Preliminary Function Analyses Of Nitrate Transporter Genes CmNRT2s And Transcription Factor CmLBD38 In Chrysanthemum

Chrysanthemum(Chrysanthemum morifolium Ramat.)is one of the top ten most popular flowers in China and top four cut flowers in the world.Nitrogen(N),an essential nutrient for fundamental biological molecules,such as nucleotides,amino acids and protein,plays a crucial role in plant growth and development.To improve the yield and quality,considerable amounts of N fertilizers are applied.However,excess use of N fertilizer and low nitrogen use efficiency(NUE)not only decrease the crop yields,but also cause pollution of soil and rivers,leading to eutrophication.Mechanisms of nitrate transport in chrysanthemum should be understood in depth to reduce the consumption of N fertilizers and incresse NUE.Based on the genes cloned from CmNRT2s gene family in Chrysanthemum before,we isolated CmNRT2.5b and CmNRT2.7b genes and promoters of CmNRT2s.We also obtained transgenic Arabidopsis and tobacco lines through the construction of expression vectors to perform the analysis of CmNRT2.5b and CmNRT2.7b.we also analyzed the function of transcription factor CmLBD38.The main results were as follows:1.Isolation and analyses of promoters of CmNRT2sNRT2s family plays an important role in nitrate transport.We isolated the promoters of CmNRT2.2,CmNRT2.3,CmNRT2.5,CmNRT2.6 and CmNRT2.7 by chromosome walking.Their length were 1597bp,1231 bp,949bp,2206bp and 1567bp seperately.Using the Softberry database and PLACE database,we found many cis-elements in promoters responsed to nitrate?carbon and lights which contributed to understanding the transcripts of CmNRT2s and searching for new nitrate resopnsive elements.2.Cloning and function analyses of CmNRT2.5b and CmNRT2.7bFull-length cDNA fragments of high affinity nitrate transporter genes CmNRT2.5 and CmNRT2.7 were cloned from Cut Chrysanthemum 'Nannong Xuefeng',named CmNRT2.5b and CmNRT2.7b,by using the RT-PCR technology.Sequence alignments of amino acids between cloned and obtained before found that the similarities are 98.41%and 99.34%.We constructed expression vectors pMDC43-CmNRT2.5b and pMDC43-CmNRT2.7b and transformed them into Arabidopsis thaliana ending up with 2 and 3 transgenic lines seperately.Meanwhile,promoters of CmNRT2.5b and CmNRT2.7b isolated from chrysanthemum were constructed into pORER2 vector containing GUS gene and transformed into tobacco.Selected by Kanamycin,we obtained the genetic stability of tobacco plants via tissue culture.These transgenic plants have laid the foundation for the further study of function of genes and promoters.3.Cloning and function analyses of CmLBD38Using the EST sequences in transcriptome database,RT-PCR and RACE methods,full-length cDNA sequence of LBD gene family were cloned from Cut Chrysanthemum'Nannong Xuefeng',designated CmLBD38.Sequence analysis showed that:CmLBD38,845bp at length,contained an 525bp open reading frame(ORF)and encoded a protein of 175 amino acids with a LOB family domain consisting of 107 amino acids.Hence,we suggested it belong to the LBD family.Phylogenetic tree analysis showed that CmLBD38 has a close relationship with Arabidopsis LBD class II genes.Recombinant vectors pMDC43-CmLBD38,pGBKT7-CmLBD38 and 35S::GAL4-DB-CmLBD38 were constructed to analyze subcellular localization and transactivation of CmLBD38.The results showed that CmLBD38 localized in the nucleus without transcription activation or inhibition activity.Besides,transgenic Arabidopsis lines of CmLBD38-overexpression were obtained.