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为了研究α–甘露糖苷酶(α-Man)基因在甜瓜果实成熟过程中的功能,应用农杆菌介导的瞬时表达技术,分别将含有甜瓜α–甘露糖苷酶基因超表达载体pPZP221-Man、两个RNAi载体pART-Man1和pART-Man2的农杆菌菌液注射至甜瓜绿熟期果实,通过观察果实成熟表型初步鉴定该基因的功能。结果表明,菌液浓度OD600=1.2时果实表型比率最高,在果实蒂部表型居多。α–甘露糖苷酶活性测定结果显示,与非注射部位相比,注射超表达载体部位的组织α–甘露糖苷酶活性较高,而注射RNAi载体的组织α–甘露糖苷酶活性较低。RT-qPCR结果显示,与非注射部位相比,注射pPZP221-Man的部位α-Man基因表达约是对照的1.2倍,而注射pART-Man1和pART-Man2的部位α-Man基因的表达均有所下降,分别是对照的47%和37%。同时对甜瓜果实成熟相关的6个候选基因在注射部位的表达情况进行了分析,结果显示,这些基因在注射超表达载体的果实部位均上调表达;而在注射两种RNAi载体的果实部位均下调表达。这些结果表明甜瓜α–甘露糖苷酶基因在果实成熟过程中具有促进成熟的作用。
In order to study the function of α-mannosidase (α-Man) gene in melon fruit ripening, Agrobacterium tumefaciens-mediated transient expression technique was used to amplify α-mannosidase gene overexpression vector pPZP221-Man Agrobacterium tumefaciens were inoculated into the green melon fruits of melon by RNAi vector pART-Man1 and pART-Man2, and the function of the gene was preliminarily identified by observing the fruit mature phenotype. The results showed that when the concentration of OD600 was 1.2, the phenotype of fruit was the highest, and the phenotype of fruit was the highest. The results of α-mannosidase activity showed that α-mannosidase activity was higher in the overexpression vector than in the non-injection site, while the α-mannosidase activity was lower in the RNAi vector. The results of RT-qPCR showed that the expression of α-Man gene at pPZP221-Man site was about 1.2-fold compared with the non-injected site, while the expression of α-Man gene at pART-Man1 and pART-Man2 site was The decline was respectively 47% and 37% of the control. At the same time, the expression of six candidate genes related to melon fruit ripening at the injection site was analyzed. The results showed that these genes were upregulated in the fruit of injected overexpression vector and downregulated in the fruit of both RNAi vectors expression. These results indicate that melon α-mannosidase gene has maturation promoting effect during fruit ripening.