Cloning And Expression Analysis Of Caladium Bicolor CbNAC8 Gene And Its Relationship With Drought Resistance

Caladium bicolor belongs to the perennial herb of Caladium in the Araceae family.Its leaf color is unique and bright.It is one of the important foliage plants as well as important garden ornamental and indoor potted plants.Caladium bicolor faces many environmental stresses during the planting process,such as temperature,moisture,light,etc.Drought is a relatively common way of stress,especially for potted Caladium bicolor.Therefore,it is particularly important to explore the formation mechanism of the drought resistance of the Caladium bicolor and to improve the drought resistance of the Caladium bicolor.Transcription factors respond to drought stress in plants at the transcriptional level by regulating the expression of related genes.NAC transcription factors are a type of transcription factors involved in plant drought stress.The development of molecular biology technology has led to rapid exploration and improvement of drought resistance in plants.Sex offers the possibility.In this experiment,a gene annotated as NAC8 was obtained from the transcriptome database of Caladium bicolor,which was constructed in the early stage.On this basis,the NAC8gene was cloned and isolated,and its molecular characteristics,expression characteristics and the relationship with drought resistance were analyzed.The main findings are as follows:（1）According to the sequence information in the Caladium bicolor transcriptome database,the complete ORF box sequence of the NAC8 gene of Caladium bicolor was cloned and named CbNAC8.The complete ORF box sequence of CbNAC8 is 1185 bp,the G+C content is about 42%,and the A+T content is about 58%,encoding a total of 394 amino acids.（2）The structural characteristics of the CbNAC8 protein of Caladium bicolor.The CbNAC8protein was predicted and analyzed by Prot Param.The results showed that the molecular formula of CbNAC8 protein was C₁₉₄₃H₃₀₂₆N₅₅₂O₅₉₄S₁₈,the molecular weight of the protein was 44.20 k D,and the theoretical isoelectric point was 6.06.Lysine（Lys）accounts for the highest proportion of amino acid composition at 8.4%,followed by aspartic acid（Asp）at 7.6%,and the protein instability coefficient is 37.02,which is a stable protein,with a total average hydrophilicity of-0.655 is a hydrophilic protein.Through sequence alignment analysis,it is found that CbNAC8 protein contains the typical NAM conserved domain of the NAC family,indicating that CbNAC8 belongs to the NAC family.Through the construction of a phylogenetic tree analysis,it is found that CbNAC8protein belongs to the second category of ONAC003 subfamily member in the Oo Ka classification method,closest to ANAC010.（3）In order to understand whether CbNAC8 has the basic characteristics of transcription factors,the subcellular localization prediction and transcription activity analysis of CbNAC8 were carried out.Online prediction of subcellular localization by Cell-PLoc 2.0 shows that CbNAC8 is localized in the nucleus.The yeast system was used to analyze the transcriptional activation activity of CbNAC8,and the results showed that CbNAC8 can transcriptionally activate the expression of Lac Z and HIS3 reporter genes in yeast cells,indicating that CbNAC8 has transcriptional activation activity.（4）In order to analyze the expression characteristics of CbNAC8,q RT-PCR technology was used to study the expression pattern of CbNAC8 in different tissues of Caladium bicolor and under the treatment of ABA and high temperature stress.The results showed that CbNAC8 was expressed to different degrees in different tissues of Caladium bicolor.Its expression was highest in leaves and lowest in stems.CbNAC8 was significantly down-regulated under both ABA and high temperature stress treatments.（5）In order to explore the correlation between CbNAC8 and physiological indicators of drought resistance,three Caladium bicolor materials with different drought resistance were selected to study the expression of CbNAC8 gene in different time periods of PEG simulated drought treatment,and to explore the four physiological indicators of drought resistance correlation between the expression of CbNAC8 gene and the physiological indicators of drought tolerance in Caladium bicolor.The results showed that drought stress can affect the expression of CbNAC8 gene,and the expression pattern of CbNAC8 in different varieties with different drought resistance was significantly different.Correlation analysis showed that at 24 h,CbNAC8 gene expression was significantly correlated with relative water content of leaves.Therefore,when investigating the drought tolerance of Caladium bicolor,the expression of CbNAC8 gene can be used as a reference index for drought resistance identification.（6）In order to study the function of CbNAC8 gene,a plant overexpression vector p CAMBIA2301G-CbNAC8 was constructed to transform Caladium bicolor and tobacco homologously and heterologously,and transgenic plants were obtained by PCR identification.The results of drought resistance analysis showed that the drought resistance of tobacco overexpressing CbNAC8 gene was lower than that of wild type tobacco.It preliminarily indicates that the CbNAC8transcription factor of Caladium bicolor may play a negative regulatory role in drought stress.