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园艺学报 ›› 2014, Vol. 41 ›› Issue (8): 1601-1608.

• 蔬菜 • 上一篇    下一篇

甜瓜α–甘露糖苷酶基因促进果实成熟功能的瞬时表达分析

乌斯呼吉日嘎拉 达赖胡,郝金凤*,张立全,赵爱奇,哈斯阿古拉   

  1. (内蒙古大学生命科学学院,内蒙古自治区牧草与特色作物生物技术重点实验室,呼和浩特 010021)
  • 收稿日期:2014-04-16 出版日期:2014-08-25 发布日期:2014-08-25
  • 基金资助:
    国家自然科学基金项目(31101559)

Analysis of Function of α-mannosidase Gene in Promoting Melon Fruit Ripening by Transient Expression

USUKHJARGAL Dalaikhuu,HAO Jin-feng*,ZHANG Li-quan,ZHAO Ai-qi,and HASI Agula   

  1. (College of Life Sciences,Inner Mongolia University;Inner Mongolia Key Laboratory Herbage & Endemic Crop Biotechnology,Hohhot 010021,China)
  • Received:2014-04-16 Online:2014-08-25 Published:2014-08-25

摘要: 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。

关键词: 甜瓜, α–甘露糖苷酶基因, 基因瞬时表达, RT-qPCR

Abstract: Transient expression system was used to analyze the function of α-mannosidase gene during melon fruit ripening. The full-length α-mannosidase gene was cloned into overexpression vector pPZP221,and two interfering sequences against α-mannosidase gene were constructed into RNAi vector pART27 respectively. These vectors were then transformed into Agrobacterium GV3101 and injected into mature green fruits of melon respectively.The majority of the phenotype related to ripening appeared in the fruit pedicle when the bacterial concentration is OD600 = 1.2. Analysis of α-mannosidase activity showed that the enzyme activity in the tissue transformed by overexpression vector was higher than the non-injected tissue,and it was lower in the tissue transformed by RNAi vectors. The expression levels ofthe α-Man gene and six genes related to fruit ripening were analyzed by qRT-PCR. The results showed that the mRNA level of the α-Man gene in the tissue transformed by overexpression vector was elevated by 1.2 times compared with the non-injected tissue,furthermore the steady-state mRNA levels of the six genes related to ripening also significantly increased. The expression levels of the α-Man gene in the tissue transformed by pART-Man1 and pART-Man2 were reduced about 47% and 37% respectively in comparison to the non-injected tissue,and those of the genes related to ripening were decreased. Our results indicated that the α-mannosidase gene may promote fruit ripening in melon.

Key words: melon, α-mannosidase gene, transient expression, RT-qPCR

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