葡萄SAP家族的鉴定与表达分析

Genome-Wide Identification and Expression Analysis of SAP Family in Grape

【目的】通过生物信息学分析明确SAP(Stress Associated Protein)家族在葡萄基因组中的数量、结构,利用qRT-PCR技术分析SAP家族的生物节律和在激素、非生物胁迫处理下的表达模式,为进一步探究SAP家族在葡萄中的作用奠定基础。【方法】在前期从山葡萄‘双优’(Vitis amurensis cv.Shuangyou)和欧洲葡萄‘红地球’(Vitis vinifera cv.Red Globe)0℃寒胁迫下转录组数据库中筛选出表达量明显上调的基因SAP15基础上,利用NCBI和葡萄基因组数据库中的BLAST功能,根据SAP家族的保守结构域,鉴定葡萄基因组中的SAP。利用生物信息学软件DNAMAN5.0、MEME、GSDS2.0、ExPASy和MEGA6等对VvSAPs序列、基因结构、蛋白质结构、理化性质、染色体定位和进化树等进行分析。采用qRT-PCR方法检测VvSAP家族的生物节律和在激素、逆境处理下的表达特征。【结果】从葡萄(Vitis vinifera)全基因组中鉴定得到15个SAP家族成员,均含有AN1保守结构域,大多含有A20保守结构域。按照其保守结构域和在染色体上的位置,依次命名为VvSAP1—VvSAP15,可分为ClassⅠ、ClassⅡ和ClassⅢ三类。理化性质分析表明,VvSAP家族编码氨基酸数目介于109—293,理论等电点分布在7.99—9.68。染色体定位分析发现,该基因家族的15个基因分布于葡萄的9条染色体上,其中第8条染色体上分布最多,有3个VvSAP。亚细胞定位分析表明,VvSAP家族主要在细胞核、叶绿体和细胞骨架中进行表达。VvSAP家族的二级结构主要以无规则卷曲和α-螺旋为主。基因结构分析表明,VvSAP1—VvSAP12的DNA序列中均无内含子,VvSAP13—VvSAP15的DNA序列中有1个内含子,基因结构高度保守。qRT-PCR分析结果表明,在VvSAP家族成员中,VvSAP1和VvSAP9在无处理和各处理下(激素和非生物胁迫)均表达极低或不表达,初步鉴定为假基因。除VvSAP10在400 mmol·L-1 NaCl处理下呈下调表达,其余基因均在50μmol·L-1 ABA、100μmol·L-1 SA和400 mmol·L-1 NaCl处理下呈上调表达,其中,VvSAP10—VvSAP14显著响应50μmol·L-1 ABA胁迫,处理24 h后相对表达量分别上调为0 h的37.19、36.63、21.69、58.34和267.35倍;VvSAP8和VvSAP11在NaCl处理4 h后相对表达量最高,分别为0 h的13.16和12.42倍;在4℃低温胁迫下,相对表达量上调最高的是VvSAP15,处理后8 h相对表达量为0 h的35.90倍。【结论】从葡萄基因组中共鉴定出15个SAP家族成员,初步鉴定得出VvSAP1和VvSAP9为假基因,15个基因分别分布于9条染色体上,并且结构进化高度保守。所有成员均相响应逆境,且均有昼夜节律变化,但该基因家族成员在不同逆境胁迫下的响应程度和在不同时间点的表达模式存在一定的差异。

【Objective】The aim of this paper was to identify the Stress Associated Protein(SAP)family genes of grape(Vitis vinifera L.)genome and to know the profile of SAP family regarding the number and structure of genes.The biological rhythm of SAP family and its expression pattern under different hormones and abiotic stress were analyzed by qRT-PCR.The study would provide theoretical basis for further exploring the roles of SAP family genes in grapevine.【Method】SAP15,a gene selected from the transcriptome database of Vitis amurensis cv.‘Shuangyou’and Vitis vinifera cv.‘Red Globe’,had a significantly up-regulated expression under the cold treatment.According to the conserved structural domains,the SAPs in grape genome-wide were identified by BLAST of NCBI and grape genome database.DNAMAN5.0,MEME,GSDS2.0,ExPASy and MEGA6 software were used for various bioinformatics analysis of VvSAPs,including the study of nucleotide sequences,gene structure,protein structure,physical and chemical properties,chromosome localization and phylogeny.Quantitative real-time PCR(qRT-PCR)was employed to detect the circadian rhythm of VvSAPs and their relative expression levels under hormone and abiotic stress.【Result】In total,15 SAPs with conservative domains of AN1 were identified from grape genome and most of them contained A20 conservative domain.They could be classified into ClassⅠ,ClassⅡand ClassⅢbased on their conservative structure domain and the location on chromosome in grape.Analysis of physical and chemical properties of VvSAP family showed that the numbers of amino acids were between 109 and293,and the theoretical equivalence points were between 7.99 and 9.68.Localization of genes on the chromosome revealed that 15VvSAPs were distributed on 9 chromosomes of the grape,and there were 3 SAPs on chromosome 8.By the sub-cellular location analysis,we noticed that VvSAPs were mainly expressed in nucleus,chloroplast and cytoskeleton in grape.The secondary structure of VvSAP were mainly irregular crimp andα-helix.The gene structures were highly conserved,and VvSAP1-VvSAP12 had no intron while VvSAP13-VvSAP15 contained one intron.The results of qRT-PCR analysis showed that VvSAP1 and VvSAP9 had extremely low expression or no expression under hormone and abiotic treatment,so they were preliminarily identified as pseudogenes.While VvSAP10 was proved to be down-regulated under 400 mmol·L-1 NaCl treatment,the remaining genes were up-regulated under 50μmol·L-1 ABA,100μmol·L-1 SA and 400 mmol·L-1 NaCl treatment,respectively.VvSAP10-VvSAP14 were highly responsive to 50μmol·L-1 ABA treatment,and their relative expressions were improved by 37.19,36.63,21.69,58.34 and 267.35 times,respectively after 24 hours treatment.After 4 hours under NaCl treatment,VvSAP8 and Vv SAP11 had the highest relative expression level,which were 13.16 and 12.42 times higher than those without NaCl treatment,respectively.The VvSAP15 had the highest response towards4℃t reatment,and its relative expression level was 35.90 times after 8 hours treatment comparing to the 0 hour.【Conclusion】15VvSAPs family were identified from the grape genome,of which VvSAP1 and VvSAP9 were preliminarily identified as pseudogenes.These members were localized across 9 chromosomes of the grape,and the evolution of VvSAP was highly conservative.All members of VvSAP family responded to adversity and had circadian rhythm,However,the expression pattern of VvSAPs was varied under different stress.