between ?-catenin and Wnt signaling pathways,the ?-catenin gene was cloned using the rapid amplification of cDNA ends technique.The full-length ?-catenin gene was 4051 bp,with a 2409 bp open reading frame encoding 829 amino acids.RT-PCR showed that Wnt4 mRNA expression was detected in all tissues and the highest expression was found in gill and digestive gland,indicating that ?-catenin gene may play an important role in ingestion.Relative gene expression of ?-catenin during larval development stages including trochophore,D-shaped,umbo,pediveliger and the juvenile stage was also detected.The ?-catenin mRNA was highest expressed in juvenile and umbo stage larvae,suggesting that ?-catenin may participate in larval development and in the process of metamorphosis in the mussel M.coruscus.In addition,they play a role of feedback regulation in the metamorphosis of larvae.Comparing with previous researches,?-catenin and Wnt6 play a role of feedback regulation,deduced that Wnt signaling molecules were passed to downstream factors to mediate larvae metamorphosis through ?-catenin.As the classic Wnt signal pathways are made of many molecules,therefore,the way those molecules coordinate or interact in this physiological process remains to be further studied.In conclusion,Wnt6 and Wnt7 b genes belong to classical Wnt signal pathway can regulate the larval settlement and metamorphosis of the M.coruscus.However,Wnt4 belong to non-classical Wnt signal pathway cann't regulate the larval settlement and metamorphosis of the M.coruscus.Moreover,the ?-catenin genes can regulate the larval settlement and metamorphosis of the M.Coruscus,which proven that the mechanism of Wnt signal pathway regulate the larval settlement and metamorphosis of M.coruscus through the ?-catenin gene to transfer the signals to the downstream factor. |
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