Study On The Color Mechanism Of Cymbidium Ensifolium With Deep-claw Covered Wheel Leaf Art Characteristic

The phenomenon of white or yellow patches or stripes on the leaves of Chinese orchid are called‘Yeyilan’(Orchid with leaf art).The common leaf art includes deep-claw covered art,patches art,crystal art and so on.Orchid with leaf art is of great ornamental and economic value,but the reasons of its formation is not clear.Gene mutation,nutritional deficiency,virus infection or fungal parasitism can cause albinism or etiolation of local leaf.The progress of Orchid with leaf art breeding is slow due to the unclear formation mechanism.At the moment,the breeding of leaves art orchids mainly relies on wild domestication,which makes wild resources face the danger of stealing and digging overly.Cymbidium ensifolium‘Jin Qi’is a natural variation of C.ensifolium which belongs to the Orchidaceae.It is one of the evolution varieties of “Xiaotaohong”(a type of leaves art orchids),with a typical deep-claw covered wheel art characteristic and a bright color like a banner.In this study,the leaf color variation mechanism of C.ensifolium‘Jin Qi’was studied from the perspectives of cytology,physiology and molecular biology.All the above fundamental researches will provide the foundation for the cultivation of new varieties of Cymbidium and other orchid genera,and meanwhile offer a reference for the research of other leaf color mutant ornamental plants.The main results are as follows:1.Cytology mechanism analysis of C.ensifolium‘Jin QiThere was no significant difference in stomatal structure between yellow part and green part.The decrease of photosynthetic rate may be related to the photosynthetic activity of mesophyll cells rather than stomatal closure.The chloroplast microstructure was observed by transmission electron microscope.It was found that the chloroplast morphology of green part was normal with a spindle shape,and a large number of grana stacks formed grana lamellae and arranged in order.While the chloroplast structure of yellow part was incomplete,various and nearly round,and grana had no obvious stacking with a large number of osmioplilic granules gathering.The results of transmission electron microscope observation and virus inoculation experiment showed that the leaf color mutation of C.ensifolium‘Jin Qi had nothing to do with virus infection.2.Physiological mechanism analysis of C.ensifolium‘Jin Qi The contents of chlorophyll a,chlorophyll b and carotenoids in yellow part were significantly lower than those in green part.It can be preliminarily judged that the production of leaf art was related to the decrease of chlorophyll.There was no significant difference in anthocyanin data between yellow leaves and green leaves,and the content of total flavonoids in yellow leaves was lower than that in green leaves.Both of them indicate the total flavonoids and anthocyanins are not the reasons of leaf color change.The chlorophyll fluorescence parameters of F0,FM,Fv / FM,PSII,ETR,QN and QP in the yellow part were significantly lower than those in the green part,indicating that the photosynthetic structure was destroyed with a relatively lower photochemical efficiency.The data analysis showed that chloroplast synthesis was blocked in the stage of coprogen III→proto IX.In summary,the variation of the leaf color of the C.ensifolium ‘Jin Qi’may be caused by abnormal chloroplast development and obstruction of a certain stage of the chlorophyll synthesis pathway.3.Molecular mechanism analysis of C.ensifolium‘Jin QiAfter transcriptome sequencing,the total raw data measured was 249.87 Mp raw reads.After data filtering,247.47 Mp clean reads were obtained,with an average length of 856 bp.Among them,200-500 bp was the largest,accounting for56.69%.Compared with the public database,there were 71602 unigenes annotations.Among the most annotated NR databases,Dendrobium catenatum accounted for59.94%,and Cymbidium ensifolium accounted for 1.44%.Both of them were orchids,and the others accounted for only 3.49%.There are 2997 differentially expressed genes in the green part(DZ)and yellow part(HY),of which 1197 are up-regulated and 1800 down-regulated.The Go database annotated 53 sub functional groups distributed in three main categories.The number of differential genes in biological process,cellular component and molecular function were 1328,1125 and 1168 respectively.Compared with the GOG database,20763 unigenes were annotated,with a total of 25 types of function annotation information.The most commonly annotated type was R(general function prediction only),with 4291 unigenes,accounting for20.67%.There were 6163 unigenes annotated to 118 pathways in KEGG public database,and 2681(43.5%)annotated to metabolic pathways.The differentially expressed genes were enriched in the pathway related to leaf color formation was flavonoid biosynthesis,carotenoid biosynthesis and Porphyrin and chlorophyll metabolism,which annotated 61、26、56 unigenes,accounting for0.99%、0.42%、0.91% respectively.Combined with the results of cytological and physiological experiments showed that chlorophyll synthesis was blocked in the coprogen III→proto IX stage,and chloroplast structure was abnormal.We focused on three ways and the 16 candidate genes were screened,including GLK1 、ARC3、ARC6、CPOX、DVR、CLH2、SGR、CYP73A、CHS、CHI、F3H、CYP75B1、PSY、LCY-B、CCD7 and NCED.The expression of 16 genes was verified by real-time fluorescent quantitative PCR.The expression trend of 10 genes was the same as that of transcriptome analysis,which were GLK1 genes related to chloroplast synthesis;ARC3 and ARC6 genes related to the chloroplast distribution;The pathway of chlorophyll synthesis is related to CPOX and DVR1;CYP73A16、CHS3、F3H-1 were involved in the synthesis of total flavonoids;The genes related to carotenoid synthesis: PSY、LCY-B.Based on the results of cell,physiological data and transcriptome analysis,we further analyzed and screened the key genes: GLK1,ARC3 and ARC6 that determine the formation of chloroplast and division,and CPOX that determine the chlorophyll synthesis stage from coprogen III to proto IX.