甘蔗B12D基因全长cDNA克隆与表达分析

Cloning and Expression Analysis of Full cDNA of B12D Gene in Sugarcane

对甘蔗（Saccharum officinarum L.）茎全长cDNA文库进行大规模测序,获得了B12D基因的全长cD-NA序列,命名为ScB12D（GenBank Accession number：KF714497）.生物信息学分析表明,该基因全长771 bp,编码87氨基酸（ORF,104 ～367 bp）;该基因编码的蛋白无信号肽,为亲水性非分泌型蛋白;有1个B12D家族保守结构域,二级结构为无规则卷曲;基因定位于质膜,参与能量代谢.同时,甘蔗SeB12D基因在不同物种间具有一定的保守性,在近缘植物中具有高度的保守性,与单子叶禾本科植物玉米、粟米、高粱及水稻的同源性超过90％,与双子叶甘薯和山茶的同源性在70％左右.荧光定量PCR表达分析结果表明,甘蔗ScB12D基因在根、蔗芽、茎（包括蔗皮和蔗髓）、叶片和叶鞘等组织中组成型表达,其中在叶鞘和根中表达量较高;在生物胁迫黑穗病胁迫下,该基因的表达量在所有时间段均呈下调趋势;在非生物胁迫时,该基因的表达受NaCl胁迫后表达量最高,在ABA胁迫下表达量次之,推测该基因的表达可能与甘蔗响应生物和非生物胁迫的机制有关.

A full length cDNA sequence of sugarcane gene was obtained from sugarcane （Saccharum officinarum L.） stem full length cDNA library through large-scale sequencing and validated by the corresponding bioinformatics analysis,termed ScB12D （Genbank Accession number：KF714497）.The full length of ScB12D gene was 771 bp,with a 264 bp open reading frame （ORF）,encoding 87 amino acids residues.The ScB12D of sugarcane was a basic protein,which has a conserved functional domain with the main function for energy metabolism,and this protein was located in plasma membrane.The main secondary structure element was random coil.At the same time,the ScB12D genes is conservative in different species,especially highly conservative in kindred plants.The B12D gene in sugarcane has a homology of more than 90％ with the same genes in the monocotyledonous gramineous plants of maize,millet,sorghum and rice,and around 70％ with the same genes in the dicotyledonous plants of sweet potatoes and camellia.Real-time quantitative PCR （RT-qPCR） analysis revealed that the expression of ScB12D was higher in root and sheath than in leaf sheath,stem （contain pith and skin）,lateral buds and leaf.Moreover,after inoculation with Sporisorium scitamineum,the expression of this gene decreased in the different time points.Meanwhile,the expression of ScB12D in the NaC1 stress was the highest in abiotic stresses and slightly lower under the abscisic acid stress.It can be inferred that the sugarcane B12D gene be associated with the reaction mechanism of sugarcane to biotic and abiotic stresses.