Tomato LYC-β RNAi Vectors Construction And Transformation Into Different Fruit Colour Tomatoes By Fruit Injection

Tomato carotenoids have great value to both itself and human being. Lycopene-β-cyclase controls the flux of lycopene to different carotenoids. When the enzyme gene wassilent in red or pink fruit color tomato, lycopene content increased. No similar research existsin other fruit color tomato. Most vectors been used former expressed constitutively, while fruitis the edible organ in tomato, so we intended to construct both constitutively and fruitspecifically expressing lycopene-β-cyclase RNAi vectors, transform them into yellow, pink,purple, green and red fruit colour tomatoes S1, S2, S3, S5and S77, and get RNAi transgenetictomato plants to make foundations for further study of the effect of the enzyme on the wholecarotenoids metabolism pathway. Main results are as follows：Two different tomato lycopene β-cyclase fragments LYC-B1and LYC-B2and tomatofruit specific promoter E8were cloned. According to X86452.1, two fragments LYC-B1from61bp to861bp and LYC-B2from480bp to781bp were cloned, and connected. When theconnection transcripts, there will form a dsRNA. The constitutive expression LYC-B RNAivector pBI121-B1B2was constructed. Tomato fruit-specific promoter E8was clonedaccording to X13437.1and fruit-specific vector of E8-pBI121was constructed. E8-pBI121was transferred into agrobacterium GV3101with freeze-thaw method, and agro injected intoS2, S3, S5and S77tomato fruits. Vector transient expression showed E8-pBI121expressed inall fruits. Then the fruit specific LYC-B RNAi vector E8-pBI121-B1B2was constructed.pBI121-B1B2and E8-pBI121-B1B2were transferred into GV3101, and agro injected intoS77leaves and fruits. Vector transient expression showed that they both expressed in S77peel,flesh and seed, whereas the former expressed in leaves while the latter didn’t. That traitvalidated E8-pBI121-B1B2as a fruit specific RNAi vector.Agrobacterium-mediated leaf disc transformation system was optimized. Hypocotyl andcotyledon survival rates in S5varied widely from11.54%to81.82%after being infected byagrobacterium with different concentrations and different infection time. Explants survivalrates were higher when OD600was0.760compared with0.971.20minutes infection was bestto hypocotyls while15minutes to cotyledons. Hypocotyl and cotyledon with36hoursco-culture time with agrobacteria had higher survival rates in S1and S2than48hours, andmore hypocotyls survived than cotyledons at the same conditions.A fruit injection transformation system was established. The influence of fruit stage being injected on fruit injection process was evaluated. Green mature, turning mature andripe mature fruits of S1, S2, S3, S5and S77was injected by GV3101, and the ratio of theharvested fruits to the total injected fruits were calculated respectively. To S3and S5the ratiochanged little between stages, about54%and36%respectively. To S1, S2and S77the ratiofluctuated, the highest ratio occurred at green mature, green mature and ripe mature with65%、75%and60%respectively. Also the number of seeds per harvested fruits was calculated.In addition to S1at green mature, the rest at ripe mature stage, fruits had the most number ofseeds per harvested fruits, in order38,34,50,43and25.The influence of injection quantity on fruit injection process was evaluated.0.2ml,0.4ml,0.6ml and0.8ml agrobacteria liquid （OD600=0.5±0.05）were injected into S1, S2, S3, S5andS77fruits at their green mature stage, and the ratio of the harvested fruits to the total injectedfruits and the number of seeds per harvested fruits were calculated respectively. To S1, thetwo values fluctuated, and the best volumes according were0.6ml and0.4ml. To S2, the twovalues had a converse trend with the volumes, and the best volumes were both0.2ml. To S3,the two values were relatively stable, and the best volume was0.6ml. To S5, two values had aconsistent trend with the volumes and the best volumes were both0.8ml.The seeds harvested from fruit injection transformation was selected by kanamycin.Seeds harvested were sterile and sowed in1/2MS solid culture containing100mg/Lkanamycin. Increasing population seeds germinated for the first13days. As the extension ofincubation time, growth of non-transgenic plants was hampered by kanamycin, resulting inetiolated weaker and dead seedling. On45th day, seedling survival rate reduced by44.97%.To S1and S5, seeds from turning mature injection grew best and ripe mature to S2and S3,with seedling survival rate on45th day74.51%,59.79%,70.30%and24.50%in order. To S2and S3, seeds from0.2ml injection volume had the most seedling survival rate on45th daywith53.13%%and50.20%.Molecular identification of transgenic plants from kanamycin selected plants. Seeds wereharvested from plants, and selected by kanamycin. DNA were extracted from170plantssurvived seedling and specific PCR were conducted.16plants about9%of survival seedlingswere identified as transgenic plants. Among the170plants,45derived from ripe matureinjection and12plants, about27%, were transgenic. Now three S2(pink), one S3(purple)and twelve S77(red) transgenetic plants are obtained. The experiment results show that fruitinjection transformation can be used to get transgenic material.