水稻黄嘌呤脱氢酶基因OsXDH克隆及表达分析

[目的]克隆水稻黄嘌呤脱氢酶(Xanthine dehydrogenase,XDH)基因(OsXDH),分析其生物信息学特性及表达特性,为研究XDH在水稻生长发育和响应逆境胁迫中的调控机制提供理论依据.[方法]以粳稻品种日本晴为材料,采用同源克隆技术克隆OsXDH基因,应用生物信息学方法对其氨基酸序列进行分析.利用实时荧光定量PCR (qPCR)检测OsXDH基因的组织表达特性及逆境胁迫下的表达情况,并对不同转基因株系乳熟期剑叶OsXDH基因表达量、XDH活性和叶绿素含量进行比较分析.[结果]克隆获得OsXDH基因的开放阅读框序列(ORF)(GenBank登录号LOC4333171),其长度为4110 bp,编码1369个氨基酸.OsXDH蛋白分子量大小为150.23 kD,理论等电点(pI)为6.54,与小麦、高粱、玉米、谷子和油菜等作物XDH蛋白氨基酸序列的相似性分别为84.54%、84.07%、81.52%、76.35%和69.22%,表明XDH蛋白氨基酸序列具有高度保守性.OsXDH基因在水稻不同组织部位均有表达,灌浆期的表达量显著高于苗期和分蘖盛期(P<0.05),且受干旱、黑暗、高温和盐胁迫诱导高效表达.OsXDH过表达水稻转基因株系乳熟期剑叶的XDH活性和叶绿素含量高于野生型,OsXDH干扰转基因株系的XDH活性和叶绿素含量低于野生型.[结论]OsXDH基因受水稻生长发育和逆境胁迫因子诱导表达,推测其是调控水稻生长发育和响应逆境胁迫的关键基因.

Cloning and expression analysis for xanthine dehydrogenase gene OsXDH in rice

[Objective]In the paper,xanthine dehydrogenase(XDH)gene(OsXDH)was cloned from rice genome and its basic biological information and expression characteristics was analyzed to provide theoretical basis for further study on the mechanism of XDH in regulating rice growth and responding to adversity stress.[Method]Oryza sativa L.spp.ja-ponica cv.Nipponbare was used as material.Gene OsXDH was cloned from rice genome by homologous cloning.Its ami-no acid sequence was analyzed by bioinformatics.Expression specificity of gene OsXDH and its expression under adversi-ty stress were studied by real-time fluorescent quantitative PCR(qPCR).The expression levels of OsXDH,XDH activity, and chlorophyll content in flag leaf of different transgenic lines at milk-ripe stage were studied.[Result]The sequence of open reading frame(ORF)of gene OsXDH was cloned(GenBank accession No.:LOC4333171). It was 4110 bp in length,encoding 1369 amino acids.The molecular weight of OsXDH protein was about 150.23 kD,with a theoretical iso-electric point(pI)of 6.54.The similarity of protein XDH amino acid sequences with Aegilops tauschhhii,Sorghum bico-lor,Zea mays,Setaria italica,and Brassica napus were 84.54%,84.07%,81.52%,76.35% and 69.22% respectively.Ami-no acid sequence of XDH protein was highly conservative.OsXDH expressed in all tissues of rice,and the expression at pustulation stage was significantly higher than seedling stage and tillering stage(P<0.05).The expression of gene OsXDH could be highly induced by the stresses of drought,darkness,high temperature and salt.Furthermore,XDH activity and leaf chlorophyll content in flag leaf of OsXDH over-expression transgenetic lines at milk ripe stage were higher than wild type,but XDH activity and leaf chlorophyll content in OsXDH interference transgenetic lines were lower than wild type.[Conclusion]The expression of gene OsXDH is induced by rice growth and adversity stress factors.It is inferred that OsX-DH is a crucial gene in rice for regulating grow and responding to adverse environment.