| As a characteristic component in tea plant （Camellia sinensis）, theanine is anonprotein amino acid, whose synthesis mechanisms has not been clarified. RNAi isinduced by the degradation of homologous mRNA, which derived by double-strandedRNA complexes. The EST sequence of GS1-1gene （contig47） was obtained fromthe cDNA library of young roots of Camellia Sinensis constructed by our laboratory.RT-PCR （real-time quantitative PCR） result had confirmed that the GS1-1gene washighly relevant to theanine synthesis. This study mainly constructed a RNAi vectoraccording to3-UTR and its adjacent sequence of GS1-1gene and applied it ongenetic transformation of leaves of tea cv. Longjing-43mediated by Agrobacteriumtumefaciens, which would facilitate studying the function of GS1-1gene and thesynthesis mechanism of theanine.1. Construction of RNA-interference vector of theanine synthesis related geneGS1-1. The3’ end of GS1-1gene was obtained by SMART RACE technology. A350bps fragment of3’-UTR and its adjacent sequence was selected as RNAi targetsequence and a500bps intron sequence, which came from the vector pJawohl8andcouldn’t be found in tea was selected as stuffer fragment. The target fragment and thestuffer sequence were linked via the technology of Zero Background T-Vector System.The product was proved to be hairpin structure and then was inserted into the plantexpression vector pCAMBIA2301and transformed into Agrobacterium strainEHA105. The correct insertion of the exogenous gene in the vectors was comfirmedby PCR amplification, enzyme and sequencing confirmation, thus RNAi vectorpCAM-RNAi-GS was successfully conducted.2. Efficient callus induction from leaves of tea cv. Longjing-43and the screeningof the optimal concentration of antibiotics. The leaves of tea cv. Longjing-43wereused as receptor material of genetic transformation owing to the low regeneration rateof plantnet. After60days of culture, the induction of leaves was the fastest in the1/4–strength Murashige and Skoog medium （1/4MS） supplemented with0.1μmol·L-1TDZ,0.49μmol·L-1IBA and0.005mg·L-12,4-D, with the highest induction rate of96%. After the leaves were cultured in medium supplemented with kanamycin ofdifferent concentrations, the concentration of kanamycin as the screening marker forleaves of Longjing-43was100mg·L-1.3. Genetic transformation of Longjing43leaves mediated by Agrobacterium tumefaciens. After a pre-culture of3days, the acceptor leaves of tea cv. Longjing-43was infected for15min by activated Agrobacterium strain EHA105with plant RNAiexpression vector pCAM-RNAi-GS, and then co-cultured in the YMB medium for4days. After the residual bacterium were removed from the infected leaves onscreening medium with kanamycin,8kanamycin-resistant calli were obtained. Out ofthe eight calli, three calli were proved to be positive and the remaining five callinegative by GUS histochemistry staining and PCR analysis. Theanine content ofcallus-4and callus-5showed a reduction of85.09%as compared to the controls usingHPLC method with trifluoroacetic acid （TFA） elution. |
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