The Clonins Of Theanine Svnthesis Related Gene In Tea Plants And The Estabiishment Of Proliferation System Of ’Xingao’ Pear

Theanine (γ-glutamyl-ethyl-amide) is a non-protein amino acid in tea. L-theanine not only has a strong influence on the taste of tea and but also important biological activities on human, for instance, blood-pressure decreasement, anticancer ability, neuroprotection, calmness, study capacity and memory enhancing functions.Theanine synthesis related gene was cloned and the proliferation system of ‘Xingao’ pear was established in this experiment. This study provides a foundation for understanding of theanine synthesis in tea plants and set up regeneration system for pear.1.3’ end of theanine synthesis related gene was amplified the cDNA end swift amplification technique with the following proliferation system:100ng template cDNA,5μl10×Buffer(Mg2+p lus),2.5mmol/L dNTPs8μl,1μl10μmol/L GSP47-1（GSP47-2for the second-round）, adaptor primer UPM（NUP for the second-round）and2.5U LATaqDNA polymerase in a total volume of50μl.The amplification was programmed as follows:94°C for3min14cycles in94°C for30s70°C for45s,72°C for80s, and reduction0.5°C each cycle25cycles in94°C for30s63°C for45s,72°C for80s, final extension at72°c for10min. At Last the cloned2.4kb sequence has a similarity of81%to the glutamate-ammonia ligase of grapes after sequencing and further Blastx in the GenBank.2. Inverse PCR(IPCR) technique is a method by which flanking sequences adjacent to known DNA fragments can be cloned. IPCR technique was used in this experiment to clone the promoter sequence of the theanine synthesis related gene, and the reaction system was composed of20μl solution including1μg template DNA, restriction enzymes Bsp1407,2μl10×T buffer and2μl0.1%BSA.The optimal system of ligation was in a50μl reaction volume, including0.5μg digested DNA,5μl10×T4ligation buffer and5U T4DNA ligation enzymes. Digested DNA was ligated to vector overnight at22°C. The amplification system of IPCR was in a reaction volume of50μl, including2.5μl10×PCR buffer,25mmol/L Mg2+5μl,50ng template DNA,10mmol/L dNTPs1μl,2μl of double-primer and1U TaqDNA polymerase. The amplification was programmed as follows,94°C for3min,29cycles in94°C for1min,56°C for1min,72°C for1min, final extension at72°C for7min. Using the established IPCR technique, we cloned a171bp backward promoter sequence with TATA-box, which plays a vital role in the correct beginning of the gene reversal.3. This experiment researched the effects of different levers of plant growth regulators on proliferation of ‘Xingao’pear in vitro. The result showed that the suitable budding medium was1/2MS+6-BA0.5mg.L-1+IBA0.2mg.L-1+GA31.5mg.L-1. The effective multiplication coefficient can be as high as3.3.