| Oriental melon is an important species in facility melon cultivation,and has become an essential industry in Northeast China.However,grafting cultivation is adopted due to the frequent occurrence of soil-borne diseases in the cultivation of oriental melons in facilities,which seriously affect its yield and quality.The practice has shown that grafting can enhance the disease resistance and cold resistance of oriental melon and has a strong root absorption capacity(such as water and nitrogen,phosphorus,potassium,and other nutrient absorption capacity).The improvement of root absorptive capacity of grafted thin-skin melon plants is often thought to be due to the well-developed root system of rootstocks.Recent research results have shown significant differences in the nitrate uptake capacity of roots of grafted plants of the same rootstock variety and different scion varieties,but the underlying mechanism is still included.HY5 is a transcription factor of the b ZIP family,which regulates the expression of multiple genes and can also regulate nitrate uptake.This study used the oriental melon variety ‘JS-1429’ and ‘JS-1408’ and the squash rootstock variety ‘ZM1507’ as the test materials.Two different rootstock combinations JS-1429/ZM1507,were compared and analyzed first.The difference in nitrate absorption rate and absorption activity with JS-1408/ZM1507,the relationship between Cmo NRT2.1 expression and nitrate absorption in squash rootstocks,analyzed the relationship between oriental melon Cm HY5 and squash rootstock Cmo HY5 in Tissue-specific expression in different graft combinations and their interaction.Finally,the effects of the interaction between Cm HY5 and Cmo HY5 on Cmo NRT2.1 and nitrate absorption capacity were systematically analyzed to understand the melon’s molecular mechanism Cm HY5 regulating the expression of Cmo NRT2.1 in squash rootstocks and affecting nitrate absorption.The main results are as follows:1.Gene identification of oriental melon HY5 and squash HY5.HY5 genes of Arabidopsis thaliana,apple,and tomato were used to obtain candidate HY5 genes of oriental melon and squash through homology alignment.Then the protein domains of all the obtained genes were determined and gene structure analyzed,and then use the amino acid sequence of the gene to construct into a phylogenetic tree to obtain Cm HY5,Cmo HY5-1,and Cmo HY5-2.2.Comparison of nitrate absorption rate and absorption activity of different grafting combinations and analysis of Cmo NRT2.1 expression in roots.The nitrate absorption rate of grafting combination JS-1408/ZM1507 was 1.57 times that of JS-1429/ZM1507,and its nitrate nitrogen absorption rate was 1.57 times that of JS-1429/ZM1507.The nitrogen absorption activity is 1.19 times that of JS-1429/ZM1507.It can be seen that the grafted combination JS-1408/ZM1507 showed a strong nitrate absorption capacity.The results of measuring and analyzing the expression levels of Cmo NRT2.1 in the roots of the two grafted plants showed that the graft combination JS-1408/ZM1507 with higher nitrate nitrogen absorption rate and absorption activity also had higher Cmo NRT2.1 expression levels,and the graft combination JS The expression level of Cmo NRT2.1 in the roots of-1408/ZM1507 was 2.02 times that of JS-1429/ZM1507,and the difference was highly significant.At the same time,the root Cmo NRT2.1 was transiently silenced by the VIGS assay,which proved that Cmo NRT2.1 positively regulates the nitrate uptake capacity of the oriental melon/squash grafted plants.3.Comparison of tissue expression specificity between Cm HY5 and Cmo HY5 in different grafting combinations.Taq Man technology was used to detect the expression levels of Cm HY5 and Cmo HY5 in different tissues and organs of two grafting combinations.The expression levels of Cm HY5 in scion leaves and stems of JS-1429/ZM1507 were 1.11 and 1.18 times of JS-1429/ZM1507,respectively.While the expression levels of Cm HY5 in rootstock stems were significantly higher than that in JS-1429/ZM1507,3.21 times of JS-1429/ZM1507.In addition,the expression levels of Cmo HY5 in the two grafting combinations were determined and analyzed.The expression levels of Cmo HY5-1 in the rootstock stems and rootstocks of the grafting combination JS-1408/ZM1507 were 1.23 and 1.18 times higher than those of JS-1429/ZM1507,respectively.The expression levels were 1.82 times and 1.60 times that of JS-1429/ZM1507,respectively.In contrast,the expression of Cmo HY5-1 and Cmo HY5-2 was not detected in the scion.4.Interaction analysis of Cm HY5 and Cmo HY5.Through yeast one-hybrid,EMSA,and tobacco GUS reporter assays,it was shown that Cm HY5 could bind to the promoters of Cmo HY5-1 and Cmo HY5-2,respectively.It can be seen that Cm HY5 can both transcriptionally regulate the expression of Cmo HY5-1and Cmo HY5-2.In addition,we performed a yeast two-hybrid assay,luciferase complementation assay,and bimolecular fluorescence complementation assay with Cm HY5,Cmo HY5-1,and Cmo HY5-2,respectively.The results showed that Cm HY5 could interact with Cmo HY5-1 and Cmo HY5-2,respectively.5.The effect of the interaction between Cm HY5 and Cmo HY5 on the expression of Cmo NRT2.1 and nitrate absorption.The tobacco GUS reporter gene test was conducted by adjusting the ratio of different Cm HY5 to Cmo HY5-1 and Cmo HY5-2 and the promoter of Cmo NRT2.1.The results showed that Cm HY5,Cmo HY5-1,and Cmo HY5-2 have positive regulatory effects on the Cmo NRT2.1 promoter and Cmo HY5-1 has a dominant effect on the promoter of Cmo NRT2.1.Through the transient silencing test of Cm HY5 in scion leaves of grafted plants,we found the expression levels of Cmo HY5-1 and Cmo HY5-2 in the rootstock stems and roots of the silenced plants were significantly reduced,and the expression level of Cmo NRT2.1 significantly reduced.In conclusion,the oriental melon transcription factor Cm HY5 is transported into squash rootstock over a long distance and binds to the promoters of Cmo HY5-1 and Cmo HY5-2 in squash rootstock,and positively regulates the transcriptional expression of Cmo HY5-1 and Cmo HY5-2.Promote the increased expression of Cmo HY5-1 and Cmo HY5-2 in squash rootstock(and the higher the long-distance transported Cm HY5 expression,the higher the Cmo HY5-1 and Cmo HY5-2 expression in squash rootstock),Cm HY5 and Cmo HY5-1 and Cmo HY5-2 were transported into squash rootstock roots to jointly promote the transcriptional expression of Cmo NRT2.1(and Cmo HY5-1 played a leading role in transcriptional regulation of Cmo NRT2.1 expression)and improved the nitrate absorption capacity of squash rootstock roots.It can be seen that the nitrate absorption capacity of the roots of the oriental melon/squash grafted seedlings is regulated by the oriental melon. |
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