| Radish (Raphanus sativus L.) is one of the most important Brassicaceae root vegetables with high nutrition and medical value. Nitrate (NO3-) is one of the important nitrogen source, which could be directly used by higher plants. NO3- is the premise of strong carcinogenic nitrosamines, exessive intake can induce cancer of the digestive organs and cause serious threat to human health. Especially for higher-nitrate content root vegetables crops, is a major source of human exposure to nitrates. At present, the domestic researches about the key gene separation identification of nitrate metabolic pathways are rarely reported in radish, which greatly limits the nitrate accumulation traits genetic improvement. Therefore, by means of breeding screen low nitrate content of radish varieties, reducing the nitrate content of radish permitted to reach the range of human health has become one of the important goals for radish breeding, which has extremely important practical significance.In view of this, based on the analysis of different varieties of radish nitrate accumulation quantity difference, we studied on the effects of different NO3--N treatments on root-shoot ratio, nitrate content and nitrate reductase activity; molecular cloning, promoter cloning and vector construction of the key genes in NO3--N metabolism pathway; expression profiling analysis of related genes in nitrate assimilation; physiological and expression characterization analysis of nitrate transporters in different NO3- treatments. The results were as follows:1. Effects of different NO3 treatments on root-shoot ratio, nitrate content and nitrate reductase activity in radish Our comparative analysis of the nitrate content between 15 radish genotypes roots and leaves, and we were screening high-nitrate content radish variety’NAU-DHP’, medium-nitrate content radish variety ’NAU-QTSH’, low-nitrate content radish variety ’NAU-XHT’. Medium-nitrate content radish variety ’NAU-QTSH’ were cultivated in soilless substrate irrigating with nutrient solution with two kinds of treatments:(Ⅰ):differentNO3--N concentration 0,5,20,30 and 50 mmol·L-1 for 24 h; (Ⅱ):different treatment time 0, 4,8,12 and 24 h under 30 mmol·L-1NO3--N, and then the root-shoot ratio, nitrate content and nitrate reductase activity (NRA) were determined. The results showed that the root-shoot ratio was increased with increasing No3--N concentration, reached maximum at 30 mmol·L-1, and then gradually decreased; With prolonged treatment time, root-shoot ratio was also increased. With increasing concentrations, nitrate content of the roots and leaves showed a decreasing trend after the first increase, and as the duration time extending, the nitrate content was also increased, but the leaves absorbing NO3--N slower than the roots, and the nitrate content of the roots was higher than leaves. With the increasing concentration, nitrate reductase activity of leaves and roots reached at minimum under 5 mmol·L-1, and reached a peak at 30 mmol·L-1 and then decreased, while with time extension, nitrate reductase activity in leaves was reached a maximum at 8 h, and then decreased rapidly, and the nitrate reductase activity in leaves was higher than roots.2. Molecular cloning, promoter cloning and vector construction of the key genes in NO3- metabolism pathwayBy homologous cloning method, the full-length cDNA and genomic DNA sequences of RsNiR & RsGS2 and the full-length cDNA sequences of RsNR, RsGDHl & RsGDH2 were isolated from radish. The genomic DNA sequence of RsNiR and RsGS2 were 1936bp and 2190bp, including 1524bp and 1287bp open reading frame, encoding 507 and 428 amino acid, respectively; The full-length cDNA sequence of RsNR, RsGDH1 & RsGDH2 were included 2742bp,1236bp and 1275bp,1524bp open reading frame, encoding 913,411 and 424 amino acid, respectively. Using the Genomic DNA Walking method, the 343bp, 843bp & 912bp 5’-flanking region upstream of RsNR, RsNiR & RsGS2 were isolated, respectively. The promoters of RsNR, RsNiR & RsGS2 contained basic cis-regulatory elements, including TATA-box and CAAT-box, light responsiveness elements and MYBHvl binding site. To further study the function and expression patterns of these genes, the studies provide a theoretical basis. Meanwhile, the construction of genes RsNiR, RsGS2 expression vectors were provided for transgenic radish studies.3. Expression profiling analysis of related genes in nitrate assimilation in radish issuesBy RT-PCR and qRT-PCR technology, we study the expression profiling of related genes in nitrate assimilation in radish different issues. The results suggested that the RsNR expression level in leaves, roots and fibrils was higher, but in petioles and stems was hardly expressed, and the leaves and roots reached a maximum under 50 mmol·L-1, the fibrils was under 20 mmol·L-1, what’s more, roots and leaves reached a maximum at 4 h, the stems and petioles were reached a peak at 12 h; The highest RsNiR was observed in leaves and roots, in the stems, petioles and fibrils only small amounts of RsNiR were found. The highest mRNA levels of RsNiR was induced by 30 mmol·L-1 NO3--N in leaves and roots, but in petioles, stems and fibrils was induced by 20 mmol·L-1. In addition, there was a rapid induction of NiR mRNA upon addition of nitrate and reached a maximum at Id in all organs, and then decreased. However, leaves were transiently induced with levels decreasing after 8 h despite the continued presence of nitrate in medium. RsGS2 gene expression reached a maximum at 30 mmol·L-1 in leaves, but roots reached the maximum at 20 mmol·L-1, the expression in fibrils reached a maximum at 4 h; RsGDH1 transcript level in roots was reached a maximum under 5 mmol·L-1, while the roots reached a peak at 12 h; RsGDH2 gene was reached a maximum under 50 mmol·L-1, while the roots reached a peak at 12 h; GLN gene family expression levels was highest in the roots. Based on the characteristics of the expression of key genes, revealing the molecular mechanisms underlying radish nitrate metabolic processes, provide the basis for screening low nitrate radish varieties.4. Physiological difference and expression characterization analysis of nitrate transporters in different NO3- treatmentsBy RT-PCR and qRT-PCR technology, we studied the physiological difference and expression characterization of nitrate transporters in different NO3- treatments of radish issues. The results demonstrated that the nitrate accumulation expression of different radish genotypes at high concentrations is more obvious, the ability of DHP transport nitrate was stronger than XHT in nitrate transport, and easier to absorb nitrate ions from the environment. The NRA of DHP was significantly higher than XHT, it has no obvious relationship to the culture conditions and plant tissues, DHP compared with XHT, the nitrate absorption capacity was stronger, the absorption rate was faster, and the reducing ability was also stronger than XHT. The xpression level of RsNRTl.l in DHP was higher than XHT, and the difference was more significant at low concentrations. RsNRT1.5 expression in accordance with order that the expression of fibrils, roots, stems, petioles, leaves, and DHP expression was higher than XHT; The expression level of RsNRTl.6 was decreased in XHT; RsNRTl.7 was mainly expressed at lower concentrations in DHP leaves and roots, but mainly expressed in the XHT petioles and stems; RsNRT2.1 gene have high expression levels, and promote the growth of lateral roots; RsNRT2.2 is a high-affinity transporter protein, and the induction of nitrate was played a significant role; RsNRT2.3 was mainly expressed in roots, inhibiting the expression in roots. |
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