Functional Analysis Of RBL And Downstream Chk1 Gene During Ovary Development Of Penaeus Monodon

Penaeus monodon,also known as the black tiger shrimp,is one of the world’s three major cultured shrimp.It is the world’s general breeding of fine species that has good quanlity of individual large,shell thick,delicious meat,nutrient-rich.As in wild environment,the female gonads of shrimps are so difficult to focus on a large number of mature for the production of seedlings that aquaculture industry development has been a lot of constraints.So far,unilateral eyestalk ablated technology is still most commonly used and practical technology to promote female shrimp ovarian mature and spawning in artificial breeding.However,the technology will lead to high mortality and low spawning quality of broodstock,which is not conducive to the development of large-scale breeding of shrimps.In order to find an alternative method,it is necessary to understand the mechanism of ovarian development of Penaeus monodon,especially the molecular mechanism.In this study,PmRBL and PmChk1 cDNA fragments were screened from the transcriptome of this laboratory.The full length of the genes was cloned by molecular biology techniques such as SMART-RACE.The full length of the PmChk1 genome was also cloned.Recombinant proteins of the PmChk1 was expressed in E.coli,purified by Ni-chelating affinity chromatography,and prepared antibody.The expression of PmChBL and PmChk1 were detected by double-stranded RNA interference.The expression of PmChBL and PmChk1 after injected with 5-HT,the stages of development ovary and eyestalk ablation.We have systematically explored the biological information and protein functions of PmRBL and PmChk1,and provide a theoretical basis for further study on the mechanism of ovarian development of Penaeus monodon.The main research is summarized as follows:(1)The PmRBL cDNA fragment was screened from splicing transcriptome of P.monodon established in our laboratory.The full length of RBL gene was cloned by SMART-RACE technique.The PmRBL cDNA sequence is 4,069 bp in length,contains 5-untranslated region(UTR)of 54 bp,3’UTR of 772 bp and 3,243 bp open reading frame which can encode 1,080 amino acids.The theoretical molecular weight is 121.76 kDa.Multiple sequence alignment showed that the PmRBL gene had a conserved Rb-A domain(393-588 aa)and a CYCLIN domain(781-918 aa).The RBL amino acid sequence of Penaeus monodon has similarity of 97% with Litopenaeus vannamei.The tree diagram depicts the evolutionary relationship of Rb protein similarity based on the similarity of Rb proteins in different species.The vertebrate Rb protein is clustered into one,Penaeus monodon and invertebrates containing Litopenaeus vannamei is clustered into one.The results of qRT-PCR showed that PmRBL was widely distributed in all tissues of Penaeus monodon,which had the highest expression in gill,followed by hepatopancreas and ovaries,and the lowest in brain.The expression level of PmRBL was the highest in ovarian stage III,followed by IV and V,and I and II had the lowest expression.The expression of PmRBL was significantly higher in stage III,IV and V than I and II.After 5-HT 12-96 h injection,the expression of PmRBL in ovaries was significantly upregulated.The expression of PmRBL in ovary was significantly up-regulated at 3-96 h after eyestalk ablation.Injection of dsRNA-RBL successfully silenced PmRBL in ovaries and hepatopancreas.The expression of PmRBL in hepatopancreas was detected by in situ hybridization.The results of in situ hybridization were consistent with qRT-PCR.The expression of PmCDC2 and PmCyclin B in hepatopancreas and ovaries was significantly decreased after dsRNA-RBL 6-24 h injection.The expression of PmRBL in ovary and hepatopancreas of Penaeus monodon was significantly decreased after dsRNA-p53 12-96 h injection.The gonadal index after dsRNA-RBL injection was significantly lower than that of the control group after dsRNA-GFP injection.The results showed that PmRBL might play an important role in ovarian development of Penaeus monodon.(2)The PmChk1 cDNA fragment was screened from splicing transcriptome of P.monodon established in our laboratory.The full length of Chk1 gene was cloned by SMART-RACE technique.The PmChk1 cDNA sequence has a length of 3,334 bp,containing 249 bp 5’-untranslated region(UTR),3’UTR of 1,630 bp and 1,455 bp open reading frame which can encode 484 amino acids.The theoretical molecular weight was 54.61 kDa.The full genome of PmChk1 had a length of 11,081 bp containing 10 exons and 9 introns.Important transcription factor binding sites include: IRF1,SREBP,NF-kB,ISGF3,HNF4,STAT1,PPAR,TATA-box and CpG islands.Multiple sequence alignment showed that PmChk1 had a conserved S-TKc domain(12-270 aa).The PmChk1 amino acid sequence had similarity of 55% with Chlorella sinica.The sequence alignment analysis showed that the Chk1 amino acid sequence was similar to Daphnia pluex.The tree diagram depicts the evolutionary relationship of Chk1 protein similarity based on the similarity of Chk1 proteins in different species.The Chk1 protein of the vertebrate was clustered into one,the Chlamydos Penicillium Chk1 and the invertebrate containing Chk1 were clustered into one.The results of qRT-PCR showed that PmChk1 was widely distributed in all tissues of Penaeus monodon,which had the highest expression in ovary,the lowest in gill and brain,and the lowest in muscle.PmChk1 had the highest expression level in stage I and II,and minimal expression in stage III.The expression level of PmChk1 was the highest in postlarval and lowest in oosperm.After 5-HT and DA 24-96 h injection,the expression levels of PmChk1 in ovaries were decreased and increased,respectively.The expression of PmChk1 in ovary was significantly decreased at 3-96 h after eyestalk ablation.Injection of dsRNA-Chk1 successfully silenced PmChk1 in ovaries and hepatopancreas.The expression of PmChk1 in hepatopancreas and ovary was detected by in situ hybridization.The results of in situ hybridization were consistent with qRT-PCR.Injection of dsRNA-Chk1 successfully silenced PmChk1 in the ovary and hepatopancreas.In situ hybridization was used to detect the expression and expression of PmChk1 in hepatopancreas and ovaries.The results of in situ hybridization were consistent with those of qRT-PCR.The activity of Chk1 was significantly lower than that the control group after dsRNA-Chk1 and dsRNA-GFP.The results of immunoblotting showed that the expression of dsRNA-Chk1 at 24 and 48 h was significantly lower than that of dsRNA-GFP 24 and 48 h after injection of dsRNA-Chk1.The expression of PmCDC2 and PmCyclin B in hepatopancreas and ovaries was significantly increased after dsRNA-Chk1 6-72 h injection.The expression of PmChk1 in ovary and hepatopancreas of Penaeus monodon was significantly up-regulated after dsRNA-p53 12-96 h,and the expression of PmChk1 in ovary and hepatopancreas was significantly up-regulated after dsRNA-RBL 6-96 h.The gonadal index after dsRNA-Chk1 injection was significantly higher than that of the control group after dsRNA-GFP and PBS.The results showed that PmChk1 may play an important role in ovarian development of Penaeus monodon.In this study,the sequence characteristics of PmRBL and PmChk1 genes were analyzed.The mRNA expression of PmRBL and PmChk1 was detected by qRT-PCR.The expression of PmRBL and PmChk1 was similar in all tissues.The expression level of PmRBL and PmChk1 was different in the stages of ovarian development.The expression of PmRBL and PmChk1 was detected by dsRNA interference,in situ hybridization,histochemistry and immunoblotting.The expression of Chk1 was inhibited by RBL,and Chk1 inhibited the expression of CDC2 and Cyclin B in the signal pathway.Thus,PmRBL and PmChk1 may affect the ovarian development of Penaeus monodon by affecting the cell cycle.These results provide a theoretical basis for further study on ovarian development mechanism of Penaeus monodon.