关键词: 林烟草 , 生物信息学 , KUP/HAK/KT家族 , NsHAK11 , 亚细胞定位 , 基因表达模式

Abstract: 【Objective】The aim of this study is to try to isolate and clone new potassium transporter KUP/HAK/KT gene in tobacco through bioinformatics method, then to determine its subcellular localization and analyze its expression. Using this method so as to provide reference for study about tobacco KUP/HAK/KT potassium transporter gene family.【Method】Firstly, by searching the genome database of Nicotiana sylvestris and N. tomentosiformis, the KUP/HAK/KT family members were predicted, and then phylogenetic trees of the above two tobaccos were constructed by multiple approaches of bioinformatics. According to the above results, a new N. sylvestris K+ transporter gene, NsHAK11, was found. Secondly, subcellular localization of the protein coded by the new gene was predicted through Wolf PSORT. Then the leaves of N. benthamiana were inoculated by infiltration with culture of Agrobacterium, and the subcellular localization of the protein was observed with laser scanning confocal microscope, which was based on the fluorescence produced by fused fluorescent protein. In this way, the prediction could be verified. Lastly, the expression pattern of NsHAK11 gene was predicted through PLACE website. The fluorescent quantitative RT-PCR method was adopted to detect the expression of NsHAK11 gene in two situations. On the one hand, the differential expression of NsHAK11 gene in different organs of N. sylvestris at flourishing flowering stage was studied. On the other hand, the expression changes of NsHAK11 gene in leaves of N. sylvestris at seedling stage under low temperature, high temperature and low-K+ stress were studied by using the same method, too. Thus, the authenticity of the prediction was verified by the fluorescent quantitative RT-PCR results.【Result】These preliminary results showed that both of N. sylvestris and N. tomentosiformis KUP/HAK/KT families have 19 members, and either of the two families could be divided into 4 clusters evolutionarily. According to the predicted sequence, a new gene of N. sylvestris KUP/HAK/KT family was cloned, which belongs to Cluster III. The new gene was named NsHAK11, because the protein coded by NsHAK11 has the highest similarity with AtKUP11 in Arabidopsis thaliana. However, the similarity of the protein with NrHAK1 and NtHAK1, which were acquired in tobacco, were only 48% and 49%. This showed that NsHAK11 is a new gene in tobacco. The results of Wolf PSORT predicted that the protein NsHAK11 is located mainly on plasma membrane. The subcellular localization experiment showed that the location of NsHAK11 fused with GFP and DsRed2 separately, two reporter proteins, were both on plasma membrane, which were basically in line with the prediction. The analysis of NsHAK11 promoter through PLACE website predicted that NsHAK11 gene can be expressed in the tissues of root, flower and germ, and its expression may be influenced by low temperature, high temperature, drought and phytohormones. The results of fluorescent quantitative RT-PCR matched with the prediction: NsHAK11 was expressed in all tissues of N. sylvestris at full flowering stage but at different levels, while the expression quantity in main roots was the highest, lower in the lateral roots and the lowest in leaves. In another experiment, NsHAK11 in leaves of N. sylvestris at seedling stage expressed different types of up-regulation when treated with low temperature, high temperature and low-K+ stress. The up-regulation of expression of NsHAK11 was maximum and earliest under low temperature stress. The level of up-regulation was lower under high temperature stress, while there was an obvious suppression at early stage. And the expression showed up-regulation but not significant when treated with low-K+ stress.【Conclusion】NsHAK11, a new KUP/HAK/KT family member of tobacco was obtained. And it encodes a potassium transporter protein, which is mainly distributed on plasma membrane. Besides, NsHAK11 is extensively located in the tissues and organs, and it can be involved in response to low temperature, high temperature and low-K+ stress.

Key words: Nicotiana sylvestris , bioinformatics , KUP/HAK/KT family , NsHAK11 , subcellular localization , gene expression pattern