海带钒依赖溴过氧化物酶基因甄别和表达分析

Identification of Kelp (Saccharina japonica) Vanadium-Dependent Bromoperoxidase Encoding Genes and Their Expression Analysis

钒依赖溴过氧化物酶(Vanadium-dependent bromoperoxidase,vBPO)在富集碘的褐藻中广泛存在,催化碘化物生成,消除过量过氧化氢,保护藻体。因此,vBPO基因是褐藻天然免疫和防御应答的指示基因之一。中国广泛栽培海带(Saccharina japonica),但现行的“浮绳网藻体倒置避夏栽培”模式可能使海带孢子体持续性地处于强紫外线、强光照、高温度及频繁的光照和温度波动等环境因素的胁迫状态。这可能是导致海带病害越来越频繁的生理学原因。本研究用转录组测序方法分析了海带孢子体4个生长发育时期(薄嫩期、厚成期、成熟期和衰老期)vBPO基因的表达模式,获得了59条至少在一个生长发育时期表达的vBPO基因转录本组装物,其中的4条含有完整vBPO基因开放阅读框。用这4条序列含有的开放阅读框逆翻译成氨基酸序列并与从NCBI数据库下载的16个物种的28条vBPO氨基酸序列构建分子系统学树,发现它们的氨基酸序列与掌状海带(Laminaria digitata)的vBPO亲缘关系最近,并与所有褐藻vBPO聚为一枝。这说明这些转录本就是海带vBPO基因表达产物。研究还发现,59条海带vBPO基因转录本在4个发育时期的平均丰度是相应时期全部基因转录本的平均丰度的9~19倍,平均13倍,这说明海带天然免疫和防御应答机制之一的碘富集机制持续性处于高活性状态。研究还发现海带vBPO基因表达模式与碘含量变化呈正相关(r=0.974)。

Vanadium-dependent bromoperoxidase(vBPO)was widely found in brown algae enriching iodine,which catalyzes the biosynthesis of iodine,and assists brown algae to remove efficiently excessive hydrogen peroxide,thus protects the algae from damage.Therefore,vBPO gene is one of diverse genes involving in brown algal innate immunity and defense response,which may serve as one of the indicators of such defense response.Kelp(Saccharina japonica)has been cultivated in a mode called“summer-avoidingculture with inverted blade fixed below floating net”on a large scale and for a long time in China,which may have stressed kelp sporophytes continuously with strong ultra violet light and sunshine,high temperature,frequent shifts of sunshine and temperature among others,causing frequent outbreaks of physiological diseases.In this study,we determined the expression patterns of kelp vBPO genes at four growth and development stages(mushroom,adult,mature and aging)of its sporophytesthrough RNA-seq.In total,59 kelp vBPO unigenes(hereafter genes)were assembled,among them,4 contained the whole open reading frame(ORF).Twenty eight amino acid sequences of the vBPO of 16 species were retrieved from GenBank,and aligned with the deduced amino acid sequences of these 4 genes containing the whole ORF,and used to phylogenetic tree construction.The phylogenetic analysis showed that kelp vBPO were phylogenetically close to those of Laminaria digitata,forming a tight clade together with the vBPO of brown algae.This indicated that the 4 genes were truly kelp vBPO genes.In addition,the average abundance of all 59 genes was extremely higher than that of all kelp genes,which varied between 9 and 19 folds with an average of 13 folds,indicating that one of the innate immunity and defense response mechanisms of kelp,iodine enrichment,functions continuously and intensively.Our findings included also that the average abundance of vBPO gene transcripts was positively proportional to the iodine content of kelp sporophytes(r=0.974).