De Novo Transcriptome Sequencing And Related Fountional Genes Analysis Of Narcissus Tazetta L.

Narcissus tazetta var.chinensis,a variety of Narcissus tazetta L.,is subordinate to Amaryllidaceae.It is one of the top-ten traditional flowers and has a long cultivation history over one thousand years in China.Due to the limited species,flower color and breeding resources of Chinese Narcissus,it is difficult to cultivate new varieties by conversional breeding.Currently,to improve the floral color of Chinese Narcissus is still one of the focus of researchers.In this study,the researchs are mainly divided into two parts.The first part is concentrated on the De nove transcriptome sequencing and metabolic pathways analysis of Yunxiang(Narcissus tazetta L.).The second part is about the cloning and expression analysis of WRKY transcription factor gene from'Zhangzhou Narcissus' and 'Huanghua Narcissus ?'.The results of this research would searve as a valueable genetic resources as well as a theoretical foundation for the breeding of new varities of Chinese Narcissus.The main results were as follows:1.Construction of the databases in the early flowering stage of white perianths and yellow coronas of Yunxiang variety of N.TazettaIn the present study,to reveal presumptive genes that are relevant to flower color and to uncover the molecular mechanisms that determine the color differences between white perianths and yellow coronas,RNA-Seq analysis were performed using the paired-end Illumina sequencing platform.Each library of the two samples produced 52,862,192(perianths)and 51,330,652(coronas)clean reads.After the filtration of the contigs,60,684 and 53,504 unigenes from the perianths and coronas,were identified.In total,we identified 8,866 DEGs s with 2263 up-regulated DEGs(399 were only expressed in coronas)and 6603 down-regulated DEGs(903 were merely expressed in perianths),were mapped to 124 KEGG pathways.The DEGs related to floral color were further verified by qRT-PCR,ten DEGs(GGPS,PSY.ZEP,PAL;4CL,F3H2,F3'H,FLS,MYB and WD40)were obtained,however,F3'5'H,ANS and ANR were not found in our databases.2.MYB and WD40 were cloned in Yunxiang variety of N.TazettaMYB and WD40 were cloned and named NtMYB and NtWD40,their ORF are 612bp and 1020bp(accession number:KT951723 and KU986509),encoding 203 and 339 amino acids,respectly.Blastn analysis of NtMYB of Yunxiang(Narcissus tazetta L.)showed that the similarity with 'Zhangzhou Narcissus',Musa acuminata subsp.Malaccensis,Glycine max and Arabidopsis thaliana were 89%,78%,79%,73%and 64%.Blastn analysis of NtWD40 of Yunxiang(Narcissus tazetta L.)showed that the similarity with Arabidopsis thaliana,Malus domestica,petunia hybrid and Medicago truncatula were 68%,68%,68%and 69%.3.qRT-PCR analysis of NtMYB and NtWD40The expression levels of NtMYB and NtWD40 were detected by qRT-PCR in the flowering phase including the flower bud stage,the alabastrum stage,the early flowering stage and the full-bloom stage with Actin as reference gene.The results showed that NtMYB and NtWD40 were expressed during flowering progress.The relative expression of NtMYB were higher in the first two stage than the early flowering stage.In the full-bloom stage,the expression levels of NtMYB in perianths were higher than coronas,and were exhibited an increasing tendency compared to the early flowering stage.As to NtWD40,the expression trends were increased from the flower bud stage to the early flowering stage,and the relative expression of NtWD40 were lower in the full-bloom stage than the early flowering stage,therefore,it was assumed that the NtMYB and NtWD40 may be associated wiih the color formation of Yunxiang(Narcissus tazetta L.).4.Construction of plant expression vectors of pCAMBIA-1301-NtMYB-GUS and pMDC44-GFP-NtMYBIn this study,the vectors of pCAMBIA-1301-NtMYB-GUS and pMDC44-GFP-NtMYB were constructed using double enzyme digestion method,this will contribute to the later study focusing on the transgene and improving the color characteristics of Yunxiang(Narcissus tazetta L.).5.WRKY gene were cloned in 'Zhangzhou Narcissus' and 'Huanghua Narcissus ?'WRKY gene were cloned and named NtWRKY40a and NtWRKY40b from'Huanghua Narcissus ?' and 'Zhangzhou Narcissus',their ORF were 945bp and 951bp(accession number:KM516168 and KM516169),encoding 314 and 316 amino acids,respectly.Blastn analysis of NtWRKY40a and NtWRKY40b showed that there were missing and replacement amino acid.Inaddition,the phylogenetic tree was constracted with other plants.6.qRT-PCR analysis of NtWRKY40a and NtWRKY40bThe expression levels of NtWRKY40a and NtWRKY40b were detected by qRT-PCR in the flowering phase including the alabastrum stage,the early flowering stage,the full-bloom stage and the faded stage with Actin as reference gene.The results showed that NtWRKY40a and NtWRKY40b were expressed during flowering progress.In 'Huanghua Narcissus ?',NtWRKY40a in the full-bloom stage and the faded stage showed the highest expression levels,and were significantly differential expression compared to the alabastrum stage and the early flowering stage.NtWRKY40b showed a significantly differential expression in the faded stage,while no differential expressions were existed among the first three stage.Therefore,it was assumed that the NtWRKY40a and NtWRKY40b have a possibility to play a role in the flower senescence in Narcissus tazetta L.