Research On The Analysis Of Transcriptome And Cold-tolerance Of The Leaf Color Mutants In Anthurium Andraeanum ’Sonate’

Anthurium andraeanum Lind.is one of the most popular tropical evergreen perennial flowers because of its brilliant color and fantastic spathe shape.At present,improving the spathe size and color,leaf shape and color,and plant shape are goals for breeding of A.andraeanum,few study on leaf color mutants were reported.To research the mechanism of leaf color mutation and cold resistance of the leaf color mutant in A.andraeanum Lind.’Sonate’,the wild type and leaf color mutants（dark green,rubescent,etiolated,albino）in A.andraeanum ’Sonate’ were used as research materials in the study.Leaf transcriptomes from wild type and rubescent were sequenced by next generation sequencing technology and differently expressed genes in the two groups of tissues were identified.Expression of genes involved in chloroplast development and division,chlorophyll and anthocyanin biosynthesis were validated by quantitative reverse transcription polymerase chain reaction（qRT-PCR）.In addition,the cold resistance of wild type and the dark green were identified by valuating the leaf,physiological and biochemical characters and the gene expression level in the wild and the dark green after exposed to 6℃ low temperature stress.The major results were as followed:1.Leaf transcriptomes from wild type and rubescent were sequenced by next generation sequencing technologylllumina HiSeq^TM 2000,a total of 39,119,350and 45,807,980 raw reads were obtained,respectively.After trimming of the adapter sequences and de novo assembly,48,284 contigs were produced,with an average length of 772 bp and N50（represents median length of all contigs）of 1,155 bp.At last,all of the assembled contigs were further scaffolded into 41,017 Unigenes（the mean size was 768 bp）,length of 50.3%Unigene were longer than 500 bp.This Transcriptome Shotgun Assembly project has been deposited at DDBJ/EMBL/GenBank under the accession GBKP00000000.Then all of the Unigenes were searched in theNCBI protein databases,a total of 27,539（67.1%）Unigenes matched a sequence in at least one of the public databases（E-value<1.0e-5）;further homology analysis showed that some of the annotated sequences share identical fragments with genes from Vitis vinifera（32.5%）,Populus trichocarpa（7.8%）,Ricinus communis（7.8%）and Prunus persica（7.6%）.GO category were classified into 41 functional groups,COG category were grouped into 24 functional categories and KEGG pathway were assigned to 261 KEGG pathways.2.The FPKM method was used to analyze the gene expression in the wild type and rubescent libraries.As a result,37,199 and 39,302 Unigenes were identified respectively in the cDNA library from wild type and rubescent mutant leaves,1,387 and 3,490 genes expressed specifically in leaves of the wild type and rubescent respectively,858 genes differently expressed（DEGs）（|log2（Ratio）|≥1 and FDR≤0.001）,including 446 up-regulated genes and 412 down-regulated genes.The functions of DEGs were classified according to GO database、COG category and KEGG pathways,the results showed that 102 DEGs were assigned to 17 COG functional groups,66 DEGswere assigned to 161 KEGG pathways,and the mainly enriched pathways were in ’Metabolic pathways’,’Biosynthesis of secondary metabolites’ and ’Microbial metabolism in diverse environments’.And the genes related to chloroplast development and division,chlorophyll and anthocyanin biosynthesis were further analyzed,the results showed that some of these genes had different expression style in the wild and rubescent.qRT-PCR was performed to validate the differential expression level of target genes in wild type and mutant plants,and we concluded that AaGLK and AaARC5are closely related with chloroplast development and division,AaDFR is a key gene in the anthocyanin biosynthesis in A.andraeanum’Sonate’.Based on results from this study,we can conclude that in leaf color mutants of A.andraeanum ’ Sonate,’ the abnormal chloroplast development and division and anthocyanin biosynthesis directly or indirectly affected the chlorophyll and anthocyanin biosynthesis,resulting in distinct content of chlorophyll and anthocyanin,at last forming different leaf colors.3.During 6℃ low temperature stress,the wild typeshowed serious symptoms of chilling injury first with leaf blade wilting and petioles softening,while the dark green showedslight symptoms of chilling injury;the chlorophyll content were both decreased,and chlorophyll degradation speed in the wild type was faster than that in the dark green type,the decreasing range was larger than that in the dark green type;the anthocyanin content were both increased,and the increasing range in the dark green type was higher than that in the wild type;the activity of superoxide dismutase（SOD）、catalase（CAT）and ascorbate peroxidase（APX）were also increased as time went on,and the increasing rate in the dark green type was higher;the expression level of AaCAT,AaAPX and AaAOX rose at first and then decreased,AaAPX wasup-regulated generally,AaAOX was up-regulated sharply after 12 h,and the expression level of AaAPX and AaAOX was higher in the dark green type than that in the wild type during low temperature stress;the expression level of AaCBF was higher after low temperature stress in both plants,however there was no obvious change rule.Based on the comprehensive analysis of the leaf,physiological and biochemical characters and the gene expression style,the cold resistance of the dark green type was stronger than the wild type in A.andraeanum ’Sonate’.