Functional Analysis Of Tea Plant NAC Transcription Factor CsNAC168 In Response To Drought Stress

Drought is one of the most common and widely distributed environmental stresses faced by plants.As an important tea-producing region in China,Yunnan has been experiencing drought in recent years,which has seriously affected the yield and quality of tea.Therefore,it has become an urgent need to explore the molecular mechanisms of drought stress in tea plants,to improve the drought tolerance and water uptake efficiency of tea plants,and to cultivate tea plants varieties with stress tolerance.Plants have developed a series of regulatory and response mechanisms in the process of gradual adaptation to complex and variable biotic and abiotic stresses.Transcription factors play an important role in regulation of plant stress tolerance and environmental adaptation.A large number of studies have shown that members of the NAC transcription factors are involved in the regulation of plant responses to both biotic and abiotic stresses,enhancing the resistance of plants in various aspects.In recent years,many studies have been reported on the involvement of NAC transcription factors in regulation of drought or salt resistance in rice,maize,soybean and other crops,but studies on the functions of NAC transcription factors in response to stress in tea plant have not been conducted indepth.In this paper,we cloned and analyzed a tea plant NAC transcription factor,named Cs NAC168,in response to drought and salt stress,and the main results were as follows:1.Bioinformatics analysis revealed that the full length of Cs NAC168 gene was3931 bp,with a CDS sequence of 1041 bp,encoding 346 amino acids.Its promoter region contains elements responsive to plant hormones such as abscisic acid and jasmonic acid.Sequence analysis of Cs NAC168 protein showed that Cs NAC168 is an unstable lipid-soluble protein without transmembrane structure,with an instability coefficient of 50.87 and a lipid-soluble index of 67.34.2.Cs NAC168 overexpression vector was constructed into Agrobacterium tumefaciens,and the transgenic Arabidopsis thaliana overexpressing Cs NAC168 ge-ne were obtained by the floral dip method.Six transgenic seedlings containing hypomycin resistance were obtained by PCR.The expression of Cs NAC168 in Arabidopsis thaliana transgenic plants was further detected by q PCR.3.Transgenic Arabidopsis thaliana seeds were treated with different concent rations of mannitol（0m M,100 m M,150 m M,250 m M）and sodium chloride（0mM,50 m M,100 m M,150 m M,200 m M）to mimic osmotic stress and salt stress,a-nd seed germination and root length were observed.The results showed that th-e seeds of Cs NAC168 overexpressed transgenic plants revealed greatly improvedtolerance to salt stress and osmotic stress.And the transgenic Arabidopsis seed s showed higher germination rate and more developed roots under stress than WT plants.4.Three-week-old transgenic Arabidopsis plants and wild-type plants were irrigated with different concentrations of PEG6000（0,10%,20%,30%）and sodium chloride（0m M,100 m M,150 m M,200 m M）.The Arabidopsis plants were detected for physiological data after two days of stress treatment.The results showed that transgenic Arabidopsis plants maintained higher structural integrity of cell membrane under osmotic stress and salt stress,had lower levels of reactive oxygen species and MDA than the wild type,and higher chlorophyll content,proline content and SOD and POD enzyme activities in leaves compared with the wildtype plants.Overall,transgenic Arabidopsis plants showed higher salt stress tolerance and greater drought resistance.5.Treatment with different concentrations（0μM,0.5μM,1.0μM）of ABA revealed that the transgenic Arabidopsis plants were significantly more sensitive to ABA,and their seed germination rate decreased and the root length became shorter.In stomatal working solutions containing different concentrations（0μM,0.5μM,1.0μM,1.5μM）of abscisic acid,leaf stomata of transgenic Arabidopsis plants induced by ABA were more tightly and completely closed relative to wild type plants.6.Cs NAC168 overexpression lines and the WT during drought stress treatment in soil revealed that the survival rate of transgenic Arabidopsis plants was 91.66%,95.83%,and the wild-type Arabidopsis plants was 9.72%.The transgenic Arabidopsis plants was much higher than wild type Arabidopsis plants,indicating that overexpression of Cs NAC168 enhanced the tolerance of Arabidopsis thaliana to drought stress.In conclusion,the tea plant NAC transcription factor Cs NAC168 is involved in ABA signaling pathway to regulate plant stress responses to drought and high salt.By increasing proline content and antioxidant enzymes activity in transgenic plants,the closure of leaf stoma was regulated,thus conferring drought and salt tolerance of the plants.This paper provides a reference for the study of the molecular mechanism of drought tolerance in tea plants,and lays a foundation for the breeding of new resistant species of tea plants.