摘要
苹果(Malus domestica Borkh.)是世界上最重要的鲜食和加工果品之一。香气作为苹果品质的重要指标在一定程度上影响了消费者的购买欲望。挥发性酯类物质是构成成熟苹果特征香气最重要的成分之一。为了研究苹果酯类合成与调控机制,我们从金冠苹果中分离出酯类合成途径最末端的关键酶-醇酰基转移酶基因(MdAAT2),并对它的表达模式和调控机制进行了深入研究。主要结果如下:
1、苹果酯类挥发性成分提取与分析
对比了三种不同类型的SPME纤维头提取苹果酯类物质的效果。结果发现,PDMS/DVB 65μm Bonded SPME纤维头较适合苹果酯类挥发性成分的萃取,优化萃取条件为:50℃萃取30分钟。SPME-GCMS分析发现:丁酸己酯、乙酸己酯、乙酸丁酯、2-甲基丁酸己酯和己酸己酯是采后成熟金冠苹果中较重要的酯类物质。
2、MdAAT2基因分离及其特征
利用同源序列设计简并引物,通过RT-PCR和RACE-PCR技术分离了乔纳金苹果和金冠苹果果实中的醇酰基转移酶基因,分别命名为MdAAT1和MdAAT2。其全长分别为:1,639bp和1,628bp,二者在氨基酸水平上的同源性为94.26%,GenBank注册号分别为AY512893和AY517491。序列分析表明,MdAAT2大部分氨基酸区域为亲水结构,其中含有2个推测的跨膜区,分别在148aa和174aa。序列比对发现MdAAT2也含有BAHD基因家族和其它已知醇酰基转移酶基因共有的氨基酸保守区:HXXXD和FGWG。聚类分析发现,MdAAT2蛋白与同属蔷薇科仁果类的梨果实醇酰基转移酶蛋白亲缘关系最近,而与柠檬、草莓、玫瑰花等醇酰基转移酶蛋白亲缘关系较远。结果表明,我们已经分离到苹果醇酰基转移酶基因。
3、MdAAT2基因在E.coli中的表达
将MdAAT2基因编码区克隆到原核表达载体pET32a-c(+)在Origami B(DE3)中表达了部分可溶的融合蛋白,酶活检测发现重组表达载体菌株醇酰基转移酶活性显著高于对照,表明MdAAT2基因参与了苹果酯类物质合成。
4、MdAAT2基因的表达分析
RT-PCR发现MdAAT2在金冠果实中特异表达,在红星果实和花中特异表达,表明MdAAT2在不同器官的表达特性有品种间差异。免疫荧光原位杂交和Western blot
Apple (Malus domestica Borkh.) is the most important processing and table fruit all over the world. Aroma compounds are among the factors that determine fruit quality and influence on the final consumer acceptance of the commodity. Volatile esters are important aromatic components of apple fruit. In order to investigate the mechanism of ester biosynthesis and regulation, we isolated the gene (MdAAT2) of apple alcohol acyltransferase, which is the key enzyme that catalyzes the last step in ester formation by linkage of an acetyl moiety from acetyl CoA to appropriate alcohols. Results are as follows:
1. Extraction and analysis of apple volatile esters
Three types of SPME fibers were used and compared in esters extraction from apple fruit. The result showed that the PDMS/DVB 65μm Bonded SPME fiber is the best suitable fiber for esters extraction, and the optimal extraction is at 50℃for 30 minutes. SPME-GCMS analysis showed that the butyl acetate, hexyl acetate, hexyl 2-methylbutyrate, and hexyl butanoate are the most important characteristic ester compounds in ripened Golden Delicious apple fruit.
2. Isolation and characterization of MdAAT2 gene
According to the homologous sequences from other plants, degenerate primers were designed to amplify specific DNA fragment using cDNA prepared from apple fruit. By RT-PCR and RACE-PCR, we isolated two AAT genes from Jonagold and Golden Delicious apple fruit named MdAAT1 and MdAAT2, with the accession numbers AY512893 and AY517491, respectively. The full-length cDNA of MdAAT1 and MdAAT2 are 1,639bp and 1,628bp, respectively, and the deduced amino acid sequence of the two genes showed high identities, which is 94.26%. After analyzed by software, most of the deduced protein sequences of MdAAT2 are hydrophilic regions, which contain two speculated transmembrane(TM) segments at 148aa and 174aa, respectively. Sequence analysis shows that MdAAT2 exhibits the features of BAHD superfamily and other plant alcohol acyltransferases, including HXXXD and FGWG motifs. Further, phylogenetic analysis of various alcohol acyltransferases indicated that the apple AAT share one cluster with pear AAT, while have more distant with that of lemon, strawberry, and rose. Thus, it is most likely that we have isolated the apple alcohol acyltransferase gene.
3. Expression of the MdAAT2 gene in E.coli
After cloning the encoding region of MdAAT2 into prokaryotic expression vector pET-32a-c(+), we obtained the partial soluble fusion proteins in Origami B(DE3) strain.
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