基于转录组分析筛选凡纳滨对虾低温胁迫下的差异表达基因

Screening of differentially expressed genes related to the cold tolerance in Litopenaeus vannamei based on high-throughput transcriptome sequencing

凡纳滨对虾(Litopenaeusvannamei)是中国主要的对虾养殖品种之一,对低温的耐受性较差,低于18℃就会停止摄食。为了探究凡纳滨对虾耐低温性状相关基因,选用凡纳滨对虾低温胁迫组(18℃)和常温组(24℃)肝胰腺组织为实验材料,进行Illumina HiSeq 2500测序,对测序原始数据进行拼接、注释,以及筛选分析低温胁迫下的差异表达基因。结果显示,测序共获得50921条基因(unigene),平均长度为828 bp, N50为1589 bp,其中28.13%为已知基因。基因差异表达分析共筛选得到243个低温胁迫相关基因,其中89个上调表达, 154个下调表达。功能富集分析发现,差异表达基因多富集在生物结合和催化过程、过氧化物酶、溶酶体、精氨酸和脯氨酸的代谢以及氨基酸的生物合成途径中。进一步根据Q-值共筛选出10个差异表达最显著的基因,其中ATP结合盒B亚家族6转运蛋白基因(ATP-binding cassette sub-family B member 6, ABCB6)、C型凝集素基因(C type lectin)、谷氨酰胺合成酶基因(glutamine synthetase, GS)在低温胁迫下均呈下调表达,推测可能参与了凡纳滨对虾低温应答反应。利用Real time RT-PCR验证转录组数据,结果证明基于转录组测序筛选低温胁迫下的差异表达基因是可行的。本研究为揭示凡纳滨对虾耐低温分子调控机制提供了基础数据和理论依据。

Litopenaeus vannamei is native to the northern waters from the Pacific West Coast to Sonora,Mexico.Since its introduction to China from Hawaii in 1998,its area and product have increased every year and have become one of the most widely produced shrimp species in China.It is suitable for growth at temperatures of 25–35℃;below 18℃,it stops feeding.Low temperature limits the farming season and area of L.vannamei,thus affecting the economic benefits.In order to explore the genes related to the low temperature tolerance of L.vannamei,hepatopancreases were chosen from the low temperature stress group (18℃) and the normal temperature group (24℃) of three families to conduct Illumina HiSeq 2500 sequencing.The analysis of the sequencing data via splicing,annotation,and differential expression revealed that a total of 214.6 million clean reads were obtained and assembled into 50921 final unigenes with an average length of 828 bp (N50=1534 bp).The assembled unigenes contained 14329 significant unigenes (28.13% of all unigenes) after BLASTX against the Nr database (E-value cut-off of 10^-5).Seven databases,Nr,Nt,GO,KEGG,Swiss-prot,KOG,and Pfam,were used to annotate 1573 unigenes,accounting for 3.08% of all unigenes.The results comparing the digital gene expression data between the challenged and control shrimp showed that under low temperature stress,the expression of 641 genes was up-regulated and 1036 genes were down-regulated in family 1;630 genes were up-regulated and 1343 genes were down-regulated in family 2;and 212 genes were up-regulated and 475 genes were down-regulated in family 3.Furthermore,243 genes were differentially expressed in all three families,including 89 and 154 genes whose expressions were up- and down-regulated,respectively,under low temperature stress.The functional enrichment analysis revealed that the differentially expressed genes were more abundant during binding,catalytic activity,the biosynthesis of amino acids,and peroxisome,lysosome,arginine,and proline metabolism.According to the Q-value,three of the top 10 genes included the ATP-binding cassette subfamily B member 6 (ABCB6),C-type lectin,and glutamine synthetase (GS).The ABCB6 gene encodes the ABC transporter and participates in the ABC transport mechanism.It has been found that the ABC transporter is involved in many abiotic stresses in plants.C-type lectin,as an immunological factor,plays an important role in the innate immune defense,and it has been demonstrated that temperature can affect its expression in both Psetta maxima and Meretrix meretrix.Glutamine synthase (GS) is involved in the regulation of ammonia nitrogen metabolism in crustaceans.Studies have shown that temperature can affect the metabolism of ammonia nitrogen in L.vannamei.C-type lectin and GS are both down-regulated under low temperature stress,so they may participate in the low temperature response mechanisms of L.vannamei,but further verification is needed.In this study,we selected 10 genes from 243 differentially expressed genes;five of the genes were up-regulated and five were down-regulated.Real time RT-PCR was used to verify the transcriptome sequencing results.The results showed that RNA-seq and real time RT-PCR produced similar expression patterns for the 10 different genes,which indicates that the differential gene expression results based on the transcriptome sequencing were credible.This study laid the foundation for the discovery of low temperature-related genes and molecular markers.It also provides a theoretical basis for in-depth discussion on the molecular determinant mechanism of low temperature resistance in L.vannamei and may guide the molecular breeding of L.vannamei in future studies.