The Molecular Mechanism For Flowering Regulation Of CmNLP7 In Chrysanthemum

Chrysanthemum(C.morifolium),which is originated in China,is considered as one of the top ten traditional famous flowers in China and one of the four most popular cut flowers in the world.It has a long history of cultivation in China.It is favored by a large number of consumers because of its rich colors,varied flower types and long vase life.It also provides high ornamental and economic value.The majority of chrysanthemum belong to short-day plant,with flowering from October to November.It is difficult to meet the market demands.Therefore,the regulation of chrysanthemum flowering can improve the market utilization value of chrysanthemum so as to increase the income.During the cultivation of chrysanthemum,the use of nitrogen fertilizer and the regulation of flowering attract the most concerns of the growers.NLP 7 encodes the nodule inception(NIN)transcription factor.It shows two typical structure domains including RWP-RK and PB1 and is conserved among different species and genera.In Arabidopsis,AtNLP7 has been identified as the key transcription factor of primary nitrate reaction,which can be combined with nitrogen response cis elements to control the nitrate absorption and the transcription of assimilated genes.In addition to its role in nitrate reaction,the nlp7 mutant of Arabidopsis has the late flowering phenotype,while the mechanism was unkowm.In the present study,CmNLP7 was cloned from Chrysanthemum ’Jinba’ and its flowering function and mechanism was analyzed.The obtained results are shown as follows.1.The total length of CmNLP 7 is 2658 bp,encoding 885 amino acids were cloned from chrysanthemum ’Jinba’.The phylogenetic tree showed high homology with AtNLP7,CmNLP7 has typical RWP-RK and PB1 domains.Subcellular localization and transcriptional activation activity experiments showed that CmNLP7 were localized on the nucleus,and has no transcriptional activation activity.Tissue quantitative analysis found that CmNLP7 were expressed in all tissues,with the highest expression in roots and leaves,followed by stems and flowers,and the overall expression in reproductive growth stage was higher than that in vegetative growth stage.It was speculated that this gene could regulate the growth and development of chrysanthemum.2.The transgenic chrysanthemum used in the present study was transformed with the over-expression vector pORE-R4-CmNLP7 and artificial miRNA plant expression vector pORE-R4-amiR-CmNLP7 respectively.It can be observed that the flowering time of the CmNLP7 knock-down transgenic Chrysanthemum was 7 to 9 days earlier than that of the wild plants.The flowering time of the over-expression lines was delayed by 3 to 4 days compared with the wild variety.The over-expression lines of CmNLP 7 transgenic chrysanthemum,the knock-down lines and WT were treated with high nitrogen and low nitrogen respectively.It was shown that the flowering time of the over-expression lines and WT was delayed about 21 to 28 days under low nitrogen condition compared with those under high nitrogen condition.Furthermore,the flowering time of the knock-down lines of CmNLP7 transgenic chrysanthemum was not affected by high nitrogen and low nitrogencondition.The results show that the knock-down CmNLP7 in chrysanthemum can reduce the effect of nitrate concentration on flowering time.Based on the differences in the flowering phenotype of the transgenic plant,the transcriptome was sequenced.Analysis of the transcriptome data revealed that the nitrate transporters NRT2.5,NPF3.1,and NPF4.6 were significantly up-regulated in the interfering lines,and the calcium ion binding protein CML23 and calcium-dependent protein kinase genes CPK3 and CPK27 were also significantly up-regulated in the interfering lines,indicating that interference with CmNLP 7 can promote the expression of these genes.The flower-promoting gene Gibberellin 20 oxidase 1 in the gibberellin pathway,the gibberellin receptor gene GID1,and SPL 7,SPL9,and SPL12 in the age pathway,and the flowering integration factor FT are up-regulated in the interfering lines.It’s consistent with its early flowering phenotype.