Characterization And Functional Analysis Of HaNAC20 Transcription Factor And HaLEA28 Protein In Haloxylon Ammodendron

Haloxylon ammodendron is widely distributed in desert and semi-desert regions,forming a unique molecular mechanism of stress resistance under long-term extreme conditions.It has the characteristics of drought resistance,high temperature resistance,severe cold resistance,salt tolerance and so on.It is an excellent resource for exploring stress resistance genes,and also an excellent tree species for sand fixation and afforestation in arid and desert regions,with great ecological benefits.NAC transcription factor is a kind of unique transcription factor in terrestrial plants,which participates in many processes such as plant growth and development,and can improve the ability of plants to resist biological and abiotic stresses to varying degrees.Late embryogenesis abundant protein（LEA）is a kind of functional protein,which can improve plant resistance under many stress conditions.The results of this study can provide the basis for revealing the molecular mechanism of Haloxylon ammodendron stress resistance from the aspect of Haloxylon ammodendron NAC transcription factor,provide theoretical guidance for screening Haloxylon ammodendron stress resistance materials,and also provide new candidate genes for plant stress resistance genetic engineering.At the same time,this study also provides the basis for further study on the function of LEA protein and the relationship between variable splicing and stress resistance.In this study,the regulatory gene Ha NAC20 and functional gene Ha LEA28 up-regulated under drought stress were screened from the sequencing results of Haloxylon ammodendron drought transcriptome.The characteristics and functions of Ha LEA28a and Ha LEA28b,two transcripts of Ha NAC20 and Ha LEA28,were studied.The main conclusions are as follows:（1）The bioinformatics analysis of the Ha NAC20 transcription factor showed that the protein has a typical NAC domain,the protein is located in the nucleus,and there is a nuclear localization signal in the sequence,which belongs to the stress-related SNAC subfamily member of the NAC family.The p CAMBIA1304-Ha NAC20-GFP fusion expression vector was constructed,and the white onion epidermal cells were transformed by Agrobacterium-mediated method.The localization of Haloxylon ammodendron Ha NAC20 protein in the nucleus was observed under the laser confocal microscope.The transcriptional activation activity of Ha NAC20 transcription factor was verified by yeast one-hybrid assay.The results showed that Ha NAC20 had transcriptional self-activation activity and the transcriptional activation domain was located at its C-terminal.（2）The plant expression vector p CAMBIA3301-Ha NAC20 was constructed and transformed into wild-type A.thaliana by Agrobacterium-mediated floral dip method.Five homozygous lines of T₃generation Ha NAC20 transgenic A.thaliana were screened and identified.The seed germination of wild type and transgenic Arabidopsis under normal growth conditions showed that the seed germination rate of Ha NAC20 transgenic Arabidopsis homozygous lines was not significantly different from that of wild type.The root length growth of at least three lines of Ha NAC20 transgenic A.thaliana was significantly higher than that of the wild type after 7 d.There was no significant difference in the size of the first flower and the first pod between the transgenic lines and the wild type.In terms of bolting number and pod number,at least three transgenic lines were significantly higher than those of wild type,but there was no significant difference in plant height between transgenic lines and wild type.Germination experiments were carried out under mannitol simulated drought stress and salt stress,and the results showed that the overexpression of Ha NAC20 gene improved the drought resistance and salt tolerance of Arabidopsis during seed germination,at the same time,the water loss rate of 3-week-old rosette leaves was measured,and the results showed that transgenic Arabidopsis was more drought tolerant.（3）Ha LEA28a gene of 831 bp and Ha LEA28b gene of 723 bp were cloned.Bioinformatics analysis showed that the encoded proteins of Ha LEA28a and Ha LEA28b in Haloxylon ammodendron were hydrophilic proteins as other LEA proteins.The 36 amino acid sequences of Ha LEA28a were a hydrophilic sequence more than Ha LEA28b,so it was speculated that Ha LEA28a was more hydrophilic than Ha LEA28b.The p CAMBIA1304-Ha LEA28a/b-GFP fusion expression vector was constructed and transformed into onion epidermal cells by Agrobacterium-mediated transformation.Both proteins were localized in the nucleus under laser confocal microscope.（4）The plant expression vector p CAMBIA3301-Ha LEA28a/b was constructed and transformed into wild-type A.thaliana by Agrobacterium-mediated floral dip method.The T₂generation transgenic A.thaliana was obtained and subjected to natural drought stress.The results showed that the survival rate of transgenic lines was significantly higher than that of wild-type after rewatering,indicating that overexpression of Haloxylon ammodendron Ha LEA28a and Ha LEA28b genes could improve the drought resistance of transgenic plants.