日本对虾热休克蛋白基因家族的鉴定及在发育时期的表达

Genome-Wide Identification of the HSP Gene Family in Penaeus japonicus and Their Expression Characteristics During Development Stages

热休克蛋白(HSPs)是一类广泛存在于原核和真核生物细胞的亚细胞区室中具有高度保守性的蛋白质,具有参与蛋白质折叠、蛋白质修复和免疫等多种功能,在机体生长发育过程中发挥重要作用。为进一步研究日本对虾(Penaeus japonicus)hsp基因家族特征及在胚胎发育时期的表达特征,本研究通过生物信息学技术对日本对虾hsp进行鉴定,并对其结构域、基因结构、染色体定位、编码蛋白的理化性质和在胚胎不同发育时期的表达特征进行分析,鉴别出15个Pjhsp基因,包括1个Pjhsp10、2个Pjhsp20、2个Pjhsp40、1个Pjhsp60、6个Pjhsp70和3个Pjhsp90,其中,7个蛋白的理化性质较稳定,不稳定系数小于40;亚细胞定位结果显示,7个Pj HSP定位到细胞质,2个定位到细胞核,2个定位到细胞外基质,2个定位到内质网,2个定位到线粒体。Pjhsp基因家族中Pjhsp40-15.349、Pjhsp70-39.287和Pjhsp70-1.298等在胚胎时期大量表达;Pjhsp20、Pjhsp70-3.662、Pjhsp70-15.369、Pjhsp70-32.916和Pjhsp90-12.759等在幼体发育时期大量表达。本研究明确了日本对虾hsp家族的基因特征、系统进化关系及其在发育时期的表达规律,丰富了日本对虾hsp家族的基础信息,为进一步探讨该家族在日本对虾生长发育中的作用提供了参考。

Heat shock proteins(HSPs),also known as heat stress proteins,can be divided into five main families,including HSP100,HSP90,HSP70,HSP60,and small heat shock proteins(sHSPs),according to their molecular weight.Further studies have shown that HSPs represent a class of highly conserved proteins that widely exist in the subcellular compartments of prokaryotic and eukaryotic cells.They have many functions,such as protein folding,repair,and immunity,and play important roles in organism growth and development.For example,Dmhsp70-A and Dmhsp70-B showed similar trends in the development of asexual embryos in Daphnia magna.However,they exhibited different trends in the development of sexual embryos despite high expression in both embryo types.Penaeus japonicus is one of the most important commercial shrimp species in China,Japan,and Southeast Asian countries.Relevant studies have shown that hsp genes widely exist in P.japonicus and play important roles in growth and development.For example,hsp10 and hsp60 were expressed in all tissues of P.japonicus,and the differential expression of hsp60 was identified in the early development stages.However,previous studies were based on single genes,and more systematic studies based on the role of the hsp gene family in the growth and development of P.japonicus have not been published.Our laboratory has completed the genome sequence of P.japonicus,which provides basic data for analyzing the molecular mechanism of hsp in the growth and development of P.japonicus.To further study the characteristics of the hsp gene family in P.japonicus and the expression characteristics of hsp genes during different development stages,the hsp gene family of P.japonicus was identified and analyzed using bioinformatics methods,and variation in its expression during the development of P.japonicus was measured using reverse transcription and quantitative real-time(RT-qPCR).Fifteen members of the hsp gene family were identified from the whole genome of P.japonicus via bioinformatics analysis,and their gene structures,motif compositions,chromosome localizations,and phylogenetic characteristics were analyzed.The expression of hsp genes in maternal-to-zygotic cells,blastocysts,gastrulae,limb buds,intramembranous nauplii,stage 1 nauplii,stage 1 zoeae,stage 1 myses,and stage 1 post-larval juveniles of P.japonicus was measured using qPCR.A total of 15 hsp genes were identified,including 1 hsp10,2 hsp20,2 hsp40,1 hsp60,6 hsp70,and 3 hsp90 genes.The physicochemical properties of seven proteins were stable,with instability coefficients less than 40.Subcellular localization results showed that most of the hsp gene family members were located in the cytoplasm,as seven hsp genes were localized to the cytoplasm,two to the nucleus,two to the extracellular region,two to the endoplasmic reticulum,and two to the mitochondria.Isoelectric point(pI)is related to the number and proportion of acidic and basic amino acids contained in a protein.Most HSPs exhibited a pI<7,suggesting they were acidic proteins.The 15 hsp genes were located on 11 chromosomes.Phylogenetic tree analysis showed that most of the HSP family members were first clustered with HSP family members from species such as Penaeus monodon,Litopenaeus vannamei,and Fenneropenaeus chinensis and then with HSP family members of other species,which was consistent with the traditional taxonomic status.A few HSP family members were clustered first with HSP family members from Eriocheir sinensis,D.melanogaster,and other insects,and then with HSP family members from other species.Motif analysis showed that the hsp gene family was highly conserved;however,the functions of different families were quite different.Structural domain analysis showed that the HSP family included cpn10-,metazoan ACD-,DnaJ bact superfamily-,GroEL-,PTZ00009 superfamily-,dnaK-,HSP70 superfamily-,HSP90 superfamily-,PRK14083 superfamily-,and HATpase_HSP90-like domains.Each gene family contained corresponding domains and was important in molecular chaperone processes.hsp genes were expressed in different developmental stages of P.japonicus and played important roles in growth and development.The trend of Pjhsp10-19.517 was similar to that of Pjhsp60-19.518,which was highly expressed during embryonic development and significantly downregulated during larval development.This is consistent with the conclusion of previous studies that HSP60 and HSP10 are molecular chaperones for each other to ensure the correct folding of the target protein.The hsp genes such as Pjhsp40-15.349,Pjhsp70-39.287,Pjhsp70-1.298,and Pjhsp90 were highly expressed in the embryonic period;Pjhsp20,Pjhsp70-3.662,Pjhsp70-15.369,Pjhsp70-32.916,and Pjhsp90-12.759 were highly expressed during the larval period.The hsp genes expressed at high levels during the embryonic period satisfy the high protein requirements of embryos by playing the role of molecular chaperone and regulating the transcription of vitellogenin to meet the embryonic need for vitellin.The hsp genes expressed at high levels during the larval period protect the body from environmental pressure,pathogens,and xenobiotics through a synergistic immune effect.The authors believe that hsp genes play important roles in the growth and development of P.japonicus,and the specific pathways and mechanisms should be further studied.This study provides basic data for further investigations of the role of hsp genes in the growth and development of P.japonicus.