Transcriptome Analysis Of Ovary And Functional Analysis Of RDH13 Gene In Red Swamp Crayfish Procambarus Clarkii

Procambarus clarkii,commonly called red swamp crayfish or freshwater lobster,belongs to order Arthropoda,Crustacea,Decapoda,Cambaridae,Procambarus.The red swamp crayfish Procambarus clarkii has become an important freshwater economic crayfish in Chinese inland due to its tasting delicious,rich nutritious and inexpensive.With the increase in consumption,the demand for these commodities exceeded supply,but the lack of high-quality young shrimp,sexual precocity,low fecundity,low hatching rate and low survival rate are frequent in cultured shrimp and have caused huge loss.The lack of high-quality seed has become a major factor restricting the development of aquaculture industry.understanding the molecular regulation mechanism of ovary and embryo development is believed to be helpful to solve these problems.And provide a theoretical to the development control of the artificial shrimp breeding process.At present,the research about ovary and embryo development of P.clarkii is mainly concentrated in the field of morphology and physiology,the development process and related mechanism are limited.Here we present the first in-depth analysis of the transcriptional of ovary in P.clarkii using RNA-Seq technologies.Further research revealed that a gene RDH13 associated with development in oogenesis and embryogenesis plays an important role,the results were reviewed as follows.Limited genetic information is available for P.clarkii and many other cultured shrimp species.In this study,we present the first in-depth analysis of the transcriptional of ovary in P.clarkii.A total of over 9.28Gb reads were obtained from illumina sequencing platforms,resulting in an assembly of 69,261 Unigenes with an N50 of 1,129bp and mean length of 634.31 bp.A total of 24,014 putative protein sequences obtained from the assembled transcripts were annotated using public protein sequence databases.The deduced protein sequenceswere assigned to 11,436 GO terms and 25 KOG categories.Based on transcriptome sequencing analysis of P.clarkii,Using BLASTx and bioinformatics methods,the sequences of deduced RDH13 gene related to development were obtained.And its function in the development of Procambarus clarkii was systematically studied.Function analysis of RDH13 gene during oogenesis:The length cDNA of the RDH13 mRNA consisted of 1229 bp containing an ORF of 978 bp corresponding to a deduced polypeptide of 325 amino acids.The deduced RDH13 protein contained one putative adh-short domain(positions 37-183),RDH13 was specifically expressed in ovary,and RDH13 mRNA was significantly up-regulated in stages IV ovaries(P<0.05).In situ hybridization exhibited positive signal of RDH13 in follicular cells around the oocytes during early development of the ovaries and exhibited specific expression of RDH13 in oocytes during near mature period.Knockdown of RDH13 by RNAi technique,the transcripts of vitellogenin(Vg)and vitellogenin receptor(VgR)were significantly decreased(P<0.05).exogenous progesterone administration(0.1μg/g body weight)resulted in an increase of RDH13 mRNA in ovaries of stage III shrimp at 72 h post-injection(hpi)(P<0.05).and the expression level of VgR mRNA in ovaries of stage III shrimp was down-regulated by progesterone injection(P<0.05).It speculated that progesterone may participate in the ovarian development of P.clarkii by regulating the expression of RDH13.Our results suggested that RDH13 may promote ovarian development by facilitating the absorption and processing of vitellogenin.Function analysis of RDH13 gene during embryonic development:RDH13 mRNA was abundantly expressed in fertilized eggs,cleavage stage and nauplii stage.Knockdown of RDH13 in cleavage stage,the embryo can not complete the normal cleavage into the gastrula,a large number of embryonic died after soaking.It also plays an important role in the early embryonic development stage.In conclusion,transcript sequences presented in this study could provide a lot of sequence information for future studies on molecular biology of P.clarkii.Moreover,identification and functional analysis of RDH13 are helpful to understand the molecular regulation mechanism during the development of P.clarkii.Which also can provide important reference date for their basic research and genetic breeding of crustaceans.it has important scientific value and practical significance.