Functional Analysis Of 14-3-3 Protein Gene HaFT-3 And Its Promoter In Haloxylon Ammodendron

Haloxylon ammodendron is a super xerophytic small tree of Chenopodiaceae.It is one of the main constructive species in desert and semi desert areas of Northwest China.It has strong resistance to high temperature,drought,cold,salinity and wind erosion.Exploring the molecular mechanism of stress resistance about H.ammodendron have great significance for breeding excellent stress resistant plants and ecological restoration.Transcriptome analysing of H.ammodendron after high temperature and drought stress,14-3-3 protein genes HaFT-1～HaFT-9 were isolated.After expression analysis,we found that the expression level of HaFT-3 gene was related to stress.Taking the HaFT-3 gene as the research object,from the perspectives of the HaFT-3 gene sequence and the HaFT-3 promoter,the method of functional analysis of transgenic Arabidopsis thaliana was used to further explore the resistance mechanism of H.ammodendron.The results are as follows:（1）The T₁ transgenic Arabidopsis thaliana of HaFT-3 were resistant to herbicides and verified by PCR,and 18 homozygous positive plants were initially obtained;T₂ generation lines were identified 9plants with a 3:1 separation ratio;T₃ generations F3-1,F3-2,F3-3 were screened out of positive plants using resistance plates and RT-PCR.Transgenic Arabidopsis thaliana was treated different abiotic stress and then analyzed the germination rate,root length,plant height,survival rate,growth status,malondialdehyde content and proline content.Compared with wild Arabidopsis thaliana,HaFT-3transgenic Arabidopsis thaliana had lower germination rate after drought and high temperature stress;higher germination rate,survival rate and proline content after salt stress;and significantly increased root length,survival rate,growth status and physiological indexes after high temperature and low temperature stress.（2）Bioinformatics analysis shows that the 1340 bp HaFT-3 promoter contains basic cis-acting elements TATA-box,CAAT-box,etc.,other elements,such as salt response element,dehydration response element,abscisic acid response element.The p CAMBIA3301-HaFT-3::Intron-GUS plant expression vector was successfully transformed into Arabidopsis thaliana through agrobacterium mediated,multiple generations of screening to obtain positive plants and then analyze the GUS activity after different stress treatments at different growth stages.Under normal conditions,lower level of GUS activity could be observed in the leaves and roots of transgenic Arabidopsis thaliana at 28 days;treated with IAA in 5 days,there is a higher level of GUS accumulation in roots;treated with high temperature stress in 14 days,there is a lower level of GUS accumulation in leaves and roots;28 days after seedlings,ABA treatment has higher GUS activity in leaves and roots;after IAA treatment,there has a higher level of GUS accumulation in leaves and lower GUS activity in roots;after high temperature treatment,leaves and roots have lower GUS activity;after 4℃low temperature stress,there is a higher level of GUS accumulation in leaves,and a lower level of GUS accumulation in roots.After 28 days seedling stress,GUS activity test results showed that GUS activity increased significantly after ABA,IAA,high temperature,and low temperature treatments.In summary,HaFT-3 gene can improve the resistance of transgenic Arabidopsis thaliana to salt stress;improve the tolerance to high and low temperature stress at transgenic Arabidopsis thaliana seedling stage.HaFT-3 promoter has space-time expression specificity and has regulatory activity under exogenous hormones ABA,IAA,high temperature and low temperature stress.