Establishment And Optimization Of Tomato Transformation With Anthocyanin5-O-Glucosyltransferase Gene From Blue-flowered Gentian By Agrobacterium Rhizogenes

Tomato(Lycopersicon esculentum Mill), belonging to Solanaceae, Lycopersicon, anannual or perennial herbaceous dicotyledonous plant, has some advantages such as rich invarious nutrients, easier to survival, shorter cultivating time, clearer genomic DNAcomposition, easier genetic transformation. As a model plant in genetic transformation,research achievements of tomato are not only of important theoretical significance, but also ofgreat application value.In our research, pCMBIA1304was used as a vector, the UDP-glucose: anthocyanin5-O-glucosyltransferase gene from blue-flowered gentian was used as target gene, andexpression vector was constructed. Gt5GT7genes were transferred into tomato plants of10genotypes mediated by Agrobacterium Rhizogenes strain A4, aiming at establishing ahigh-frequency transformation system of tomato. Results are mainly as follows:1. According to the DNA sequences of Gt5GT7(AB363839) published by GenBank, itsprimer was designed and BglII and BstEII restriction site were added at both ends, then aPCR reaction of pCR4TOPO-Gt5GT7was proceeded with. Gt5GT7gene was recycled,extracted and purified by reagent kit. Then a double digestion of Gt5GT7target gene obtainedby PCR and pCMBIA1304was conducted. Recycled Target gene Gt5GT7and largefragments of pCMBIA1304then linked Gt5GT7gene and large fragments of pCMBIA1304with TaKaRa Rapid DNA Ligation Kit, transferred the expression vector into E.coli DH5α.At last, plasmids of resistant colonies were extracted. The result showed that plant expressionvector pCAMBIA1304-Gt5GT7was successfully constructed.2. The expression vector pCAMBIA1304-Gt5GT7was transferred into AgrobacteriumRhizogenes A4using Electroporation. Then plant expression vector pCMBIA1304-Gt5GT7was transferred into tomato by recombinant Agrobacterium Rhizogenes strain A4mediatedsystem using leaf disc method so that high frequency regeneration of genetic transformationsystem of tomato were established.3. The experiment concluded that the optimal medium for callus induction wasMSB5+6-BA2.0mg/L+IAA0.2mg/L-0.5mg/L; the optimal medium for bud differentiation was: MSB5+6-BA2.0mg/L+IAA0.15mg/L-0.5mg/L; the optimal medium for rootingwas:1/2MS+IBA0.5mg/L.4. PCR analysis was used to detect genomes of transgenic regeneration plant withplasmid pCAMBIA1304-Gt5GT7as positive control and wild-type tomato plant genome asnegative control. Result showed that2regeneration plants had amplified1892bp DNA band,which had the same location as amplified band with positive plasmid but no target band wasdetected in negative control. It preliminary showed that exogenous Gt5GT7gene wasintegrated into tomato genome.5. Phenotype changes among those Ct5GT7-transgenic tomato plants: mature fruit wasgreen of the non-transformed in compared with the transgenic tomato was light orange.