Preliminary Functional Analysis Of PdbCML48 Gene Of Populus Davidiana×P.bolleana

In the long process of evolution,plants have formed a series of self-protection mechanisms in morphology,physiology and molecular adaptation.These protective mechanisms include a series of ingenious mechanisms for sensing internal and external signals and stimuli.Studies have shown that Ca²⁺,as a multifunctional signal molecule,is the core of complex anti-stress signal pathway in response to adverse environmental conditions and plays an important role in various biological functions.The CML（Calmodulin-like protein）gene family is one of the major Ca²⁺receptor proteins,but at present,most of the studies on the CML gene family are concentrated in Arabidopsis thaliana,and few reports are reported in woody plants.Poplar is a model plant for forest research,and the analysis of its CML gene function will provide new information for a comprehensive understanding of the gene function of this family.In this study,a CML gene named PdbCML48（Populus davidiana×P.bolleana calmodulin-like protein 48）was screened from the differential gene expression profile of Populus davidiana xylem before and after salt stress treatment,and its expression pattern and biological function were preliminarily analyzed.The work and results of this paper are summarized as follows:1.In this study,a CML gene was obtained from Populus davidiana,which was100%identical with PtCML48 gene of Populus trichocarpa,so it was named PdbCML48,and its CDS sequence was 744 bp,encoding 247 amino acids.The gene is located on chromosome 9,including 4 exons and 4 introns.The results of sequence alignment and phylogenetic tree showed that PdbCML48 contained multiple calcium ion binding sites and five EF-hand motif,which suggested that it belonged to EF-hand superfamily,and had high similarity with CML genes of Hevea brasiliensis,Prunus persica and Armeniaca mume,all of which were above 70%.2.Semi-quantitative PCR was used to detect the expression pattern of PdbCML48in different tissues.The results showed that PdbCML48 was expressed in different tissues of Populus davidiana,such as roots,leaves,xylem and phloem.Furthermore,quantitative PCR was used to analyze the response pattern of PdbCML48 to abiotic stress and various hormones.The results showed that apart from PEG,NaCl and various plant hormones including NAA,GA₃,ABA,Eth and BR could induce the up-regulation of PdbCML48 in different degrees,among which NAA had the most significant induction effect.These results suggest that PdbCML48 is involved in the signal transduction process of NaCl and various plant hormones.The subcellular localization of PdbCML48 protein was carried out by transient transformation of Nicotiana tabacum.The results showed that PdbCML48 protein existed in both cytoplasm and nucleus.3.In order to identify the function of PdbCML48 gene,we constructed PdbCML48-P2301 overexpression vector and transformed Populus davidiana by leaf disk method.Nine transgenic lines were obtained by resistance and real-time quantitative PCR screening.Phenotypic analysis showed that there was no significant difference between transgenic lines and wild-type lines under normal conditions.However,under the treatment of 100μM/L GA₃,the rooting of transgenic and wild-type plants was inhibited,but the root system of transgenic plants was inhibited to a lesser extent than that of wild-type.When the concentration of GA₃ reached 500μM/L,the growth of wild-type roots and stems and leaves was completely inhibited,while the roots of transgenic lines were produced and the stems and leaves were also elongated.These results suggest that the overexpression of PdbCML48 gene may reduce the sensitivity of transgenic plants to GA.4.In order to further explore the mechanism of PdbCML48,the wild-type and overexpressed PdbCML48 strain OE2 of Populus davidiana treated with 500μM/L GA₃ for 1 d and 4 d were analyzed by transcriptome sequencing.The results showed that 1298 and 791 differentially expressed genes（DEGs）were obtained in the two control groups respectively.Through the annotation analysis of Gene Ontology（GO）,it is found that DEGs is mainly enriched in"cellular process"and"metabolic process"in biological processes.Among the cell components,the number of DEGs in"cell part"and"membrane part"is the largest;In molecular function,DEGs is mainly enriched in"binding"and"catalytic activity".According to the analysis of the enrichment of Kyoto encyclopedia of genes and genomes（KEGG）,DEGs is mainly enriched in the biosynthetic and metabolic pathways and plant hormone transduction pathways.The functional classification analysis of DEGs showed that there were 49 genes related to ion homeostasis.There are 53 genes related to antioxidant system;There are 13 genes related to gibberellin.