Mechanism Of Transcription Factor CmHY5 Affecting Root Growth And Nitrate Uptake Of Oriental Melon/Squash Grafted Seedlings

At present,grafting technology has been widely used in the production of melon in China.The grafted cultivation of melon can not only effectively prevent soil-borne diseases and overcome continuous cropping obstacles,but also can significantly promote the absorption and utilization of nitrate nitrogen in grafted melon plants.The level of nitrogen content has a significant effect on the yield and quality of grafted melon,but the mechanism of nitrate absorption under the interaction of oriental melon and squash rootstocks has not been clarified.In the previous screening of different oriental melon and squash grafting combinations,our research group found that the stock-spike combination with high nitrate nitrogen absorption rate showed higher expression of nitrate transporter CmoNRT2.1 and transcription factor CmHY5.In view of this,this study use oriental melon cultivars ’JS1408’and ’JS1429’ as scions,and squash cultivars ’ZM1519’ and ’ZM1507’ as rootstocks to explore the interaction of HY5 in oriental melon scions with NRT2.1 in squash rootstocks,combined with the morphological characteristics of root system,the molecular mechanism of CmHY5 regulating nitrate nitrogen uptake by grafted melon seedlings was analyzed,in order to provide theoretical basis and practical basis for the screening of high nitrate nitrogen utilization stock-spike combinations.The main findings are as follows:1.Using bioinformatics software,through amino acid sequence alignment and phylogenetic tree construction,melon genes CmHY5 and squash CmoHY5-1 and CmoHY5-2,which are highly homologous to HY5,and squash gene CmoNRT2.1,which were identified are highly homologous to NRT2.1.Amino acid sequence analysis showed that CmHY5,CmoHY5-1 and CmoHY5-2 all contained bZIP protein domain,while CmoNRT2.1 contained MFS₁ protein domain,which was consistent with the structural characteristics of these two kinds of proteins.Subcellular localization showed that CmHY5 and CmoNRT2.1 are located on the nucleus and cell membrane in tobacco epidermal cells,which was consistent with their biological functions.2.Through 15N stable isotope tracing and root scanning analysis of the grafted seedlings of different melons grafted with the same squash rootstock,it is found that the grafted seedlings with melon ’JS1408’ as the scion had significant nitrate uptake activity and absorption rate,as well as root phenotypes of the rootstocks,including root length,root surface area and root forks were significantly higher than ’JS1429’,while the average root diameter was not significantly different;CmHY5 was further detected in squash rootstocks by qRT-PCR technology,CmHY5,CmoHY5 and CmoNRT2.1 genes in roots are detected.There are significant differences in expression.The expression patterns of CmHY5 in different tissues of the oriental melon self-rooted seedlings are analyzed.The results showed that CmHY5 is express in the roots,stems and leaves,and the expression of CmHY5 in the leaves and stems of the melon scion ’JS1408’ is significantly higher than that of ’JS1429’,indicating that the cultivar characteristics oriental of melon scion affecte the nitrate uptake activity,uptake rate and root phenotype of grafted seedlings.3.The CmHY5 silent grafted seedlings are obtained by agrobacterium-mediated VIGS transient silencing,and irrigated with 15N isotope.The analysis results showed that the expression of CmoNRT2.1 in the root system was significantly down-regulated;Compared with the wild type,the root length,root surface area and root fork of VIGS plants are significantly reduced,while the average root diameter is not significantly different.This indicated that CmHY5 is a positive regulator involved in the regulation of CmoNRT2.1 on root phenotype and nitrate uptake.4.The yeast expression vectors of CmoNRT2.1,CmHY5,CmoHY5-1 and CmoHY5-2 are constructed by DNA recombination technology.The results of yeast one-hybrid experiments showed that the transcription factors CmHY5,CmoHY5-1 and CmoHY5-2 all had significant interactions with CmoNRT2.1.GUS experiments and tobacco dual luciferase experiments further verified that CmHY5,CmoHY5-1 and CmoHY5-2 can transcribe activates CmoNRT2.1 expression.