Cloning And Functional Analysis Of LpDREB9 And LpDREB12 Genes In Lilium Pumilum

Lilium pumium is a plant of the Liliaceae Lilium with high ornamental value,homology of medicine and food,and strong resistance.It is an important parent for plant resistance breeding.DREB transcription factor is the main member of AP2/ERF family,which widely exists in Arabidopsis thaliana,Oryza sativa,Nicotiana tabacum and other plants,and plays an important role in responding to abiotic stresses.In this study,12 DREB transcription factors were screened and named Lp DREB1-Lp DREB12 based on the transcription data of L.pumium(root,bulb and leaf),and their bioinformatics and expression patterns in different tissues under stress were analyzed.The Lp DREB9 and Lp DREB12 genes were cloned,the overexpression vector was constructed by homologous recombination,the N.tabacum was transformed,and the transgenic plants were obtained by screening.The drought stress was carried out on the transgenic N.tabacum Lp DREB9 and Lp DREB12 genes,which their drought resistance was identified,so as to provide high-quality gene resources for resistance breeding.The main research achievements are as follows:1.The bioinformatics analysis of DREB transcription factors of L.pumium showed that the number of amino acids encoded by 12 DREB genes was 120-215,and the encoded proteins were mostly unstable proteins with a certain hydrophilicity,alpha helices and random coils are the main components of protein secondary structure.Phylogenetic analysis showed that 12 L.pumium DREB transcription factors clustered with 61 A.thaliana DREB transcription factors,indicating that L.thaliana and A.thaliana DREB family members are highly conserved.Conserved motif analysis showed that there were differences in the number,types and positions of conserved motifs in different Lp DREB proteins,but motif 1 and motif 3 were highly conserved.2.Expression pattern analysis showed that the expression of 12 L.pumium DREB genes were tissue specific under drought,salt,low temperature and abscisic acid stress.Under drought stress,Lp DREB1 gene was down-regulated in all tissues,11 Lp DREB genes were up-regulated,of which 3 were up-regulated in roots,bulbs and leaves,7 genes were up-regulated in two parts,and 2 genes were only up-regulated in roots or leaves.Under low temperature stress,12 Lp DREB genes were up-regulated,one gene was up-regulated in roots,bulbs and leaves,8 genes were upregulated in two parts,and the rest were only up-regulated in leaves.Under salt stress,all genes were up-regulated,of which 4 genes were up-regulated in all tissues,4 genes were only upregulated in two parts,and the rest were only expressed in bulbs or leaves.Under ABA treatment,12 Lp DREB genes were up-regulated,but only 1 gene was up-regulated in all tissues,6 genes were up-regulated in 2 sites,and the rest were up-regulated in bulb or leaf.3.The cloned Lp DREB9 gene was 462 bp in ORF length,encoding 153 amino acids,and the Lp DREB12 gene was 648 bp in ORF length,encoding 215 amino acids.Bioinformatics analysis showed that both Lp DREB9 and Lp DREB12 proteins were hydrophilically unstable and non-secreted proteins.The GV1300-Lp DREB9 and GV1300-Lp DREB12 overexpression vectors were constructed.Subcellular localization analysis by injection method showed that both Lp DREB9 and Lp DREB12 genes were expressed in the nucleus.The leaf disc method was used to transform N.tabacum,and 6 transgenic N.tabacum Lp DREB9 and Lp DREB12 plants were finally obtained by screening and identification.4.Overexpression of both Lp DREB9 and Lp DREB12 genes enhanced the sensitivity of transgenic N.tabacum to ABA.Under ABA stress,the seed germination rate,root length and fresh weight of transgenic N.tabacum Lp DREB9 and Lp DREB12 were significantly lower than those of wild type.5.Overexpression of Lp DREB9 gene enhanced the drought resistance of transgenic N.tabacum.Under drought stress,seed germination rate,root length and fresh weight of seedlings of transgenic N.tabacum Lp DREB9 were significantly higher than those of wild type.The growth state of transgenic N.tabacum.seedlings under stress was better than that of the wild type.The activities of SOD,POD,CAT and the contents of Pro and chlorophyll were significantly higher than those of the wild type,and the content of MDA was lower than that of the wild type.The drought tolerance of the plant was improved.Overexpression of Lp DREB12 gene weakened the drought resistance of transgenic N.tabacum.Under drought stress,the seed germination rate,root length and fresh weight of seedlings of transgenic N.tabacum Lp DREB12 were significantly lower than those of the wild type.The growth state of transgenic N.tabacum Lp DREB12 seedlings under stress was inferior to that of the wild type.The activities of SOD,POD,CAT,and the contents of Pro and chlorophyll were significantly lower than those of the wild type,and the content of MDA was higher than that of the wild type,and the drought resistance of the plants was reduced.