Cloning And Functional Identification Of Alanine Decarboxylase Gene And Alanine Aminotransferase In Tea Plants(Camellia Sinensis)

Nitrogen is the most demanding fertilizer in the process of plant growth and development,and nitrogen deficiency decreases crop productivity heavily.Thus,excess nitrogen fertilizer is applied in modern agricultural production.For most crops can't use nitrogen fertilizer efficiently,large amount of the fertilizer is lost that caused serious environmental pollution.Therefore,it is the fundamental way to reduce the application of nitrogen fertilizer and the environmental pollution by cultivating cultivars with high Nitrogen Use Efficiency?NUE?by means of genetic engineering.Tea plant?Camellia sinensis?harvested for leaves and the nitrogen use efficiency of which affect the tea yield directly.Thus it has been one of the most important targets for tea breeders to cultivate varieties with high nitrogen use efficiency.The nitrogen uptake and utilization of tea plants depends heavily on their ability to absorb,assimilate,transport and distribute nitrogen into the harvest organs.At present,the genes related to nitrogen uptake and preliminary assimilation in tea plants have been cloned.It has been found that genes involved in alanine metabolism,which is the downstream pathway of nitrogen assimilation,played important roles in the uptake and utilization of nitrogen.In this study,two genes involved in alanine metabolism was cloned from tea leaves,and the functional identification was conducted and the transcript levels of the two genes response to nitrogen supply was studied.The main results are as follows:1.The primers were designed according to the gene sequences obtained from the transcriptome database of tea plants,and the CL4912 gene was cloned and the gene sequence was analyzed by bioinformatics.The results showed that the full-length?1716 bp?cDNA of CL4912 contained a ORF?1437 bp?encoded an amino acid sequence of 478 residues which gave an estimated protein molecular mass of 53.6 kD and a theoretical isoelectric point?pI?of 5.83.CL4912 was a stable hydrophilic protein without transmembrane structure and signal peptide.2.Homologous sequence search and phylogenetic analysis showed that CL4912 protein might functioning as a serine decarboxylase.So the CL4912 protein was prokaryotic expressed and purified and its catalytic activity was tested.The results showed that the molecular weight of the protein was about 55kD and CL4912 protein functioned as a serine decarboxylase as well as an alanine decarboxylase.Because the enzymatic activity of the protein catalyze the decarboxylation of alanine is8¹0 times of that of serine,the gene encoding CL4912 protein is named as alanine decarboxylase?CsAlaDC?.3.The transcript levels of CsAlaDC gene in different tissue of tea plants and its response to nitrogen were detected and analyzed,the results showed that the gene was expressed in shoots,mature leaves and the roots of tea plants and the relative expression level in roots was extremely high to the extent that the expression was several hundred times higher than that of leaves.After treatment with nitrogen,the expression of CsAlaDC gene in roots of different tea cultivars had similar changes.The expression of CsAlaDC gene was down-regulated in a short term?24h?after nitrogen treatment,followed by a long term up-regulation,at the 5^th day of the treatment,the gene reached the maximum response,then it down-regulated again.The extent that CsAlaDC response to nitrogen was different for nitrogen levels supplied and cultivars variety.4.A cDNA?1747bp?contained the complete ORF?1626bp?of AlaAT1 was isolated from tea plant?Camellia sinensis?.The deduced amino acid sequence of 541 residues gave an estimated protein molecular mass of 59.4 kD and a theoretical isoelectric point?pI?of 5.82.CsAlaAT1 was a hydrophilic protein without transmembrane structure and signal peptide.On line blast sequence search showed that amino acid sequence of CsAlaAT1 shared highly similarity with AlaATs from other plants.Results of phylogenetic analysis showed that CsAlaAT1 had the closest genetic relationship with cucumber AlaAT2,suggesting that the two proteins had similar biological functions.5.The expression levels of CsAlaAT1 in various tissues and its changes response to different N concentration were investigated by real-time fluorescent quantitative RT-PCR.Results of RT-PCR showed that CsAlaAT1 expressed in all tissues of tea plant and the transcript level was highest in root.The transcript abundance of CsAlaAT1 were up-regulated by N both in shoots and mature leaves and increased more greatly under the high N condition.Interestingly,the CsAlaAT1 expression levels in root under high N condition were up-regulated while levels under low N treatment after 24h was down-regulated.