甜瓜肌醇半乳糖苷合成酶基因CmGAS1的表达与功能分析

甜瓜（Cucumis melo L.）是棉子糖系列寡糖（Raffinose Family Oligosaccharides，RFOs）转运型植物。肌醇半乳糖苷合成酶（GAS）是催化RFOs合成的关键酶。甜瓜CmGAS1基因（GenBank登录号为AY077642.1）的cDNA全长为1 903 bp，开放阅读框（ORF）为996 bp，编码331个氨基酸。CmGAS1蛋白分子量约为38 kD，理论等电点（pI）为4.81。氨基酸序列比对和系统进化树分析表明，CmGAS1氨基酸序列与黄瓜（AAO84915.1）亲缘关系最近，同源性为98.79%，与西瓜（Cla009222）同源性为97.58%。荧光定量结果表明，甜瓜叶片从幼叶（库）至成熟叶（源）CmGAS1的表达显著升高，第5节位叶片达到最大值。去掉50%叶片植株与对照相比，完全展开功能叶片的CmGAS1表达量明显升高。应用CmGAS1特异性启动子构建CmGAS1的过表达载体，并采用农杆菌介导进行甜瓜遗传转化，获得了PCR阳性转化植株，转化植株完全展开功能叶片的净光合速率以及叶片中的蔗糖、棉子糖、水苏糖含量与野生型相比均有所提高。推测CmGAS1在甜瓜叶片棉子糖系列寡糖合成过程中起重要作用。

Expression and Function Analysis of CmGAS1 in Melon

Melon belongs to a group of plants that mainly transport Raffinose Family Oligosac- charides（RFOs）. Galactinol synthase（GAS）is the key enzyme of RFOs synthesis. The aim of this study was to explore the function of CmGAS1 in melon. The full-length cDNA of CmGAS1（accession number：AY077642.1）was 1 903 bp，and the open reading frame（ORF）was 996 bp，encoding 331 amino acids. The molecular weight of CmGAS1 protein was about 38 kD，and the theoretical isoelectric point（pI）was 4.81. Amino acid sequence alignment and phylogenetic analysis showed that the amino acid sequences of CmGAS1 had the closest genetic relationship with cucumber（AAO84915.1），and the homology with cucumber was 98.79%. Homology with watermelon（Cla009222）was 97.58%. RT-qPCR analysis showed that the expression of CmGAS1 gene increased significantly from young leaves（sink）to mature leaves（source）. Expression of the fifth leaf reached the maximum value. Compared with the control，gene expression of the fully expanded functional leaves increased distinctly when 50% leaves were removed. The over expression vector of CmGAS1 was constructed with the specific promoter. The genetic transformation of melon was mediated by Agrobacterium. The positive plants were obtained by PCR identification. The net photosynthetic rates，the content of sucrose，raffinose and stachyose all increased in positive plants compared with the wild type. These results suggested that CmGAS1 played an important role in RFOs synthesis in melon leaves.