| Chamaecrista nictitans is tropical perennial legume forage of Chamaecrista spp., with many distinctive characteristics such as barren-resistant, acid-resistant and drought-resistant. At present, it has been extensively planted in Fujian, Jiangxi and Hunan provinces, and it was used for improving the red soil, conserving water and soil and stock. Chamaecrista nictitans has the maximum nutrient content at the early flowering period, and especially crude protein (CP) content reaches 15.9%. However, it had not been extensively utilized in animal husbandry due to its high condensed tannins content.This experiment has seven nitrogen treatments: 0, 15, 30, 45, 60, 75 and 90 mg/kg·N (represented by the symbols CK, A, B, C, D, E and F, respectively) in this experiment were carried out. CT content in leaves and stems of Chamaecrista nictitans was determined in order to study the effect of nitrogen application on CT content. Simultaneously, the activity of dihydroflavonol-4-Reductase (DFR) was measured and DFR gene was cloned. Finnally, the expressional level of DFR gene in different tissues and treatments was analyzed, which was instrumental for us to understand the molecular mechanism of CT biosynthesis. Main results are as follows:1. By using Vanillin-HCl Assay method, the CT content of Chamaecrista nictitans in leaves and stems in the treatments was determined. The results showed that CT content in leaves was evidently higher than that in stems, and CT content had a decline trend with the increase of nitrogen. This result indicated that nitrogen has some inhibitory effect on CT biosynthesis. Among 7 different nitrogen concentrations, CT content in leaves was decresed from 14.78% to 1.14% gradually. However, CT content in stems was increased at low nitrogen treatment and reached its peak value (4.62%) at B treatment, then sharply decreased to a stability level (0.98%～1.14%).2. Kjeldahl Method was used to determine crude protein. The results showed that CP content had not a significant difference in different nitrogen concentrations. CP content in leaves increased from 18.52% to 22.30%, and that in stems was in the range of 5.73%-7.98% in stem.3. Activity detection by continuous monitoring method was carried out to determine the enzyme activity of DFR. The results showed that there had a positive correlation between CT content and DFR activity in stems and leaves, and the correlation coefficients were 0.991 and 0.945 respectively. DFR activity in leaves decreased from 310.1U/g to 31U/g. However, its activity in stem had a ascent trend at the low nitrogen concentration, and then dropped from 103.69 U/g (B treatmet) rapidly and kept at a stable level (50 U/g) at last.4. By RT-PCR method, the cDNA fragments of DFR gene in Chamaecrista nictitans was obtained. It was named CnDFR, 600bp length, including a reading frame encoding 200 amino acids. Alignment analysis showed that CnDFR had a high homology with DFR gene in other plants. Analysis of DFR protein in Chamaecrista nictitans indicated that its theoretical molecular weight is 22421.6 KDa, predicted pI value is 5.23, theoretical formula is C1014H1568N254O301S9, and instability coefficient is 39.74. So, the protein encoded by CnDFR gene is speculated a stable protein.5. By Real-time PCR method, expressional level of DFR gene in leaves and stems different nitrogen treatment was detected. The results showed that the general trend was increased at first then decreased, finally rising again. The variation trendency of DFR gene at low nitrogen concentration was consistent with the change of CT content and DFR activity. |
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