Cloning Of The Related Drought-resistant Gene From Psammochloa Villosa(Poaceae) And Its Expression Pattern Analysis

Drought stress is one of the most important abiotic stresses affecting plant growth and development.When plants are short of water,cell expansion and growth will be hindered,making plants grow short or even die.Normally,plants respond to and adapt to drought by regulating the expression of drought-tolerance-related genes in a series of “defensive responses”.P.villosa is an endemic desert herb of the Inner Mongolia Plateau and its adjacent areas,which is extremely adaptable to arid environments and is ideal material for mining drought-tolerant genetic resources.Therefore,it is of great theoretical and practical significance to clone drought tolerance related genes and study their functions for improving plant stress resistance.In this study,based on the full-length transcriptome data of three generations,8drought-tolerant related genes of P.villosa were cloned by PCR technology,and their expression levels were quantitatively analyzed to obtain the drought-tolerant genes with the highest expression level.The function was verified by drought-related physiological index detection,which provided a theoretical basis for the future study of the molecular mechanism of drought tolerance in P.villosa.The main results are as follows:⑴We obtained the drought-tolerant gene sequences of 8 related species of P.villosa by consulting the literature,and then compared to the full-length transcriptome of the three generations of P.villosa using Bio Edit software to obtain the 8 sequences with the highest homology.8 drought tolerance-related genes of P.villosa were obtained by PCR amplification using c DNA as template and named as Pv DREB,Pvb ZIP,Pv MYB,Pv WRKY,Pv NAC,Pv PP2 C,Pv AQP and Pv P5 CS.⑵Bioinformatics analysis showed that the length of coding regions of the 8drought tolerance genes ranged from 819 to 1 890 bp,encoding a total of 272 to 629 amino acids,and all of them contained a complete conserved domain.Hydrophilic and hydrophobic analysis showed that except that Pv AQP belonged to hydrophobic protein,the rest were hydrophilic proteins.Pv DREB and Pv AQP had transmembrane structure,and the proteins encoded by other genes do not have transmembrane structure.The prediction of signal peptide showed that the proteins encoded by the 8drought tolerance-related genes did not have signal peptide structure.At the same time,glycosylation and phosphorylation site prediction found that Pvb ZIP,Pv MYB and Pv AQP did not have possible glycosylation sites,and the proteins encoded by the other 5 genes had glycosylation sites,while all proteins had possible phosphorylation sites.⑶The 20% PEG-6000 was used to simulate drought stress treatment(0,6,12,24,48 and 72 h).The results showed that the expression trends of Pv DREB,Pv WRKY and Pv PP2 C genes in roots were the same.The expression level decreased first and then increased at 0-12 h,increased to the peak at 12 h,and increased first and then decreased at 24-72 h.In terms of stems,the expression trends of Pv DREB,Pvb ZIP,Pv MYB,Pv WRKY and Pv PP2 C genes were similar.The expression levels increased rapidly at 0-6 h,began to decrease at 6-12 h,and increased again at 12-48 h.In terms of leaves,the expression levels of Pv DREB,Pvb ZIP,Pv MYB,Pv WRKY and Pv P5 CS genes peaked at 12 h of drought stress.8 drought-tolerant genes were induced by drought in roots,stems and leaves.⑷By comparing the expression levels of 8 drought-tolerant genes in leaves at 12 h of simulated drought stress,it was found that the expression level of Pv DREB gene was the highest.On this basis,the Pv DREB gene was cloned by PCR,and the p C2300s: Pv DREB expression vector was constructed by double enzyme digestion technique.The recombinant vector was transferred into tobacco by Agrobacterium-mediated method,and 8 positive tobacco plants were obtained.The physiological analysis of drought tolerance of transgenic and wild-type tobacco showed that under certain drought stress intensity,with the increase of drought stress time,the activities of SOD,POD and CAT and the content of PRO in transgenic tobacco were significantly higher than those in wild-type(P<0.01),while the content of MDA was significantly lower than that in wild-type(P<0.01),indicating that the scavenging ability of reactive oxygen species was relatively higher,and the degree of membrane lipid peroxidation of transgenic tobacco was lower.It was considered that the overexpression of Pv DREB gene could improve the drought tolerance of transgenic tobacco.