Stress-resistance functional validation of transcription factor HaNAC12 of Haloxylon ammodendron

【Objective】To verify the stress-resistance function of the NAC transcription factor gene（HaNAC12）of Haloxylon ammodendron，to analyze the molecular mechanism of H.ammodendron in response to stress，so as to provide theoretical reference for the genetic improvement of stress-resistance in H.ammodendron and other crops.【Method】Expression pattern of HaNAC12 gene under drought，salt and ABA treatments by quantitative real-time PCR（qRT-PCR）.HaNAC12 transgenic positive plants of Agrobacterium tumefaciens were cultivated and screened through homologous recombination method，A.tumefaciens-mediated flower spraying method and herbicide spraying method.Difference of germination rate，stomatal openness，relative water content，plant height，growth rate and physiological indexes after different stress treatments between the positive plants and wild type plant（WT）were determined and plants without treatments were taken as control to verify the stress-resistance function of HaNAC12 gene in A.tumefaciens under stresses.【Result】 Relative expression of HaNAC12 gene was extremely significantly higher at 12 h of drought stress than that at 0 h （P<0.01，the same below）.At 1 h of salt stress，relative expression of the gene was extremely significantly higher than that at 0 h.Relative expression of the gene was extremely significantly higher than that at both 3 and 12 h of ABA treatment（P<0.05，the same below）.Three high expression T₃ generation transgenic lines HaNAC12-1，HaNAC12-2 and HaNAC12-3 were obtained by screening.Mean final germination rate of WT after seed drought stress was 45.00% and the final germination rate of the 3 transgenic lines was 66.11%-85.00%.Final germination rate of WT after seed salt stress was 36.42% and the final germination rate of the 3 transgenic strains was above 49.38%-58.64%.WT turned dry and yellow under natural drought and salt stress，while the 3 transgenic lines did not turn dry.The 3 transgenic lines showed seedling in accelerated development such as early bolting and early flowering under natural drought stress，and their growth rate was faster than WT.Stomatal opening was reduced by 67.54% in WT and 91.08%，83.14% and 85.04% in the three transgenic strains after natural drought stress at seedling stage，and relative water contents of HaNAC12-1，HaNAC12-2 and HaNAC12-3 strains were significantly or extremely significantly higher than that of WT.Malondialdehyde content，proline content，hydrogen peroxide content and catalase activity of the 3 transgenic strains increased significantly after drought and salt stress compared with those before stress，with greater increases of proline content and catalase activity than WT and smaller increases of malondialdehyde content and hydrogen peroxide content increasing than WT.【Conclusion】 Overexpression gene of the HaNAC12 in A.tumefaciens enhances its resistance to drought and salt stress at the germination and seedling stages，and promotes flowering in Arabidopsis after drought stress，indicating that HaNAC12 gene can improve the drought and salt tolerance of transgenic Arabidopsis.