Studies On The Cloning Of Lycopene Gene From Tomato

The leaves of tomato were token as test materials in the study,extracted RNA from tomato leaves,used the RNA for RT-PCR.Then cloned two important genes of lycopene ,which were named lycb and lyce.After that constructed two antisense vectors of them.Based on the constructed two antisense vectors ,constructed a antisense fusion vectors of them.At the same time , the high efficient systerm was established on plant regeneration.Above all ,gived a good preparation for the next step.1 Extracted the RNA of tomato by the Urea-licl method .Based upon the known lycopene cyclase genes,the specific primers were designed, extracted RNA frome tomato leaves was used for RT-PCR,then the 723bp and 569bp gene fragments were amplified,and were connected with Peasy-T1 Cloning Vectorand identified by sequencing. Through the Genebank sequence analied,the two cloned genes were found the right genes which we needed.2 After cloned two genes,used prmer 5.0 analied them, selected the genes which we needed. Analied the enzyme digestion sites,selected the right enzyme digestion sites, made the selected genes insert to PROK2 by reversion. Lycb gene was digested by BamHI and SmaI. The gene was inserted into PROK2 expressing vector in order to construct antisense vector ,which was named pro-b. In the same way,Lyce gene was digested by BamHI and Xbaâ… .The gene was inserted into PROK2 expressing vector to construct antisense vector and named pro-e.Then we constructed their antisense expression vectors of lycopene cyclase genes. On the basis, constructed a fusion gene of them.When constructed,the first maked the two genes together which we called a fusion gene. In this process used a intermediate vector. Subcloned gene we named PUC-b+e.Then made the fusion gene linke PROK2,so constructed a antisense expression vector of the fusion gene which was named Pro-b+e.3 Based on the previous studies,Take tomato cotyledon as receptor materials. Studied the induction effects of the callus and the buds induetion were quite complex according to diffcrent hormone categories,concentrations and their eombinations. The right medium of regeneration was MS+ZT1.5(mg/L)+IAA0.2(mg/L). Found the effection of cotyledon age of 12d on callusand buds induction is much higher.The right medium of root duction was 1/2MS(MS)+IAA0.2(mg/L),root earlier,the number of root was much bigger. In addition,the problem of browning in tomato had improved. Through a great deal of trials,we founded 30mg/L citric acid not only prevent browning,but also promoted regeneration,25mg/L citric acid mark down browning while rooting.