Studies On The Changes Of Structure,Physiology And Gene Expression In Chromolaena Odorata Adventitious Rooting

Chromolaena odorata(L.)RM King and H.Robinson is a perennial member of the Compositae family.It is also known as a Eupatorium odoratum,airplane plant or Siam weed.The species is a native of Central and South America.In recent decades,this species was widespread over many parts of the world including Southeast Asia,Africa and the Pacific Islands.In China,C.odorata can be found in the provinces of Yunnan,Guangxi,Guangdong,Hainan and Guizhou.The species is considered to be one of the world's worst invasive alien species.It has been showed that C.odorata has strong adventitious root formation ability,which is closely related to its high invasion ability.However,the mechanism of adventitious root formation in C.odorata has not been studied.Hence,the changes of structure,physiology and gene expression in C.odorata adventitious rooting were researched in the paper.The main results were showed as follows:1.The structure and physiological changes of C.odorata in adventitious root formationThe changes of structure and physiology of C.odorata were studied by cutting test.Adventitious roots were induced in C.odorata cutting within a week,and 100%adventitious rooting rate was gained in different base materials including vegetable soil,red soil and sand.It was easy to induec adventitious root at water content from 20%to 60%in base materials.It showed that C.orodata has strong environment adaptality.C.orodata stem has thin cortex,big pith,and lacks thick wall cells.Latent root primordium was not found in the stem,and the primordium was initialed from fascicular cambium cells.There was not callus formation in adventitious rooting of C.orodata.The formation of root primordium and adventitious root was found on third day and 7th day,respectively.The contents of IAA,ZT,and POD activity showed a rising tendency overall,but those of ABA and flavonoids showed a downtrend in adventitious rooting of C.orodata.2.RNA-seq analysis of C.odorata in adventitious rootingThe stem bases of C.odorata cuttings were collected for transcriptome sequencing at different stages:0,3 and 7 days after cutting.180,805 unigenes were generated by RNA-seq,and about 90,000(nearly 50%)were annotated.These unigenes were assigned to different functional classification by COG and GO analysis such as secondary metabolites biosynthesis,transport and catabolism,signal transduction mechansisms,et al.It shows that C.odorata's rooting process needs multiple genes to participate in encoding various proteins.A total of 40,348 annotated sequences were mapped to the reference canonical pathways in the KEGG database,and they were assigned to 121 KEGG pathways.Among them,1,562 unigenes were involved in hormone-related pathways.They were cysteine and methionine metabolism,tryptophan metabolism,carotenoid biosynthesis,diterpenoid biosynthesis and brassinosteroid biosynthesis.Several integrated secondary metabolic pathways were also discovered.It was indicated that these pathways correlated closely with rooting mechanisms in the cuttings.In short,the transcriptome database contained abundance informations on rooting,so they may serve as public data sets for future genetic research on this topic.3.Differential expressed gene analysis of Chromolaena odorata in adventitious rootingThe stem bases of C.odorata cuttings were collected at different stages:0,3 and 7 days after cutting.The first stage and the mix sample of last two stages were using for DGE analysis,and the DGE tag sequences were mapped to the C.odorata reference transcriptome dataset.FDR?0.001 and absolute value of log2ratio?1 were used as the threshold for significant differences in gene expression.Compared with before cutting,4,392 unigenes exhibited significant differences after cutting.Beside those genes that sepecially expressed only in befre or after cutting,and their genetic fragments detected were comparatively rare,2,062 unigenes were found to be significantly up-regulated,while 1,238 unigenes were significantly down-regulated.Among these,87 unigenes were found to be significantly regulated by more than 50times.These genes may be the key factors in Chromolaena odorata rooting.The differentially expressed genes were performed further analysis.The results showed that the differentially expressed genes were arranged into 10 categories according to functional classification,including hormones,growth and developmental process,defense,cytochrome P450,ubiquitin,peroxidase calcium ion,zinc finger protein,elongation factor and aspartate-related,besides those essential genes for maintaining the life.The analysis of metabolic pathway showed that Cytokinins,abscisic,ethylene,gibberellin and flavonoids may be the main secondary metabolite during in the process of rooting in C.odorata.4.Gene Cloning and its expression in tobacco of flavonoid 3'-hydroxylase in C.odorataA flavonoid 3'-hydroxylase gene cDNA sequence was cloned by RACE and RT-PCR techniques from C.odorata.The obtained full-length cDNA was named CoF3'H with GenBank accession number HQ268505.1.It is 1628 bp in length,containing a 1524 bp open reading frame,encoding 507 amino acid residues.The amino acid sequence contain cytochrome P450 domain and cysteine heme binding region(F××G×R×C×G).Homology analysis showed that the deduced CoF3'H protein was highly homologous to F3'H proteins from different species.CoF3'H gene was transferred into tobacco,it was highly expressed in tobacco,and the transcript of CoF3'H in transgenic tobacco plants was 69 times high than that in wild plants.Moreover,transgenic plant had higher content of flavonvoids than wild type plant.This result shows that CoF3'H plays an important role in flavonoid biosynthesis.The CoF3'H expression in transgenic tobacco can inhibit the growth of this plant by studying the T2 phenotypes.