Cloning And Functional Identification Of CLH Gene In Populus Deltoides Marsh From Bud Mutation

As important deciduous tree,Populus deltoides Marsh possesses a high ornamental value for its leaves remaining yellow in spring,summer and autumn.The yellow leaves of Populus deltoides Marsh（mutant）and green leaves of its wild-type were used as materials.The differences of pigment content and gene expression between wild type and mutant were analyzed to study the molecular mechanism underlying the leaf color of Populus deltoides Marsh.The transcriptome sequencing was used to find the differences of m RNA transcription level in yellow leaves and green leaves,and the CLH gene associated with chlorophyll biosynthesis pathway was cloned and functional identified.The results are as follows:1.The content of flavonoids and photosynthetic pigments in yellow leaves and green leaves was determined for finding the main pigment that result in yellow phenotype of Populus deltoides Marsh.The results showed that the difference between the total flavonoid contents of the green leaves and yellow leaves was not significant,while the contents of chlorophyll a and chlorophyll b in green leaves were 3 and 6 times higher than that of yellow leaves,respectively,which suggested that the yellow leaf phenotype in mutant is a result of a lack of chlorophyll.2.To find the differentially expressed genes related to leaf color,the transcriptomes of green leaves and yellow leaves were sequenced by Illumina Hi Seq ^TM4000,a new generation of high-throughput sequencing technology.The results showed that 40,779,290and 41,776,346 clean reads in sequence information of green leaves and yellow leaves were generated,respectively.Additionally,73.79%or 71.67%of reads of each samples were mapped to the Populus trichocarpa Torr.&Gray genome sequence.A total of 153differentially expressed genes（DEGs）were identified.In total 26 DEGs were assigned to the GO database,52 DEGs were annotated to 31 KEGG pathways,and 3 DEGs were involved in the porphyrin and chlorophyll metabolic pathways.Among these,two DEGs encoding geranylgeranyl diphosphate（CHLP）are down-regulated in the yellow leaves,and DEG encoding chlorophyllase（CLH）is up-regulated in the yellow leaves.Quantitative real-time PCR（RT-q PCR）further confirmed the accuracy of the transcriptome sequencing results.3.Pd CLH gene was cloned from yellow leaves using PCR technology.The full-length c DNA of Pd CLH gene was 1089 bp which encodes 326 amino acids with the main features of the lipase GHSRG conservative amino acid sequence.According to the bioinformatics analysis,the molecular weight of Pd CLH protein was 39155.18 and its isoelectric point was 5.03,which belonged to hydrophobic unstable protein.The anlysis also shown that the protein contains a signal peptide and 30 phosphorylation sites,but it doesn’t contain transmembrane region,which was predicted to belong to secretory protein.The secondary structure of Pd CLH protein included 14α-helixes and 31β-sheets.Phylogenetic analysis indicated that the protein was closely related to Populus euphratica CLH.4.The Pd CLH gene was ligated with the Pcambia 1301-E vector to obtain a plant overexpression vector p CAMBIA1301E-Pd CLH.The positive transgenic tobacco were obtained using agrobacterium EHA105 competent cells mediated infection.Overexpression of Pd CLH could result in yellow true leaves and weak plants.Study found that the chlorophyll content was significantly lower in CLH overexpressing tobacco plants than in wild type plants.The Pd CLH is the rate-limiting enzyme of the chlorophyll degradation pathway in Populus deltoides Marsh,which accelerates the chlorophyll degradation and results in yellow phenotype of Populus deltoides Marsh.