Cloning And Functional Verification Of The Drought-Tolerant Candidate Gene CBF1 In Psammochloa Villosa(Poaceae)

Psammochloa villosa is a perennial herb,which belongs to Psammochloa of tribe Stipeae in Poaceae.In this study,based on the third-generation full-length transcriptome data of P.villosa,we cloned the CBF1 gene of P.villosa by RT-PCR;In order to verified the function of CBF1 gene of P.villosa,we constructed two plant overexpression vectors and transformed them into tobacco and barley by Agrobacterium-mediated method,respectively.The study provided a theoretical basis for further exploring the molecular mechanism of PvCBF1 gene responding to drought tolerance.Main results are as follows:1.Full-length transcriptome sequencing was performed on the PacBio platform with the mixed samples of root,stem and leaf of P.villosa.A total of 184076 highquality transcripts were finally obtained after removing the redundancy sequences.Among them,97892 transcripts were annotated in KOG database and divided into 26 categories according to their functions;87964 transcripts were annotated in GO database and divided into 51 functional groups of three categories including cell components,biological processes and molecular functions;In the KEGG database,89086 transcripts were annotated to five metabolic pathways,including cell processing,environmental information processing,genetic information processing,metabolism and biological system.2.Based on CBF1 gene sequences of Brachypodium distachyon,a relative species of P.villosa,drought-tolerant candidate gene was obtained by homologous comparison in the full-length transcriptome CDS library of P.villosa using BioEdit software and named as PvCBF1.According to the sequence of PvCBF1 gene,we designed a pair of specific primer to amplify the coding sequence of PvCBF1 gene of P.villosa by PCR.Bioinformatics analysis showed that the encoding region was 690 bp in length of PvCBF1 gene,encoding 229 amino acids which contained a AP2 domain and two typical conserved motifs: PKRPAGRTKFRETRHP and DSAWR;Phylogenetic analysis showed that PvCBF1 was closely related to CBF1 transcription factor of Triticum aestivum.The q PCR results indicated that PvCBF1 gene was expressed in roots,stems,leaves and seeds without tissue specificity.Under drought stress,PvCBF1 gene was rapidly expressed and peaked at 6 h;Subcellular localization experiments showed that PvCBF1 transcription factor was mainly located in nucleus.3.Two plant expression vectors pC2300s: PvCBF1 and pLGY-02: PvCBF1 were constructed and transformed into tobacco and barley via Agrobacterium-mediated genetic transformation system,respectively.Transgenic tobacco and barley seedlings were obtained through infection,screening,rooting and seedling refining.Ten positive tobacco plants and one positive barley plant were obtained by further PCR identification.In order to evaluate the effects of PvCBF1 gene in the transgenic plants to drought tolerance,the transgenic tobacco and barley were treated by drought stress condition for 0,3,6,9,12 and 24 h.Six related physiological indexes including SOD,POD,CAT,PRO,MDA and relative electric conductivity were measured at each time points.The results showed that the antioxidant enzymes such as SOD,POD and CAT were maintained high activities,the content of PRO was significantly increased,the content of MDA was relatively small,and the relative electric conductivity was lower compared with the wild type tobacco and barley,indicating that the scavenging ability of reactive oxygen was improved,and the degree of membrane lipid peroxidation was relatively low in transgenic tobacco and barley.Thus,the PvCBF1 gene could increased the drought tolerance of transgenic tobacco and barley.