Inhibition Of Caffeine Synthase Gene In Tea Plant And Its Expression In Other Organism

Caffeine (1, 3, 7-trimethylxanthine) is the major purine alkloids in tea plant [Camellia sinensis(L.) O. kuntze], and its content in tea leaves ranges from 2% to 5% of dry wt. Caffeine is one of the most popular drug on the globe. A moderate caffeine intake can cause mild stimulation that may be beneficial in terms of increased alertness, concentration, improved performance and decreased fatigue. However, high intakes may have unexpected short-term side effects, including palpitations, gastrointestinal disturbances, anxiety, tremor, insomnia, arrhythmia, increased blood pressure and respiratory rate, etc. Moreover, it also could increase the risk of coronary heart disease and some cancers like urethra cancer, bladder cancer, breast cancer etc. These adverse side-effects account for the demand for decaffeinated tea and tea drink. In this paper, regeneration system of tea plant was studied, and the TCS gene cDNA was cloned and expressed in E.coli and tobacco for the first time. We also employing the technologies of antisense RNA and double-stranded RNA(dsRNA) to inhibit the expression of TCS gene in tea plant for the first time.At present, the decaffeinated tea was obtained by developing lower-caffeined tea cultivar or supercritical fluid extraction. But a breeding program to obtain low-caffeined tea plants would probably take 20 years or more, and the developed tea species may not suit for processing high-quality tea. Using supercritical fluid extraction in the process of tea would lead to the lost of the volaties and other functional compounds. These methods could not ultimately solve the problem of decaffeination, and the large-scale production of transgenic caffeine-deficient tea plant may be a more practical alternative way. Antisense RNA technology was an effective way to silencing specific gene, and was widely employed in plant breeding. In this paper, a 800bp DNA fragment including 5'untranslation region and part of code region of TCS cDNA was amplified by RT-PCR. The fragment was placed under the control of promoter 35S of binary vector pBI121 in the antisense orientation, forming a vector that could express the antisense TCS RNA. Then the vector was introduced into Agrobacterium tumefaciens LBA4404(pAL4404) by triparental mating. The calli induced from cotyledon was transformed, and some resistant calli was obtained. The analysis of these resistant calli by PCR and RT-PCR suggested that the antisense TCS gene was integrated into the genomic DNA and antisense RNA was expressed. In the field of tea breeding, this study was the first attempt of employing the antisense RNA technology to improve tea plant, and these resistant calli was expected to develop into caffeine-free tea plantlet.dsRNA technology was discovered recently, and many studies demonstrated that it could suppress genes more effectively than antisense technology. Inverted repeats of 800 bp TCS cDNA fragments sandwiching a 400 bp GUS gene fragment was inserted into the binary vector pBI121 and placed under the control of 35S promoter, forming a dsRNA expression vector. The calli induced from cotyledon was transformed by the Agrobacterium tumefaciens carrying this vector, and some resistant calli was aquired. The reseach set a basis to develop decaffeinated tea plant. However, the resistant calli grew very slowly, so the identification of resistant calli has not been carried out. Calli induction, somatic embryogenesis and differentiation were systemically studied in this paper. The calli could be induced from leaflet, stalk cutting and petiole. As far as the effects on inducing callus were concerned, 2,4-D was superior to NAA, and high contention of 2,4-D could also cause the occurrence of adventitious roots from callus. The results of this study indicated that the explants from seed (e.g. mature cotyledon and immature cotyledon) could easily differentiate into somatic embryo and adventitious buds, and in most cases, the explants from nutrition organs could only differentiate into roots but could not generate buds. Furthermore, a feasib...