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
|
摘要
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.
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.
引文
[1]WangXQ,MaS,DongSL.Studiesonthebiologyand culturalecologyofLitopenaeusvannamei:areview[J].TransactionsofOceanologyandLimnology,2004(4):94-100.[王兴强,马甡,董双林.凡纳滨对虾生物学及养殖生态学研究进展[J].海洋湖沼通报, 2004(4):94-100.]
[2]Fisheries Bureau of the Ministry of Agriculture. China FisheryStatisticalYearbook2017[M].Beijing:ChinaAgriculturePress,2017.[农业部渔业局.2017年中国渔业统计年鉴[M].北京:中国农业出版社, 2017.]
[3]Ponce-PalafoxJ,Martinez-PalaciosCA,RossLG.The effectsofsalinityandtemperatureonthegrowthandsurvivalratesofjuvenilewhiteshrimp,Penaeusvannamei,Boone, 1931[J]. Aquaculture, 1997, 157(1-2):107-115.
[4]Zhao Y Z. Litopenaeus vannamei “Guihai No. 1”[J]. Ocean andFishery,2013(3):54.[赵永贞.凡纳滨对虾“桂海1号”[J].海洋与渔业, 2013(3):54.]
[5]MengXH,LuanS,LuoK,etal.Litopenaeusvannamei“Renhai No. 1”[J]. Chinese Fisheries, 2016(3):49-52.[孟宪红,栾生,罗坤,等.凡纳滨对虾“壬海1号”[J].中国水产,2016(3):49-52.]
[6]KongJ,HeJG,JiangXW,etal.Litopenaeusvannamei“Haixingnong No. 2”[J]. Ocean and Fishery, 2017(6):67-71.[孔杰,何建国,江谢武,等.凡纳滨对虾“海兴农2号”[J].海洋与渔业, 2017(6):67-71.]
[7]Anonymous.Litopenaeusvannamei“GuangtaiNo.1”[J].Ocean and Fishery, 2017(6):34.[佚名.凡纳滨对虾“广泰1号”[J].海洋与渔业, 2017(6):34.]
[8]Anonymous. Litopenaeus vannamei “Kehai No. 1”[J]. Ocean andFishery,2012(10):58.[佚名.凡纳滨对虾“科海1号”[J].海洋与渔业, 2012(10):58.]
[9]Anonymous.Litopenaeusvannamei“ZhongkeNo.1”[J].Ocean and Fishery, 2012(4):50.[佚名.凡纳滨对虾“中科1号”[J].海洋与渔业, 2012(4):50.]
[10]Anonymous.Litopenaeusvannamei“ZhongxingNo.1”[J].Farmers Science and Technology Training, 2013(12):38.[佚名.凡纳滨对虾“中兴1号”[J].农民科技培训, 2013(12):38.]
[11]Chun S X, Yao L K, Xing Y H, et al. Advances on the study ofmulti-geneexpressionsystem[J].ChinaBiotechnology,2011, 31(6):116-123.[楚素霞,姚伦广,邢延豪,等.多基因表达系统研究进展[J].中国生物工程杂志,2011,31(6):116-123.]
[12]Zeng D G, Chen X L, Xie D X, et al. Deep sequencing-based transcriptome analysis of Litopenaeus vannamei[J]. Genomics and Applied Biology, 2013, 2(3):308-313.[曾地刚,陈秀荔,谢达祥,等.基于高通量测序的凡纳滨对虾的转录组分析[J].基因组学与应用生物学, 2013, 2(3):308-313.]
[13]Yang M, Yu Y, Zhang X J, et al. Development of microsatellitemarkersfromthetranscriptomesequencesofPacific whiteshrimp(Litopenausvannamei)[J].MarineSciences,2017,41(2):96-102.[杨铭,于洋,张晓军,等.基于转录组数据的凡纳滨对虾微卫星标记开发[J].海洋科学, 2017,41(2):96-102.]
[14]Huang W, Ren C H, Li H M, et al. Transcriptomic analyses on muscle tissues of Litopenaeus vannamei provide the first profile insight into the response to low temperature stress[J].PLoS ONE, 2017, 12(6):e0178604.
[15]Grabherr M G, Haas B J, Yassour M, et al. Full-length transcriptome assembly from RNA-Seq data without a reference genome[J]. Nature Biotechnology, 2011, 29(7):644-652.
[16]PerteaG.Transcriptassemblyandquantificationby RNA-Seqrevealsunannotatedtranscriptsandisoform switchingduringcelldifferentiation[J].NatureBiotechnology, 2010, 28(5):511-515.
[17]AndersS,HuberW.Differentialexpressionanalysisfor sequence count data[J]. Genome Biology, 2010, 11:R106.
[18]Young M D, Wakefield M J, Smyth G K, et al. Gene ontology analysis for RNA-seq:accounting for selection bias[J].Genome Biology, 2010, 11:R14.
[19]Kanehisa M, Araki M, Goto S, et al. KEGG for linking genomestolifeandtheenvironment[J].NucleicAcidsResearch, 2008, 36:480-484.
[20]LivakKJ,SchmittgenTD.Analysisofrelativegeneexpression data using real-time quantitative PCR and the 2–ΔΔCTmethod[J]. Methods, 2001, 25(4):402-408.
[21]Kong X H, Wang G Z, Li S J, et al. Antioxidant effects and ATPaseactivitychangesinhepatopancreasofmudcrabScylla serrata under low temperature acclimation[J]. Journal of Fishery Sciences of China, 2005, 12(6):708-713.[孔祥会,王桂忠,李少菁,等.低温驯化下锯缘青蟹肝胰腺的抗氧化效应及ATPase活性变化[J].中国水产科学, 2005, 12(6):708-713.]
[22]SongZM,LiuJY,ZhuangP,etal.Influenceof low-temperature stress on the antioxidant enzymes activities andmalondialdehydecontentsinliverofjuvenileSiganus guttatas[J]. Marine Fisheries, 2015, 37(2):142-150.[宋志明,刘鉴毅,庄平,等.低温胁迫对点篮子鱼幼鱼肝脏抗氧化酶活性及丙二醛含量的影响[J].海洋渔业,2015,37(2):142-150.]
[23]Lynch M, Kuramitsu H. Expression and role of superoxide dismutases(SOD)in pathogenic bacteria 1[J]. Microbes and Infection, 2000, 2(10):1245-1255.
[24]Xu Q Q, Han B S, Luo J T, et al. Effects of cold stress on ROSproductionandexpressionofMAPKproteinsinzebrafishZF4cells[J].JournalofFisherySciencesofChina,2016, 23(4):771-776.[许琼琼,韩兵社,罗军涛,等.低温胁迫诱导斑马鱼ZF4细胞ROS及MAPK相关蛋白表达的影响[J].中国水产科学, 2016, 23(4):771-776.]
[25]Jiang W W, Fang J G, Li J Q, et al. Effects of temperature changeonphysiologicalandbiochemicalactivitiesofHaliotis discus hannai Ino[J]. Journal of Fishery Sciences of China Sciences, 2017, 24(2):220-230.[姜娓娓,方建光,李加琦,等.温度胁迫对皱纹盘鲍生理和生化活动的影响[J].中国水产科学, 2017, 24(2):220-230.]
[26]Rauen U, Tittel A, Kerkweg U, et al. 164. Role of lysosomes incold-inducedapoptosisofhepatocytes[J].Cryobiology,2006, 53(3):436-436.
[27]KlímaM,VítámvásP,ZelenkováS,etal.Dehydrinand proline content in Brassica napus and B. carinata under cold stress at two irradiances[J]. Biologia Plantarum, 2011, 56(1):157-161.
[28]Javadian N, Karimzadeh G, Mahfoozi S, et al. Cold-induced changes of enzymes, proline, carbohydrates, and chlorophyll inwheat[J].RussianJournalofPlantPhysiology,2010,57(4):540-547.
[29]Wang Y B, Miao J L, Jiang Y H, et al. Roles of proline and soluble sugar in the cold-adaptation of Antarctic ice microalgae[J]. Biotechnology Bulletin, 2016, 32(2):198-202.[王以斌,缪锦来,姜英辉,等.脯氨酸和可溶性糖在南极冰藻低温适应机制中的作用[J].生物技术通报,2016,32(2):198-202.]
[30]Davies P L, Hew C L. Biochemistry of fish antifreeze proteins[J]. FASEB Journal, 1990, 4(8):2460-2468.
[31]PengJX,YinQ,CuiL,etal.MolecularcloningofLitopenaeus vannamei TCP-1-Beta gene and analysis on its relationship with cold tolerance[J]. Acta Hydrobiologica Sinica,2011, 35(4):604-609.[彭金霞,殷勤,崔亮,等.凡纳滨对虾TCP-1-Beta基因的克隆及其与耐寒性状的相关性[J].水生生物学报, 2011, 35(4):604-609.]
[32]Peng J X, Fang Z F, Wei P Y, et al. Sequence and expression analysisofmetallothioneinfromLitopenaeusVannamei[J].Acta Hydrobiologica Sinica, 2013, 37(4):678-683.[彭金霞,房振峰,韦嫔媛,等.凡纳滨对虾MT基因序列及其在卵巢发育和低温胁迫中的表达分析[J].水生生物学报, 2013,37(4):678-683.]
[33]Peng J X, Lyu L H, Wei P Y, et al. Cloning characterization and expression analysis of a cold-inducible DEAD-box RNA helicasegeneinLitopenaeusvannamei[J].ActaHydrobiologica Sinica, 2016, 40(3):474-480.[彭金霞,吕丽虹,韦嫔媛,等.低温诱导型凡纳滨对虾DEAD-boxRNA解旋酶基因的克隆与表达分析[J].水生生物学报,2016,40(3):474-480.]
[34]Peng J X, Jiang X Z, Fang Z F, et al. Sequence and expression analysis of COPE gene from Litopenaeus vannamei[J].SouthwestChinaJournalofAgriculturalSciences,2013,26(1):371-376.[彭金霞,蒋小珍,房振峰,等.凡纳滨对虾COPE基因序列及低温表达分析[J].西南农业学报,2013, 26(1):371-376.]
[35]Yin Q, Cui L, Peng J X, et al. Molecular cloning of LVANT2geneanditsexpressionpatternbycoldinduction[J].Acta Hydrobiologica Sinica, 2012, 36(1):24-28.[殷勤,崔亮,彭金霞,等.凡纳滨对虾ANT2基因的克隆及低温表达谱分析[J].水生生物学报, 2012, 36(1):24-28.]
[36]Peng J X, Wei P Y, Yin Q, et al. Sequence of HSP10 gene inLitopenaeusvannameianditslowtemperatureexpression analysis[J].SouthwestChinaJournalofAgriculturalSciences,2013,44(5):838-843.[彭金霞,韦嫔媛,殷勤,等.凡纳滨对虾HSP10基因序列与低温表达分析[J].两南农业学报, 2013, 44(5):838-843.]
[37]Yu K, Ye H H, Huang C C, et al. Effects of different temperature and salinity on the expression of adenine nucleotide translocase2(ANT2)m RNAinthemudcrab,Scyllaparamamosain[J]. Journal of Fishery Sciences of China, 2014,21(6):1172-1180.[于坤,叶海辉,黄陈翠,等.拟穴青蟹ANT2基因在不同温度和盐度条件下的应激表达[J].中国水产科学, 2014, 21(6):1172-1180.]
[38]Liu B, Wang M Y, Xie J, et al. Effects of acute cold stress onserumbiochemicalandimmuneparametersandliver HSP70geneexpressioninGIFTstrainofNiletilapia(Oreochromisniloticus)[J].ActaEcologicaSinica,2011,31(17):4866-4873.[刘波,王美垚,谢骏,等.低温应激对吉富罗非鱼血清生化指标及肝脏HSP70基因表达的影响[J].生态学报, 2011, 31(17):4866-4873.]
[39]Chen Z Z, Cheng C H C, Zhang J F, et al. Transcriptomic andgenomicevolutionunderconstantcoldinAntarctic notothenioidfish[J].ProceedingsoftheNationalAcademy of Sciences of the United States of America, 2008, 105(35):12944-12949.
[40]Stéphane G, Hélène J, Alain V, et al. At MRP6/AtABCC6, an ATP-Bindingcassettetransportergeneexpressedduring earlystepsofseedlingdevelopmentandup-regulatedby cadmiuminArabidopsisthaliana[J].BMCPlantBiology,2008, 8:22.
[41]Fang C W, Huang Q Y, Ling X P, et al. Stress proteins of gill tissue in Patinopecten yessoensis exposed to cadmium salt[J].ChemicalJournalofChineseUniversities,2010,31(3):507-513.[方财王,黄清育,凌雪萍,等.在镉盐胁迫下扇贝鳃组织应激蛋白的研究[J].高等学校化学学报,2010,31(3):507-513.]
[42]Xia D D, Ma A J, Huang Z H, et al. Effect of environmental stress on the function of C-type lectin in turbot(Scophthalmus maximus)[J]. Journal of Fisheries of China, 2017, 41(2):161-169.[夏丹丹,马爱军,黄智慧,等.环境胁迫对大菱鲆C-型凝集素功能的影响[J].水产学报,2017,41(2):161-169.]
[43]Li M, Zhou S M, Liu L, et al. Molecular clone and expression of C-type lectin in Meretrix meretrix[J]. Oceanologia et Limnologia Sinica, 2015, 46(5):1186-1192.[李猛,周素明,刘璐,等.文蛤(Meretrix meretrix)C-型凝集素基因的分子克隆及表达分析[J].海洋与湖沼, 2015, 46(5):1186-1192.]
[44]ZhangT,SunCB,GuanRL.Effectofmulti-factorson energymetabolismofjuvenileLitopenaeusvannamei[J].Journal of Tropical Organisms, 2012, 3(1):11-15.[张特,孙成波,关仁磊.多因子对凡纳滨对虾仔虾能量代谢的影响[J].热带生物学报, 2012, 3(1):11-15.]
[45]Lu B B, Zhou W, Zhang J, et al. Effect of temperature on expressionofglutaminesynthetaseandNADH-glutamate synthaseinriceplants[J].JournalofWuhanUniversity(Science Edition), 2002, 48(2):239-242.[陆彬彬,周卫,张吉,等.温度对水稻谷氨酰胺合成酶和NADH-谷氨酸合酶表达的影响[J].武汉大学学报(理学版),2002,48(2):239-242.]
[46]He P P, Wei P Y, Zhao Y Z, et al. Effects of different low temperaturestressonactivitiesofdigestiveenzymesin Penaeusvannamei[J].Southwest China Journal of Agricultural Sciences, 2017, 30(1):233-237.[何苹萍,韦嫔媛,赵永贞,等.不同程度低温胁迫对凡纳滨对虾主要消化酶活性的影响[J].西南农业学报, 2017, 30(1):233-237.]
[47]Zhu M K, Yao C L. The impact of temperature stress on the oxygenmetabolismandenergymetabolisminthehepatopancreasofshrimpLitopenaeusvannamei[J].Journalof Fisheries of China, 2015, 39(5):669-678.[朱孟凯,姚翠鸾.温度胁迫对凡纳滨对虾肝胰腺氧代谢及能量代谢的影响[J].水产学报, 2015, 39(5):669-678.]
[2]Fisheries Bureau of the Ministry of Agriculture. China FisheryStatisticalYearbook2017[M].Beijing:ChinaAgriculturePress,2017.[农业部渔业局.2017年中国渔业统计年鉴[M].北京:中国农业出版社, 2017.]
[3]Ponce-PalafoxJ,Martinez-PalaciosCA,RossLG.The effectsofsalinityandtemperatureonthegrowthandsurvivalratesofjuvenilewhiteshrimp,Penaeusvannamei,Boone, 1931[J]. Aquaculture, 1997, 157(1-2):107-115.
[4]Zhao Y Z. Litopenaeus vannamei “Guihai No. 1”[J]. Ocean andFishery,2013(3):54.[赵永贞.凡纳滨对虾“桂海1号”[J].海洋与渔业, 2013(3):54.]
[5]MengXH,LuanS,LuoK,etal.Litopenaeusvannamei“Renhai No. 1”[J]. Chinese Fisheries, 2016(3):49-52.[孟宪红,栾生,罗坤,等.凡纳滨对虾“壬海1号”[J].中国水产,2016(3):49-52.]
[6]KongJ,HeJG,JiangXW,etal.Litopenaeusvannamei“Haixingnong No. 2”[J]. Ocean and Fishery, 2017(6):67-71.[孔杰,何建国,江谢武,等.凡纳滨对虾“海兴农2号”[J].海洋与渔业, 2017(6):67-71.]
[7]Anonymous.Litopenaeusvannamei“GuangtaiNo.1”[J].Ocean and Fishery, 2017(6):34.[佚名.凡纳滨对虾“广泰1号”[J].海洋与渔业, 2017(6):34.]
[8]Anonymous. Litopenaeus vannamei “Kehai No. 1”[J]. Ocean andFishery,2012(10):58.[佚名.凡纳滨对虾“科海1号”[J].海洋与渔业, 2012(10):58.]
[9]Anonymous.Litopenaeusvannamei“ZhongkeNo.1”[J].Ocean and Fishery, 2012(4):50.[佚名.凡纳滨对虾“中科1号”[J].海洋与渔业, 2012(4):50.]
[10]Anonymous.Litopenaeusvannamei“ZhongxingNo.1”[J].Farmers Science and Technology Training, 2013(12):38.[佚名.凡纳滨对虾“中兴1号”[J].农民科技培训, 2013(12):38.]
[11]Chun S X, Yao L K, Xing Y H, et al. Advances on the study ofmulti-geneexpressionsystem[J].ChinaBiotechnology,2011, 31(6):116-123.[楚素霞,姚伦广,邢延豪,等.多基因表达系统研究进展[J].中国生物工程杂志,2011,31(6):116-123.]
[12]Zeng D G, Chen X L, Xie D X, et al. Deep sequencing-based transcriptome analysis of Litopenaeus vannamei[J]. Genomics and Applied Biology, 2013, 2(3):308-313.[曾地刚,陈秀荔,谢达祥,等.基于高通量测序的凡纳滨对虾的转录组分析[J].基因组学与应用生物学, 2013, 2(3):308-313.]
[13]Yang M, Yu Y, Zhang X J, et al. Development of microsatellitemarkersfromthetranscriptomesequencesofPacific whiteshrimp(Litopenausvannamei)[J].MarineSciences,2017,41(2):96-102.[杨铭,于洋,张晓军,等.基于转录组数据的凡纳滨对虾微卫星标记开发[J].海洋科学, 2017,41(2):96-102.]
[14]Huang W, Ren C H, Li H M, et al. Transcriptomic analyses on muscle tissues of Litopenaeus vannamei provide the first profile insight into the response to low temperature stress[J].PLoS ONE, 2017, 12(6):e0178604.
[15]Grabherr M G, Haas B J, Yassour M, et al. Full-length transcriptome assembly from RNA-Seq data without a reference genome[J]. Nature Biotechnology, 2011, 29(7):644-652.
[16]PerteaG.Transcriptassemblyandquantificationby RNA-Seqrevealsunannotatedtranscriptsandisoform switchingduringcelldifferentiation[J].NatureBiotechnology, 2010, 28(5):511-515.
[17]AndersS,HuberW.Differentialexpressionanalysisfor sequence count data[J]. Genome Biology, 2010, 11:R106.
[18]Young M D, Wakefield M J, Smyth G K, et al. Gene ontology analysis for RNA-seq:accounting for selection bias[J].Genome Biology, 2010, 11:R14.
[19]Kanehisa M, Araki M, Goto S, et al. KEGG for linking genomestolifeandtheenvironment[J].NucleicAcidsResearch, 2008, 36:480-484.
[20]LivakKJ,SchmittgenTD.Analysisofrelativegeneexpression data using real-time quantitative PCR and the 2–ΔΔCTmethod[J]. Methods, 2001, 25(4):402-408.
[21]Kong X H, Wang G Z, Li S J, et al. Antioxidant effects and ATPaseactivitychangesinhepatopancreasofmudcrabScylla serrata under low temperature acclimation[J]. Journal of Fishery Sciences of China, 2005, 12(6):708-713.[孔祥会,王桂忠,李少菁,等.低温驯化下锯缘青蟹肝胰腺的抗氧化效应及ATPase活性变化[J].中国水产科学, 2005, 12(6):708-713.]
[22]SongZM,LiuJY,ZhuangP,etal.Influenceof low-temperature stress on the antioxidant enzymes activities andmalondialdehydecontentsinliverofjuvenileSiganus guttatas[J]. Marine Fisheries, 2015, 37(2):142-150.[宋志明,刘鉴毅,庄平,等.低温胁迫对点篮子鱼幼鱼肝脏抗氧化酶活性及丙二醛含量的影响[J].海洋渔业,2015,37(2):142-150.]
[23]Lynch M, Kuramitsu H. Expression and role of superoxide dismutases(SOD)in pathogenic bacteria 1[J]. Microbes and Infection, 2000, 2(10):1245-1255.
[24]Xu Q Q, Han B S, Luo J T, et al. Effects of cold stress on ROSproductionandexpressionofMAPKproteinsinzebrafishZF4cells[J].JournalofFisherySciencesofChina,2016, 23(4):771-776.[许琼琼,韩兵社,罗军涛,等.低温胁迫诱导斑马鱼ZF4细胞ROS及MAPK相关蛋白表达的影响[J].中国水产科学, 2016, 23(4):771-776.]
[25]Jiang W W, Fang J G, Li J Q, et al. Effects of temperature changeonphysiologicalandbiochemicalactivitiesofHaliotis discus hannai Ino[J]. Journal of Fishery Sciences of China Sciences, 2017, 24(2):220-230.[姜娓娓,方建光,李加琦,等.温度胁迫对皱纹盘鲍生理和生化活动的影响[J].中国水产科学, 2017, 24(2):220-230.]
[26]Rauen U, Tittel A, Kerkweg U, et al. 164. Role of lysosomes incold-inducedapoptosisofhepatocytes[J].Cryobiology,2006, 53(3):436-436.
[27]KlímaM,VítámvásP,ZelenkováS,etal.Dehydrinand proline content in Brassica napus and B. carinata under cold stress at two irradiances[J]. Biologia Plantarum, 2011, 56(1):157-161.
[28]Javadian N, Karimzadeh G, Mahfoozi S, et al. Cold-induced changes of enzymes, proline, carbohydrates, and chlorophyll inwheat[J].RussianJournalofPlantPhysiology,2010,57(4):540-547.
[29]Wang Y B, Miao J L, Jiang Y H, et al. Roles of proline and soluble sugar in the cold-adaptation of Antarctic ice microalgae[J]. Biotechnology Bulletin, 2016, 32(2):198-202.[王以斌,缪锦来,姜英辉,等.脯氨酸和可溶性糖在南极冰藻低温适应机制中的作用[J].生物技术通报,2016,32(2):198-202.]
[30]Davies P L, Hew C L. Biochemistry of fish antifreeze proteins[J]. FASEB Journal, 1990, 4(8):2460-2468.
[31]PengJX,YinQ,CuiL,etal.MolecularcloningofLitopenaeus vannamei TCP-1-Beta gene and analysis on its relationship with cold tolerance[J]. Acta Hydrobiologica Sinica,2011, 35(4):604-609.[彭金霞,殷勤,崔亮,等.凡纳滨对虾TCP-1-Beta基因的克隆及其与耐寒性状的相关性[J].水生生物学报, 2011, 35(4):604-609.]
[32]Peng J X, Fang Z F, Wei P Y, et al. Sequence and expression analysisofmetallothioneinfromLitopenaeusVannamei[J].Acta Hydrobiologica Sinica, 2013, 37(4):678-683.[彭金霞,房振峰,韦嫔媛,等.凡纳滨对虾MT基因序列及其在卵巢发育和低温胁迫中的表达分析[J].水生生物学报, 2013,37(4):678-683.]
[33]Peng J X, Lyu L H, Wei P Y, et al. Cloning characterization and expression analysis of a cold-inducible DEAD-box RNA helicasegeneinLitopenaeusvannamei[J].ActaHydrobiologica Sinica, 2016, 40(3):474-480.[彭金霞,吕丽虹,韦嫔媛,等.低温诱导型凡纳滨对虾DEAD-boxRNA解旋酶基因的克隆与表达分析[J].水生生物学报,2016,40(3):474-480.]
[34]Peng J X, Jiang X Z, Fang Z F, et al. Sequence and expression analysis of COPE gene from Litopenaeus vannamei[J].SouthwestChinaJournalofAgriculturalSciences,2013,26(1):371-376.[彭金霞,蒋小珍,房振峰,等.凡纳滨对虾COPE基因序列及低温表达分析[J].西南农业学报,2013, 26(1):371-376.]
[35]Yin Q, Cui L, Peng J X, et al. Molecular cloning of LVANT2geneanditsexpressionpatternbycoldinduction[J].Acta Hydrobiologica Sinica, 2012, 36(1):24-28.[殷勤,崔亮,彭金霞,等.凡纳滨对虾ANT2基因的克隆及低温表达谱分析[J].水生生物学报, 2012, 36(1):24-28.]
[36]Peng J X, Wei P Y, Yin Q, et al. Sequence of HSP10 gene inLitopenaeusvannameianditslowtemperatureexpression analysis[J].SouthwestChinaJournalofAgriculturalSciences,2013,44(5):838-843.[彭金霞,韦嫔媛,殷勤,等.凡纳滨对虾HSP10基因序列与低温表达分析[J].两南农业学报, 2013, 44(5):838-843.]
[37]Yu K, Ye H H, Huang C C, et al. Effects of different temperature and salinity on the expression of adenine nucleotide translocase2(ANT2)m RNAinthemudcrab,Scyllaparamamosain[J]. Journal of Fishery Sciences of China, 2014,21(6):1172-1180.[于坤,叶海辉,黄陈翠,等.拟穴青蟹ANT2基因在不同温度和盐度条件下的应激表达[J].中国水产科学, 2014, 21(6):1172-1180.]
[38]Liu B, Wang M Y, Xie J, et al. Effects of acute cold stress onserumbiochemicalandimmuneparametersandliver HSP70geneexpressioninGIFTstrainofNiletilapia(Oreochromisniloticus)[J].ActaEcologicaSinica,2011,31(17):4866-4873.[刘波,王美垚,谢骏,等.低温应激对吉富罗非鱼血清生化指标及肝脏HSP70基因表达的影响[J].生态学报, 2011, 31(17):4866-4873.]
[39]Chen Z Z, Cheng C H C, Zhang J F, et al. Transcriptomic andgenomicevolutionunderconstantcoldinAntarctic notothenioidfish[J].ProceedingsoftheNationalAcademy of Sciences of the United States of America, 2008, 105(35):12944-12949.
[40]Stéphane G, Hélène J, Alain V, et al. At MRP6/AtABCC6, an ATP-Bindingcassettetransportergeneexpressedduring earlystepsofseedlingdevelopmentandup-regulatedby cadmiuminArabidopsisthaliana[J].BMCPlantBiology,2008, 8:22.
[41]Fang C W, Huang Q Y, Ling X P, et al. Stress proteins of gill tissue in Patinopecten yessoensis exposed to cadmium salt[J].ChemicalJournalofChineseUniversities,2010,31(3):507-513.[方财王,黄清育,凌雪萍,等.在镉盐胁迫下扇贝鳃组织应激蛋白的研究[J].高等学校化学学报,2010,31(3):507-513.]
[42]Xia D D, Ma A J, Huang Z H, et al. Effect of environmental stress on the function of C-type lectin in turbot(Scophthalmus maximus)[J]. Journal of Fisheries of China, 2017, 41(2):161-169.[夏丹丹,马爱军,黄智慧,等.环境胁迫对大菱鲆C-型凝集素功能的影响[J].水产学报,2017,41(2):161-169.]
[43]Li M, Zhou S M, Liu L, et al. Molecular clone and expression of C-type lectin in Meretrix meretrix[J]. Oceanologia et Limnologia Sinica, 2015, 46(5):1186-1192.[李猛,周素明,刘璐,等.文蛤(Meretrix meretrix)C-型凝集素基因的分子克隆及表达分析[J].海洋与湖沼, 2015, 46(5):1186-1192.]
[44]ZhangT,SunCB,GuanRL.Effectofmulti-factorson energymetabolismofjuvenileLitopenaeusvannamei[J].Journal of Tropical Organisms, 2012, 3(1):11-15.[张特,孙成波,关仁磊.多因子对凡纳滨对虾仔虾能量代谢的影响[J].热带生物学报, 2012, 3(1):11-15.]
[45]Lu B B, Zhou W, Zhang J, et al. Effect of temperature on expressionofglutaminesynthetaseandNADH-glutamate synthaseinriceplants[J].JournalofWuhanUniversity(Science Edition), 2002, 48(2):239-242.[陆彬彬,周卫,张吉,等.温度对水稻谷氨酰胺合成酶和NADH-谷氨酸合酶表达的影响[J].武汉大学学报(理学版),2002,48(2):239-242.]
[46]He P P, Wei P Y, Zhao Y Z, et al. Effects of different low temperaturestressonactivitiesofdigestiveenzymesin Penaeusvannamei[J].Southwest China Journal of Agricultural Sciences, 2017, 30(1):233-237.[何苹萍,韦嫔媛,赵永贞,等.不同程度低温胁迫对凡纳滨对虾主要消化酶活性的影响[J].西南农业学报, 2017, 30(1):233-237.]
[47]Zhu M K, Yao C L. The impact of temperature stress on the oxygenmetabolismandenergymetabolisminthehepatopancreasofshrimpLitopenaeusvannamei[J].Journalof Fisheries of China, 2015, 39(5):669-678.[朱孟凯,姚翠鸾.温度胁迫对凡纳滨对虾肝胰腺氧代谢及能量代谢的影响[J].水产学报, 2015, 39(5):669-678.]