| Using three varieties of grandiflora clematis with different flower types(single tepal species' Julka',semidouble tepal species 'Vyvyan Pennell' and double tepal 'Blue Light')as test materials to explore the relevant mechanism of the formation of double flower clematis.Conventional paraffin section method and microscopy were used to observe and compare the flower bud differentiation period of the three different perianth types of grandiflora clematis to clarify the origin mode of the heavy perianth type of grandiflora clematis.The contents of four endogenous hormones(IAA,GA,ABA,ZR)in three different perianth types of clematis were determined by ELISA.Using high-throughput sequencing technology to "Vivian" at the same time three different petals on the same plant type(single,semidouble,double)for the transcriptome sequencing,Refering to transcriptome data of differential expression genes of three samples and selected some key genes to further verification analysis by means of fluorescence quantitative PCR.Preliminary screening related genes which could control the clematis double flower traits.Main results are as follows:1.Observing flower bud differentiation of three varieties of grandiflora clematis with different flower types.Result showed that flower bud differentiation of large flower clematis were growed by the inside extroversion,the flower bud differentiation process can be divided into the early stage of the flower bud differentiation,early stage of flower bud differentiation,sepal primordium differentiation stage,stamen primordium differentiation stage and pistil primordium differentiation stage.So the origin of clematis was different degrees petaling of female and male stamens.In the process of petaling of female and male stamens,the petaling of male stamens usually precedes the petaling of female stamens2.LSD difference comparison showed that the mass fraction of four plant hormones in different stages of flower bud differentiation and differen tclematis varieties both have significant changes.Among them,low concentration of GA,ABA and high concentration of IAA are conducive to the transformation of clematis buds from vegetative growth to reproductive growth after breaking dormancy,while low concentration of IAA and high concentration of ZR,ABA and GA are conducive to the differentiation and morphology of flower buds to promote the development and maturity of flower organs.At the same time,clematis at double perianth period hadhigher GA content compared with other differentiation times.Rising IAA content is likely benefit to the evolution of sepals from single to double.The changes of ABA content basically had the same trend of high and low variation in different flower bud differentiation stages,and ABA content in the same differentiation stage was higher in the early double petals than in the single petals,and higher in the late single petals than in the double petals.The effect of ZR on the single and double clematis characters was not obvious.In terms of hormone balance,at the late stage of flower bud differentiation,ABA/GA and GA/ZR ratio increased,while the ratio of ZR/IAA decreased,which may be conducive to the development and maturation of flower organs and the formation of double petaling traits of clematis varieties.3.Clematis.‘Vyvyan Pennell'from the same anther(July)of the same plant with three different perianth types were sequenced by transcriptome.The cDNA library of clematis was preliminarily constructed.Transcriptome data were screened,spliced,functionally annotated,and the differential expression of Unigene were analyzed.Transcriptome sequencing produced a total of 13.8GB original data,a total of 146 036 sequences and 99 652 Unigene genes were obtained.The length of these genes ranged from 201 to 14 638 bp,with an average length of 977 bp.Homologous search and functional annotation were performed on the Unigene obtained after clustering.A total of 9 133 Unigene were homologous with seven databases(NR,GO,NT,Pfam,KEGG,KOG,Swissprot),accounting for 9.16% of the total.After SSR site search and analysis,23 035 SSR sites were identified from a total of 99 652 sequences detected,including18 243 SSR sequences,with a total length of 129 654 975 bp.A total of 3,075 differentially expressed genes(DEG)were obtained by comparing the transcripts of three different flower types in pairs,among which 134 differentially expressed genes coexisted in the three samples.There were 649 up-regulated DEG and 605down-regulated DEG in transcripts of single and semi flowering(A vs B).The comparison of semi-dead flowers with dead dead flowers(B vs C)included 1046up-regulated DEG and 721 down-regulated DEG.There were 1129 upregulation DEG and 859 upregulation DEG in the comparison of single blossom and double blossom(A vs C).4.25 differentially expressed genes were selected from 134 DEG coexistenced in three samples according to their metabolic pathways and functions.Then drawed the clustering diagram to clearly showed the expression levels of each gene in single flower,semi-double flower and fully double flower.10 high expressed genes with larger readcount,longer sequence and may be associated with flower growth were selected to fluorescence quantitative PCR.Integrated the transcriptome data and fluorescence quantitative PCR test results we can known that the gene types that affect clematis doubling are mainly related to growth and development traits such as flower development,endogenous hormones and cell wall remodeling,etc.Indoleacetic acid and abscisic acid are the major endogenous hormones involved in the doubling petal of clematis.Uunder the same growth conditions,IAA in double flower had higher quantity and ABA had higher quantity in the single flower.According to the results of the comprehensive data,the genes with significant differences in expression in clematis are mainly(1)MADS-box genes: PMADS 1,AP3,FRUITFULL and FLC.(2)Auxin reactive protein genes :IAA9,auxin input vector,indoleacetic acid-induced proteinARG7,Abscisic acid ‘8' hydroxylase,ACS,etc.Therefore,it is speculated that these key genes may be related to the regulation of double tepal traits of clematis. |
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