Functional Analysis Of Ethylene Receptor Gene OsETR4 From Rice Under Low Temperature Stress

Ethylene is a gaseous hormones, plays a huge regulating role when plants facing the biotic and abiotic stresses, also in the cell growth and development process. After combined with ethylene, the structure of ethylene receptors changed, and it will influence the expression of related genes. The ethylene receptor can also control the interaction between different signaling pathways, it determines whether a plant is sensitivity or insensitivity of ethylene, regulates the response of external environment stimulating and the process of growth and development in plants. Based on the important role of ethylene receptor in response to environmental stress, in this study,using rice ethylene receptor 4( Os ETR4) over-expressing transgenic material as the experimental project, we carried out functional analysis of Os ETR4 including phenotypic characterization, gene expression analysis, transcriptome analysis in low temperature.The results of this experiment are as follows:1. Molecular detection of genetically modified materials: Using hygromycin resistance detection and molecular analysis, two over-expressing transgenic lines,Os ETR4-1 and Os ETR4-2, were selected from T2 generation of Os ETR4 transgenic rice. Western-Blot revealed that target protein Os ETR4 was expressed in transgenic lines at the protein level.2. The cold tolerance analysis of Os ETR4 genes: The low temperature processing results of seed germination and seeding stage revealed that germination rate and seeding growth of Os ETR4-1 and Os ETR4-2 were significantly stronger than the control variety Kitaake under low temperature stress, showed that Os ETR4 gene plays an important role in rice germination and seedling growth period.3. Gene expression analysis: Real-time fluorescence quantitative QPCR( QPCR)showed that the gene expression of Os ETR4 in over-expressing lines increased rapidly at the beginning of the cold treatment, reached the peak at the 3h and then declined,became very low after the 12 h at the seedling stage. On the contrast, the control variety began a slow decline, and there was no expression after 12 h.4. Transcriptome analysis: Transcriptome analysis was conducted using the leaves after low temperature treatment for 6h. The results showed that there were5212 genes differently expressed in over-expressing lines compared to control variety, 2555 genes were up-regulated, 2657 genes were down-regulated, found out the biological process and signal pathway which the up-regulated genes mainly participated in under the low temperate.5. The preliminary screening of downstream genes: Five genes which related to stress tolerance were chosen from the up-regulated genes and confirmed by QPCR analysis. The results revealed that 2 Zinc-finger protein genes were differently expressed between the Os ETR4 lines under the low temperature treatments.