The Altered Expression Of Genes Related To Nitrogen Absorption And Utilization Of Tea Cultivars With Different Nitrogen Use Efficiency

Nitrogen（N）, an essential component of nucleic acids, proteins, hormones and chloroplast in plant, is one of the major mineral nutrients for plant growth and development. However, inefficient absorption and low conversion of the supplied N result in low nitrogen use efficiency（NUE） and adverse impacts on the environment including eutrophication, soil acidification and nitrous oxide emissions. Hence, enhancing NUE is essential for both sustainable development of agriculture and protection of environment.. Tea（Camellia sinensis）, as a perennial leaf crop, requires more nitrogen than most other crops. Its quality and yield are strongly correlated with the N-fertilizer application. Generally, the nitrogen content in tender tea leaves ranges from 40 to 50g·Kg-1.The highest nitrogen content in tender tea leaves could reach 60-70 g·Kg-1.Therefore, it is important to breed high NUE tea cultivars and carry out relative studies. Several studies were carried out as follows. First, the kinetics of NH₄⁺ uptake in ten widely-planted tea varieties of China was investigated. Second, the reliable reference genes for q RT-PCR analysis of tea plants treated under various nitrogen conditions were selected. Third, the expression patterns of genes encoding ammonium transporters and assimilatory enzymes, including Cs AMT1.1, Cs AMT1.2, Cs AMT3.1, Cs GS1.1, Cs GS1.2 and Cs GOGAT in four tea cultivars with different NUE and under different nitrogen treatments were analyzed. The main results are as follows:1. The kinetics of NH₄⁺ uptake of ten widely-planted tea varieties of China was studied by the improved conventional exhaustion method. The results showed that the uptake kinetic parameters were differed significantly by genotypes. The maximum uptake rate(V_max) and α(V_max/Km) of ‘Zhongcha302’ were much higher than others, indicating ‘Zhongcha302’ could well-adapt to both low and high nitrogen conditions. The α values of ‘Jiaming1’ was the highest and the V_max was low, which demonstrated that it had high affinity uptake of N at low N concentrations, but had poor performance at high N conditions. The V_max values of ‘Longjing43’, ‘Fudingdabaicha’ and ‘Tieguanyin’ were higher than the average indicated that these genotypes had the ability to accumulate higher biomass. The V_max and α of other varieties were lower than the average indicated that these varieties had low affinity uptake for nutrient both high and low N levels. Therefore, reasonable application of nitrogen fertilizer is very important to tea plant growth.2. We chose 6 housekeeping genes which included five commonly used and one new candidate to systematically assess their expression levels at three different tissues（young leaves, mature leaves and roots） under different nitrogen regimes by q RT-PCR. Ge Norm and Norm Finder software were used to analyze and evaluate the data for reference genes. The results indicated that GAPDH, β-actin and RPL13 are the best reference genes for normalizing target gene expression in tea plant under different nitrogen nutrition, whereas α-tubulin and Ru BP are not suitable in many experimental conditions and the best combination（GAPDH+β-actin） was recommended. Thus, validation of suitable reference genes for specific condition can guarantee the accurate quantification of the target genes in q RT-PCR analysis.3. The expression patterns of ammonium transporters genes Cs AMT1.1,Cs AMT1.2,Cs AMT3.1 and key nitrogen metabolism genes Cs GS1.1,Cs GS1.2, Cs GOGAT in the roots of four tea varieties with different NUE were detected after the resupply of NH₄⁺-N. Nevertheless, we found no significant expression differences between the varieties, except the expression level of Cs AMT1.2 showed significant positive correlations with different nitrogen treatments. It might be because these genes are not the major genes which involves in nitrogen metabolism, and nitrogen metabolism is a complicated physiological and biochemical process which includes many regulation genes and many genes may belong to multigene families. The relationship of gene expression and phenotype remains unclear. Part of the gene expression may be balanced by other genes in the same family.