森林草莓醇酰基转移酶基因FvAATW2功能研究

Studying Function of Alcohol Acyltransferase Gene FvAATW2 of Fragaria vesca by Over-expressing in Tobacco and Cultivated Strawberry

在从森林草莓(Fragaria vesca L.)成熟果实中克隆到醇酰基转移酶基因(FvAATW2)的基础上,构建由CaMV35S启动子驱动的植物正义表达载体pBI121-FvAATW2,在烟草(Nicotiana tabacum L.)和草莓(Fragaria×ananassa Duch.)‘Camarosa’品种中过量表达FvAATW2,以研究其功能。采用农杆菌介导的叶盘法转化烟草和草莓;利用PCR和Southern blot筛选出转基因株系;通过实时定量PCR和测定AAT酶活性来检测转基因植株中外源基因的表达;利用SPME/GC–MS方法检测烟草叶片和草莓果实中的挥发性成分。对转基因烟草外源基因表达和早期叶片挥发性成分的检测结果表明,构建的FvAATW2表达载体功能正常,转基因株系能够在生长早期合成酯类物质;通过检测转基因草莓成熟果实的酯类成分发现,与野生型对照相比,转基因草莓果实中酯类占挥发物的总比例以及乙酸辛酯、己酸乙酯、己酸辛酯、辛酸乙酯等挥发酯的比例显著提高,而丁酸甲酯的含量显著降低,辛酸乙酯的含量显著增加,说明外源FvAATW2在草莓中能够正常表达并影响果实酯类合成,从而通过改变挥发性酯类构成使果实香气变浓。

FvAATW2 was an alcohol acyltransferase gene,cloned from Fragaria vesca L. fruits by RT-PCR. In this study,the sense plant expression vector p BI121-FvAATW2 driven by cauliflower mosaic virus 35 S promoter was constructed through replacing GUS with FvAATW2,and then was used to genetically transform tobacco and a cultivated strawberry cultivar ‘Camarosa’,to study the function of FvAATW2. Agrobacterium tumefaciens strain LBA4404 carrying p BI121-FvAATW2 was employed to infect leaf-discs in the genetic transformation. Transgenic lines were identified by both PCR and Southern blot. The expression of exogenous FvAATW2 in transgenic in vitro plants was studied using relative enzyme activity and real-time quantitative PCR. It was found that FvAATW2 expressed in all 6 tested tobacco lines and AAT activity values of 5 lines were higher than wild-type plants. Volatiles in leaves of the transgenic tobacco with the highest AAT activity were also detected by SPME/GC-MS at 10 ~ 12 leaves phase. Octyl propanoate was found in transgenic leaves,while not any ester captured in wild-type plants at the same phase. The results showed that pBI121-FvAATW2 functioned correctly and esters could be synthesized much earlier in transgenic tobacco than in wild-type one. Four strawberry transgenic lines were obtained after PCR and Southern blot operation. A transgenic line bearing more aromatic fruits was screened out and used for the volatiles detection. Through analyzing the ester composition of mature transgenic fruits,it was found that the proportion of esters in total volatiles and respective proportions of octyl acetate,ethyl hexanoate,octyl hexanoate,and ethyl octanoate were higher significantly than those of wild-type fruits,while mass fraction of methyl butanoate decreased significantly and that of ethyl octanoate increased significantly. It was indicated that exogenous FvAATW2 expressed normally in cultivated strawberry and effected ester synthesis,and it could make fruit aroma intense by changing ester composition.