Study On The Response Mechanism Of Leymus Chinensis WRKY40 Gene To Drought Stress

Leymus chinensis is one of the most important species in grassland.It has the characteristics of high yield.high quality and animal husbandry.It is the dominant perennial grass crop and ecological grass in China,In recent years,due to the influence of global climate change,most areas of grassland in China are faced with adverse environment,especially drought,which has greatly affected the improvement of the productivity of Leymus chinensis,and then affected the economic income of herdsmen and the development of animal husbandry.1.This study was based on the label of expression sequence selected by the previous transcriptome experiment of our research group.By BLAST comparison.the gene encoded 350 amino acids and obtained by the analysis of the protein conserved structure online website:this gene encodes C2H2 zinc finger protein and contains WRKY domain with molecular weight of 38.1 kDa,named LcWRKY40.The gene was cloned from Leymus chinensis by RT-PCR method.qRT-PCR expression specificity analysis showed that the LcWRKY40 gene was expressed in the root,stem,leaf,leaf sheath,corm and anther of Leymus chinensis.Meanwhile,PEG6000 and alkaline salt(NaHCO3)were used for stress treatment of two-week-old seedlings of Leymus chinensis,it was detected that the relative expression of LcWRKY40 was induced by it and there were significant differences in leaves and roots.Therefore,it was speculated that LcWRKY40 proteins were involved in the corresponding stress response regulation pathway.2.The LcWRKY40 gene was constructed on yeast expression vector and then transferred into yeast cells.and the expression was increased by alkaline salt(NaHCO3)stress.3.To detect its functional location,we fused a green fluorescent protein(GFP)to detect the expression of this fusion protein.GFP signals were found in the nucleus,indicating that LcWRKY40 protein functions in the nucleus.4.To detect the functional role of its protein,EMSA kit was used to detect the gel block signal of LcWRKY40-GST fusion protein and biotin labeled probe.the results showed that there was a specific binding band between the fusion protein and the biotin labeled probe,indicating that the expressed LcWRKY40 protein had the effect of binding specific probe.5.LcWRKY40 transformed bensbaccos and overexpressed LcWRKY40 transgenic tobacco plants after drought stress treatment,the results showed that the overexpressed plants all had strong drought tolerance,and showed overexpression on physiological indexes such as fresh weight,malondialdehyde content LcWRKY40 plants were superior to wild-type plants.This study also found that the overexpressed LcWRKY40 gene of tobacco and wild-type tobacco had obvious differences in phenotype,including leaf morphology,leaf edge,epidermal hair,chlorophyll content and so on.6.Super-expressive transgenic rice was obtained by LcWRKY40 transformation of rice.Preliminarily speculated that LcWRKY40 gene is involved in the regulation pathway in response to drought stress stress,and it is involved in the growth and development process of plants,which may play an important role directly or indirectly in transcriptional regulation.Taken together,this study revealed that LcWRKY40 transcription factor can participate in drought stress response regulation,play an important role in enhancing crop drought tolerance,and may regulate plant phenotype and growth and development.