油茶PODs基因鉴定及在自交和异交下的表达分析

Expression Analysis under Self and Outcross Pollination and Identification of PODs Gene in Camellia oleifera Abel.

油茶(Camellia oleifera Abel.)具有自交不亲和性(SI,self-incompatibility),自然座果率低,这严重影响油茶产量,制约了油茶产业的发展。为研究过氧化物酶(POD,peroxidase)在油茶自交不亲和反应中的作用,本研究通过逆转录克隆技术从油茶中克隆出4条POD基因,分别命名为CoPOD1/2/3/4。其基因编码区长度分别为1086、1011、1020和1218 bp,编码361、336、339和405个无跨膜结构、有信号肽的蛋白质。同源序列比对显示CoPOD1/2/3/4蛋白质之间的同源性较低,但均有过氧化物酶活性位点和过氧化酶近端血红素配体序列;系统进化树显示CoPOD1/2/3/4蛋白质与茶树(Camellia sinensis(L.)O.Ktze.)POD蛋白质的亲缘关系最近。实时荧光定量结果显示,油茶PODs基因在自交授粉后24~48 h范围内呈现显著上调后又下降的趋势,在自交授粉36 h雌蕊中CoPOD1/3/4的表达量高于异交。自交授粉24~72 h油茶雌蕊内POD酶活性高于异交并在36 h达到最高值,异交授粉雌蕊内POD活性前期处于动态平衡状态,在72 h后出现明显上调,据此推测油茶PODs基因可能参与了油茶自交授粉后花粉管程序性死亡,进而参与了油茶自交不亲和反应。本研究为进一步研究油茶自交不亲和机制提供参考。

Camellia oleifera Abel.,which shows self-incompatibility(SI)and low natural fruit setting rate,has seriously affected its yield production and the following development in industry.To uncover the function of Peroxidase(POD)in self-incompatibility,four POD genes CoPOD1/2/3/4 were cloned from C.oleifera by reverse transcription cloning technology.The length of gene coding regions were 1086,1011,1020 and 1218 bp,encoding 361,336,339 and 405 amino acids non-transmembrane proteins with signaling peptides,respectively.Although lower sequence similarity among the CoPOD1/2/3/4 proteins is observed,they all contain peroxidase active sites and heme ligand sequences near peroxidase.The phylogenetic analysis revealed closely sequence homology of CoPOD1/2/3/4 to those of Camellia sinensis(L.)O.Ktze.Real-time fluorescence quantitative results showed that PODs were significantly up-regulated,followed by decreased expression within 24-48 h after self-pollination.The transcripts of CoPOD1/3/4 were higher than cross-pollinated in pistills at 36 h after self-pollination.The POD activity in the pistil of self-pollination was higher than that of cross-pollinated within 24-72 h,showing the highest expression peak at 36 h.The POD activity in the pistil of the cross-pollinated was in dynamic balance at early stage,with significantly up-regulated after 72 h.We speculate that the POD genes might be involved in the programmed pollen tube death of C.oleifera after self-pollination,thus participating in the self-incompatibility reaction.Collectively,this paper provides reference for further research on self-incompatibility mechanism in C.oleifera.