Screening And Identification Of Genes Involved In High Level Of Amino Acid From Anji Baicha(Camellia Sinensis)

Anji Baicha is a natural mutant tea plant (Camellia sinensis). It was discovered in thehigh mountain, with an altitude of 800 meters, located at Anji town of Zhejiangprovince. The leaf of Anji Baicha has stage albescent phenomenon. During thealbescent period, the content of amino acid in white leaf is 2～3 times higher than thatin green leaf. This study focused on the cloning of differentially expressed genesbetween green and white leaf to understand the molecular mechanism of amino acidincreasing during the albescent period. The main results were as follows:In order to extract the total RNA with high quality from the leaves of tea plant, thisstudy did modification that based on the tradition Tri-Reagent method, a sort ofgeneral method for RNA extraction. When the procedure was for precipitating RNA,an extra half volume high concentration salt was added before adding half volumeisoamyl alcohol for decreasing the interference of tea polysaccharides. And forprotecting the RNA extract being browned and keeping away the pollution of RNase,a quick and coherent operation under low temperature environment was undergone.With this modified method, the total RNA from tea plant showed two clear bands of28s RNA and 18s RNA after agarose gel electrophoresis, and the band of 28s RNAwas obviously brighter than the one of 18s RNA. This result showed the total RNAwith better intactness, and no degradation.To separate the differentially expressed genes between white and green period leaf ofAnji Baicha, a subtracted library was constructed by the suppression subtractivehybridization (SSH) method. Screening the forward and reverse subtraction librarywith colony PCR and dot hybridization, 80 clones were identified as the differentialexpressed cDNA fragment, and the size was between 200 bp and 800 bp.Sequencing the 80 clones and through blast analysis, the results showed the cloneswere involved in photorespiration, energy metabolism, protein metabolism,stress-response, and saccharides metabolism respectively. Amino acids coded by 1-5gene had 83％similarity with Arabidopsis thaliana serine-glyoxylateaminotransferase. Amino acids coded by 1-9 gene had 90％similarity withArabidopsis thaliana serine hydroxymethyl transferase. Amino acids coded by 2-13gene had 94％similarity with calmodulin-binding heat-shock protein. The expression level of the 1-5 gene, 1-9 gene and 2-13 gene in the leaves of whiteand green period were analyzed by real-time PCR method, and the result showed theexpression level of 1-5 gene and 1-9 gene were higher in white period and 2-13 genewas higher in green period.In terms of the research results, the stage albescent of Anji Baicha leaf was regulatedby calmodulin-binding heat-shock protein. The reason for the amino acid and theanineuncommonly enrichment may be the result of exceed expression of serine-glyoxylateaminotransferase and serine hydroxymethyl transferase genes. For the lowtemperature at the beginning of spring tea, the expression of calmodulin-bindingheat-shock protein was restrained, but the serine-glyoxylate aminotransferase andserine hydroxymethyl transferase genes could exceed expression, thus thebiosynthesis of amino acid and theanine was promoted. So the content of total aminoacid and theanine were higher in white period leaf. At the middle or last period ofspring tea, with ambient temperature rising, the expression of calmodulin-bindingheat-shock protein was activated. Thus, the metabolism change of Anji Baicha wasaccelerated and the biosynthesis of chlorophyll was speeded up, following the risingof chlorophyll content. So the leaf color was changed from cream to green. But theexpression of serine-glyoxylate aminotransferase and serine hydroxymethyltransferase genes were restrained, that caused the biosynthesis block of amino acidsand the theanine, and following the content reducing of amino acid in the leaf of greenperiod.