| Abalone is a very important marine economic shellfish and the Pacific abalone Haliotis discus hannai（H.discus hannai）is the main breed of abalone in China.The commercial growth cycle is generally 2 years,which restricts the development and economic benefits of abalone aquaculture.The molecular genetics research of H.discus hannai is currently weak and the functions of most genes unclear,which is not conducive to the genetic improvement of related traits.Therefore,it is necessary to study the molecular mechanism of the important economic character of abalone and its muscle growth,so as to provide scientific basis for the cultivation of fast growing abalone.In this paper,the molecular mechanisms of regulating the growth traits of H.discus hannai were studied at the gene,transcriptional and epigenetic levels.The differentially expressed mRNAs,lncRNAs,miRNAs and the key molecular signaling pathways affecting the growth and muscle growth of H.discus hannai were studied by using RNA-seq,miRNA-seq and various bioinformatics analysis techniques.The functions of the candidate genes,such as hdh-myostatin,hdh-BMP7,hdh-miR-1984 and hdh-MIRP1 were studied by molecular biological techniques including gene cloning,qRT-PCR,Dual Luciferase Reporter Assay,RNAi,prokaryotic expression,and GST pull down.In addition,the micromanipulation techniques and gene editing techniques suitable for abalone were constructed.The main research results are as follows:1.Study on mRNA,lncRNA and microRNA related to growth traits of H.discus hannaiThe adductor muscle of H.discus hannai with significant difference on body weight were collected for RNA-seq and miRNA-seq sequencing.A total of 11,668 mRNA transcripts,2,463 lncRNAs and 205 miRNAs were obtained.18 mRNAs（such as GDF8 and BMP7）,27 lncRNAs（such as XLOC036689）,and 7 miRNAs（such as hdh-miR-1984）,were speculated to be the key candidate genes for growth regulating of H.discus hannai.Further bioinformatics analysis found that the mRNA,lncRNA,and miRNA were mainly through the control of energy metabolism,insulin signaling pathway,TGF-beta signaling pathway and MAPK signal pathway to regulate the growth of the H.discus hannai.2.Cloning,expression analysis and functional verification of hdh-myostatin of H.discus hannaiThe CDS region of hdh-myostatin of H.discus hannai was cloned with a total length of 1,470 bp,encoding 489 amino acids.The hdh-myostatin mRNA was expressed at all growth stages and widely distributed in different tissues.9 SNPs were proven to be significantly correlated with the growth traits of H.discus hannai.These SNP sites of hdh-myostatin could be used as a molecular marker-assistant breeding for growth traits of H.discus hannai.The results of RNA interference（RNAi）showed that the shell length and body weight gain for the treatment group were significantly higher compared with the control group,and the expression levels of hdh-TβRI,hdh-ActRIIB,hdh-Smad3 and MHC all responded to some extent.Subsequently,the CDS region of df-myostatin-like of H.discus hannai ♀×Haliotis fulgens ♂（DF）was cloned and compared with hdh-myostatin.The results of qRT-PCR showed that the expression of myostatin mRNA in larger individuals were lower than that in smaller individuals of the same age in DD and DF.The expression level of myostatin mRNA in DF was lower than that in the same age abalone of DD.Generally speaking,hdh-myostatin was closely related to the growth of H.discus hannai,which could inhibit the growth of Haliotis discus hannai.3.Cloning,expression analysis and functional verification of hdh-BMP7 of H.discus hannaiThe CDS region of hdh-BMP7 of H.discus hannai was cloned with a total length of 1,251 bp,encoding 416 amino acids.The hdh-BMP7 mRNA was expressed at all growth stages and widely distributed in different tissues.There were 4 SNPs of the hdh-BMP7 were significantly correlated with the growth traits of H.discus hannai.These SNP sites of hdh-BMP7 could be used as a molecular marker-assistant breeding for growth traits of H.discus hannai.The results of RNAi show that the final live weight,the increase in shell length,the increase in shell width and the body weight gain were decreased significantly in treatment group compared with the control group.Interference hdh-BMP7 after a month,the expression level of hdh-BMPRⅠ,hdh-BMPRⅡ,hdh-Smad1,and the MHC were significantly lower compared with the control group.Then,we cloned the CDS region of df-BMP7 of DF,which obtained the same length as that of H.discus hannai.The results of qRT-PCR showed that the expression of hdh-BMP7 mRNA in larger individuals were higher than that in smaller individuals of the same age abalone in DD and DF.The expression level of hdh-BMP7 in DF was higher than that in the same age abalone of DD.In conclusion,these results suggest that hdh-BMP7 is closely related to the growth of H.discus hannai,and hdh-BMP7 promotes the growth of H.discus hannai.4.Study on the function of hdh-miR-1984 in regulating growth traits of H.discus hannaiThe results of qRT-PCR showed that hdh-miR-1984 was expressed in all growth stages of H.discus hannai,and the abundance of hdh-miR-1984 fluctuated with the age,which was widely distributed in different tissues.The DLR Assay showed that hdh-miR-1984 could bind to the 3’UTR of hdh-BMP7.The increase in shell length and the living body weight gain were increased significantly in injection antagomir group compared with the PBS control group of H.discus hannai.Interference hdh-miR-1984 after a month,the expression level of the hdh-BMP7,hdh-BMPRI,hdh-BMPRII,and the MHC were significantly increased compared with the PBS group.In conclusion,these results suggest that hdh-miR-1984 is closely related to the growth of H.discus hannai.Hdh-miR-1984 may negatively regulate the muscle growth of abalone by inhibiting the expression of target gene hdh-BMP7,and ultimately regulate the growth of abalone.5.Preliminary study on the function of hdh-MIRP1 in H.discus hannaiThe CDS region of hdh-MIRP1 was 456 bp in length and encoded 151 amino acids.The hdh-MIRP1 is mainly expressed in the cerebral ganglia of H.discus hannai,and almost not expressed in other tissues.There were one SNPs in the CDS region was significantly correlated with the meat yield of H.discus hannai.The SNP site could be used as a molecular marker-assistant breeding for growth traits of H.discus hannai.The results of prokaryotic expression,GST pull down and protein profile detection showed that hdh-MIRP1 mainly interacted with 82 proteins.Subsequently,the df-MIRP1-like was obtained by cloning and compared with hdh-MIRP1.The qRT-PCR results showed that the tissue distribution of the df-MIRP1-like was consistent with the results of the hdh-MIRP1.The expression of MIRP1 mRNA in larger individuals were higher than that in smaller individuals of the same age in DD and DF.The expression level of MIRP1 mRNA in DF was higher than that in the same age of DD,which further indicated that MIRP1 may be actively involved in the growth regulation of abalone.6.Establishment of microinjection and gene editing methods for H.discus hannaiThe microinjection technology was successfully established for the first time in abalone.For Nodal,the Golden Gate method was used to assemble TALEN,and the Nodal TALEN mRNA was micro-injected into the unfertilized eggs of H.discus hannai.After artificial insemination and mutation detection,the successful mutation of Nodal was found,indicating that a TALEN-mediated genome editing system suitable for H.discus hannai was successfully constructed.At the same time,the CRISPR/Cas9-mediated genome editing technology was explored,but no mutation was found,and more experimental exploration is further needed.All in all,this is the first study of genomic editing on specific sites of abalone. |
PDF Full Text Request