关键词: 狗枣猕猴桃, AkNAC2, 转录因子, 基因克隆, 农杆菌介导法, CBF通路

Abstract:

【Objective】The NAC transcription factors constitute the largest family of transcription factors in plants, and play an important role in plant resistance to abiotic stresses such as cold. Actinidia kolomikta is one of the most cold-resistant kiwifruits. Based on the transcriptome data of Actinidia kolomikta, a NAC transcription factor family member responding to cold-AkNAC2 was obtained. The function of AkNAC2 in cold response was further studied by constructing overexpression and inhibition expression lines, which provided available gene resources for cultivating cold-resistant kiwifruit varieties.【Method】The leaves cDNA of Actinidia kolomikta were used as experimental materials to clone AkNAC2, and its sequence information and protein structure were analyzed by bioinformatics. The subcellular localization of AkNAC2 was analyzed by GFP labeling. The tissue-specific expression information of AkNAC2 was obtained by transcriptome data, and the response of AkNAC2 to cold, salt and waterlogging stress was detected by qRT-PCR. The overexpression lines of AkNAC2 Nicotiana benthamiana and the inhibitory expression lines of Actinidia kolomikta were constructed. The phenotypic, physiological and biochemical indexes and the expression of CBF pathway related genes were compared between the transgenic lines and the control after cold treatment.【Result】The CDS length of AkNAC2 is 897 bp. and has a domain. AkNAC2 encoding 298 amino acids, was a hydrophilic acidic protein, its promoter contained multiple cis-elements related to abiotic stress. Phylogenetic analysis indicated that AkNAC2 was closely related to NAC2 of Actinidia eriantha. Subcellular localization showed that AkNAC2 is localized in the nucleus. The expression level of AkNAC2 was the highest in leaves, followed by pistillate flowers, staminate flowers, fruits and stems, and the lowest in roots. The expression of AkNAC2 could be induced by cold, salt and waterlogging stress, and the response of AkNAC2 to cold stress was the most obvious. Six Nicotiana benthamiana transgenic lines were obtained by Agrobacterium-mediated, named OE1-OE6, Among them, the gene expression levels of OE2, OE4 and OE6 were higher, which were used as follow-up research materials. Under 4 ℃, compared with wild-type Nicotiana benthamiana, the relative conductivity and malondialdehyde content of the three transgenic Nicotiana benthamiana lines were lower, the content of protective soluble sugar and soluble protein significantly increased, the activity of peroxidase and superoxide dismutase increased, and the antioxidant capacity significantly increased. The expression levels of CBF pathway-related genes (NbtICE1, NbtCOR47, NbtCBF1, NbtCBF2, NbtCBF3) were significantly up-regulated. The AkNAC2 inhibitory expression lines of Actinidia kolomikta were obtained by transient transformation. qRT-PCR showed that the expression of AkNAC2 in the inhibitory expression lines was significantly lower than that in the wild type and the empty vector group. On the 4th day of treatment, the expression level reached the lowest, which was 0.40 times that of the wild type and 0.34 times that of the empty vector group. After 4 °C low temperature treatment, the relative conductivity and malondialdehyde content of the inhibited expression lines were significantly increased, the contents of protective substances such as soluble sugar and soluble protein were significantly decreased, the activities of peroxidase and superoxide dismutase were significantly decreased, and the expression levels of CBF pathway-related genes (AkCBF1.1 and AkCBF3) were significantly down-regulated.【Conclusion】A low temperature response gene AkNAC2 of Actinidia kolomikta was cloned, it may positively regulate the cold resistance of plants by affecting the expression of key genes in the CBF pathway and reducing oxidative stress.

Key words: Actinidia kolomikta, AkNAC2, transcription factor, gene cloning, Agrobacterium-mediated, CBF pathway