Cloning And Analysis Of Five Haloxylon Ammodendron Resilience Related Genes HaNAC2~HaNAC6

As an endemic plant in desert areas, Haloxylon ammodendron has the ability to resist drought, heat and salinity, It is a very good windbreak and sand fixation plants in desert areas. It must form a complete stress resistance mechanisms in the growth process of evolution. NAC transcription factor can make responses to biotic and abiotic stress, while it also involved in the regulation in plant growth and development process, Therefore, NAC transcription factor family are often used to research on plant resistance. Currently the research about NAC genes generally concentrated on a number of strong resistance of crops, the research on Haloxylon ammodendron about molecular biology is still in its infancy.In order to explore the function of NAC transcription factors in the resilience regulation network of Haloxylon ammodendron, get stronger resistance plants, This study cloned five Haloxylon ammodendron NAC genes, expression analysis and subcellular localization analysis, the main contents are as follows:(1)Picking five Haloxylon ammodendron NAC genes from the Unigene sequences that were obtained from drought transcriptome database of our group built before, named as Ha NAC2~Ha NAC6, designed primers according to these five NAC sequences, Using RT-PCR methods to clone full length c DNA based on these five NAC sequences, the ORF length of corresponding genes are 729bp、864bp、555bp、738bp and648 bp, encoding a protein that sequentially composed by 242、287、184、245 and 215 amino acids, the results of online prediction showed that these 5 Haloxylon ammodendron NAC proteins were Nonsecreting hydrophilic proteins, and the transmembrane does not exist. Designing promoter clone primer according to the full length c DNA sequence of Ha NAC2, finally successfully cloned a 1750 bp specific promoter of Ha NAC2, and analysis the promoter elements of this promoter sequences.(2)Analysis of these five Haloxylon ammodendron NAC genes about tissue specific expression by RT-PCR, the final results show that Ha NAC2 gene has the highest expression level in roots of Haloxylon ammodendron, Ha NAC3 gene has the highest expression level in seeds of Haloxylon ammodendron,the highest expression part of Ha NAC4~Ha NAC6 is Haloxylon ammodendron assimilating branches. Using Real-time quantitative PCR to analysis the expression of Ha NAC gene under the drought stress in different period of assimilation branches, The results showed that only Ha NAC3 gene had the maximum expression under the drought stress for 24 h, the other four genes maximum expression under the drought stress appeared at 48 h, Ha NAC2~Ha NAC5 showed a significant increase after drought stress, while it changes little in Ha NAC3.(3)Designed primers by coding region of five Haloxylon ammodendron NAC genes, added Nco I and Spe I restriction sites to these primers, With the restriction enzymes Nco I and Spe I to digest Ha NAC genes and plant expression vector p CAMBIA1304, the degested target genes and vector fragment were connected by T4 DNA ligase, Finally p CAMBIA1304-Ha NAC-GFP fusion expression vectors were constructed successfully.(4)Extracting the plasmid of p CAMBIA1304-Ha NAC-GFP fusion expression vector, transformed the recombinant plasmid into protoplasts of chickpea by PEG mediated method.Using the laser confocal microscopy to observe the subcellular localization of Haloxylon ammodendron NAC proteins, the results show that Ha NAC2~Ha NAC6 were all localized in the nucleus.