Cloning Of CsNLP1 Gene In Cucumber And Its Function Identification In NO₃^- Signaling Regulation

Cucumber(Cucumis sativus L.)is one of the important vegetables cultivated in facilities in China.Due to the deterioration of soil quality in facilities year by year and the low NUE of existing varieties,the phenomenon of pursuing high yield through excessive application of N fertilizer is common in production.Improving crop NUE is one of the important means to reduce N fertilizer inputs.It has been shown that NLP(NIN-like protein)family genes play an important role as transcription factors in regulating the physiological responses of plants to adapt to changes in the external nitrogen environment,however,few studies on cucumber NLPs have been reported.In this study,we first identified the cucumber NLP family members,performed a comprehensive bioinformatics analysis to analyze their evolutionary relationships and predict their potential functions.Analysis of the expression of the family genes in different tissues and their response to the external nitrogen environment screened that the CsNLP1 gene responded most strongly to NO3-,but its response mechanism and function were not clear.Therefore,in this study,we cloned the CsNLP1 gene,transferred it into Arabidopsis by constructing an overexpression vector,analyzed the role of overexpression of CsNLP1 transgenic Arabidopsis in nitrogen metabolism,and transformed cucumber using VIGS technology to compare the growth and nitrogen utilization of control and transgenic plants.To reveal the biological functions of CsNLP1 gene in nitrogen metabolism of cucumber,the main results were as follows:(1)AtNLP7 is a key transcription factor of primary nitrate response that regulates NO3-signaling and assimilation.In this study,we found three genes homologous to AtNLP7 in cucumber using bioinformatics;evolutionary analysis of NLP in different species was performed,and the analysis revealed that most of the NLP family proteins contain three conserved structural domains:RWP-RK,PB1 and GAF-like structural domains,among which CsNLP1 has the closest affinity to AtNLP7,suggesting that CsNLP1 gene may also be involved in the regulation of N metabolism in cucumber.The cucumber NLP gene family members all contain four introns and five exons with similar motif composition and protein structure;analysis of the promoter region revealed that the cucumber NLP gene family members contain cis-acting elements related to plant growth,environmental stress and hormone signaling,and it is hypothesized that the family genes may be involved in cucumber growth and development in response to adversity.The expression patterns of cucumber NLP gene family members in root,stem,leaf,flower and fruit tissues were analyzed,and it was found that cucumber NLP family members were expressed in all tissues to varying degrees,with the highest expression of CsNLP1 gene in leaves and higher expression of CsNLP2 and CsNLP3 in leaves,male and female flowers.The cucumber NLP gene family responded to both the external nitrogen environment and drought environment,with the CsNLP1 gene responding most strongly.(2)In this study,the CsNLP1 gene was cloned and transformed into the Arabidopsis nlp7-1 mutant,and it was found that the phenotype of the transgenic Arabidopsis strain could be restored to the wild-type level;more in-depth studies revealed that after NO3-treatment,the NO3-content,NR activity,NIR activity,GDH activity,GOGAT activity,and GS activity in the transgenic strain could be restored to the wild-type level.In addition,CsNLP1 was able to regulate the relative expression of NO3-responsive genes such as AtNIR1,AtNIA2,AtNRT1.1 and AtGS2 in the plants;this indicates that CsNLP1 can affect the assimilation and regulation of NO3-.(3)CsNLP1 gene silencing vector was constructed,and cucumber was transfected with VIGS technology.After detecting the silencing efficiency by qRT-PCR,plants were treated with different concentrations of NO3-for phenotypic observation and determination of physiological indexes.The results showed that the biomass of stems,leaves and roots of VIGS-disturbed plants were significantly decreased under different concentrations of nitrogen treatment;moreover,NR activity,NIR activity,GDH activity,GOGAT activity and GS activity were also significantly lower than those of control plants;this indicated that CsNLP1 could affect the growth of plants as well as nitrogen assimilation,and had an important role in the process of NO3-uptake and assimilation.