CDNA Cloning Of Tomato Acid Invertase And Function Analysis In Cold Resistance Reaction Of Cell Wall Invertase

According to their location in cell, plant invertases include cell wall invertase, cytoplasmic invertase and vacuolar invertase, which can divide sucrose into glucose and flucrose.It is well known that invertase can regulate cell physiological condition or activate certain genes expression by changing the osmotic pressure, construction sugar concentration grads to sugar accumulation and composition or taking part in the signal transferring.In this research, we cloned two fragments of tomato acid invertase cDNA (one for cell wall invertase and one for vacuolar invertase), and constructed two plant expression vectors. In order to reseach the genes function in low temperature reaction, we obtained LIN6 antisense RNA gene transformed plants and researched those plant under low temperature.The details were as follows:1. Total RNA was isolated from tomato (cv. Zhongshu No. 4 ) leaves and a fragment about 668bp, the homology being 99.71% compared to that of tomato fruit vacuolar invertase gene TIV1, was amplified by RT-PCR. The fragment was cut by two restriction enzymes SalⅠa nd BamHⅠ,and inserted into plant expression vector pBinAR between CaMV 35S promoter and OCS terminator to form antisense plant expression vector pBinAR-aTIV1.2. Total RNA was isolated from tomato (cv. Zhongshu No. 4) leaves and a fragment about 416bp, the homology being 99.3% compared to that of tomato fruit cell wall invertase gene LIN6, was amplified by RT-PCR.. A antisense RNA expression vector pBinAR-aLIN6 through insert anti-orientationaly this cDNA into the plant expression vector pBinAR.3. The regenetation system of tomato explant in vitro was constructed.The experiment results showed that B5 +IBA0.05mg·L^-1+6-BA1.5mg.·L^-1 was the best shoot regeneration media for tomato MicroTom, and on this media the shoot regeneration frequency could reach 95.8% and 60.0% for cotyleton and Hypocotyl respectively. For tomato seed facial sterilization, 3%NaClO was better than 0.1%HgCl2.4. Antisense-LIN6 binary expression vector was transferred into tomato MicroTom mediated by agrobacterium tumefaciens strain EHA105. The transform parameters in Agrobaeterium-mediated transformation were as follows:prculture 2d on MS medium,dipped for 10min in EHA105 suspension (OD=0.5,supplied with 200umol·L^-1 AS),co-cultivated with Agrobacterium for 2 d on B5 + IBA0.05mg·L^-1 +6-BA1.5mg.·L^-1.The proper content of Kanamycin in medium was 50mg·L^-1, 300 mg·L^-1 for Cefoperazone Sodium and Solbactam Sodium to restrain bacterium grow.5. 110 of Kanamycim-resistance shoots were obtained by the procedure as above.The putative transformants were assayed by PCR analysis and Southern blot hybridization.In five plants,T42,T43,T61,T67,T92 ,were obtained the predicted result,and their cell wall invertase activity deduced by 67.9% , 13.4%,73.5%,85.6% and 58.0% respectively.LIN6 gene was down-regulated by 65.6% ,which was proved by semiquantitative RT-PCR analysis.6. Taken the nontransformants as control, transformants were treated with low temperature, 15℃for 14h in 4000lx light dense and 10℃for 10h in darkness. The result shew that the photosynthetic ratio, content of carotenoids and soluble sugar in tomato leaves (especially noninductive soluble sugar ) deduced remarkably.An correlation analysis results shew that the activity of cell wall invertase were remarkably correlated with the content of carotenoids and soluble sugar in tomato leaves, the activity of cytoplasmic invertase and vacuolar invertase and the photosynthetic ratio in tomato leaves under low temperature. Therefore, we could conclude that cell wal invertase could maintain soluble sugar and carotenoids at a certain level for holding photosynthetic ratio in low temperature.Based on this mechanism of protection reaction against low temperature for cell wall invertase, it was made clear that such measures as praying sugar solution, nutrition material and that of protecting carotenoids, were necessary to help plant grow well in low temperature.And the activity of cell wall invertase should also be a index in breeding.