Preliminary Study On The Biological Functions Of SlGS1.1 And SlAS1 In Tomato

Glutamine synthetase(GS,EC 6.3.1.2)and asparagine synthetase(AS,EC 6.3.5.4)are the most important enzymes in the process of inorganic nitrogen assimilation,having great potential in improving plant nitrogen use efficiency.At present,the expression and function of GS and AS in plants such as rice and Arabidopsis thaliana are studied in depth,some of which have obtained inspiring results.However,GS and AS have undergone different evolution processes among different species,and biological functions of some GS and AS genes are specific.Therefore,for better understanding the functions of GS and AS genes,it is important to conduct research on the GS and AS genes of other species.Tomato is one of the world's most important vegetables with rich nutrition favored by consumers.Production period of tomato is long with high yield.Its production process needs large demand for nitrogenous fertilizer,but the utilization rate is low.Excessive application of nitrogenous fertilizer not only increases production costs,but also causes an environmental pressure.Thus,increasing tomato nitrogen utilization is of great significance.The functional studies of tomato's GS and AS genes can provide important clues for the analysis of molecular mechanism of nitrogen assimilation and the improvement of nitrogen use efficiency.However,tomato GS and AS family gene function research is rarely reported.Previous results show that SlGS1.1 may play an important role in primary assimilation,storage,transport,reuse of nitrogen,and ripening and quality formation of tomato fruit.In this research,biological functions of SlGS1.1 and SlAS1 in carbon and nitrogen metabolism were prelimilarily investigated by creating and using overexpressing transgenic tomato plants,as well as identification of the modifications of these transgenic plants at transcriptional,enzymatic and metabolic levels.Major research results are as follows:1.The ORF fragment of SlGS1.1was amplified from tomato(A57)using specific primers,and was inserted into plant transformation vector pMV2 under the control of CaMV35 S promoter based on enzyme digestion and ligation method.Overexpression transgenic tomato plants were generated through Agrobacterium tumefaciens-mediated transformation method,and homozygous lines were identified through PCR-based molecular identification;2.Compared with the wildtype,the expression levels of SlGS1.1 in roots and leaves of transgenic tomato plants was significantly increased by more than 10 times,while in fruits,increase was only 5-8 times.The transcription level of SlGS2 was also increased in leaves of transgenic plants.Under the conditions of nitrogen deficiency,transgenic plants exhibited a higher expression level of SlGS1.1 than wildtype both in leaves and roots;3.Total GS activities of transgenic tomato plants were significantly elevated,about 62.0%-76.5% in leaves and 5.0%-11.6% in roots under nomal conditon.While under nitrogen deficiency conditions,differences between transgenic plants and the wild-type were become less;4.Compared with the wild-type,both total enzyme activities of some nitrogen metabolism related enzymes,such as NADH-GOGAT,GDH,AspAT and AS,and the transcription levels of genes coding these enzymes were affected in transgenic tomato plants.Under nitrogen deficiency conditions,the expression of most genes was inhibited,and the activity of enzyme was also decreased;5.No apparent phenotypic differences were identified between SlGS1.1 over expression plants and wild-type,but some the metabolite contents were significantly altered in transgenic lines.Compared with wild-type,the levels of NO3-in roots and leaves,the soluble protein levels in roots,leaves and fruits,and the total amino acid contents in fruit were increased,the NH4+ contents in leaves and fruits,the Glu conents in roots and fruits were decreased,in transgenic tomato lines.Whereas under the condition of nitrogen deficiency,SlGS1.1 transgenic plants showed lower levels of NH4+ and Glu contents and a higher level of soluble protein content than wild-type;6.Compared with wild-type,both sugar and citric acid contents in roots of SlGS1.1 transgenic plants were significantly decreased,sugars in leaves were increased.Total soluble sugar,glucose and malic acid contents in fruits of transgenic plants were decreased,while the contents of fructose and citric acid in fruit were increase;7.The expression of organism and nitrogen response showed that SlAS1 showed a constitutive expression pattern in tomato plants,and more expressed in petals and ovary,and at the roots SlAS1 was induced by nitrogen8.A vector that could constitutively express SlAS1 was constructed,and 14 T0 transgenic tomato plants were created through Agrobacterium-mediated transformation method.And then,homozygous transgenic lines were obtained through molecular seletion.9.Compared with the wild-type,the transcription levels of SlAS1 in transgenic plants were significantly increased,with 80-100 times in leaves,20-40 times in roots and 200-800 times in fruits.No apparent phynotypic differences were found between transgenic and wild-type plants.