Studies On Genetic Transformation Of Tomato With IGF-1 Gene Under The Control Of Different Fruit Specific Promoter

Tomato( Lycopersicon esculentum Mill.), belong to Solanaceae family, which is originally from South America subtropical annual plants, and one of the most important vegetable crops planted all over the world. Tomato has been developed express oral vaccine research, which can greatly improve the application value. Different types of fruit specific promoter in fruit development period and control mechanism are different,so screening ideal fruit specific promoter can be for the use of expressing foreign protein lay the preliminary foundation.IGF-1 is a key factor to regulate the growth, reproduction, immunity and metabolism of the body. It has broad application prospects in the treatment of diabetes, osteoporosis, muscular dystrophy and other diseases. The study is materials with tomatoes, have built 5 promoter expression vector, and used method of agrobacterium mediated, introduced IGF-1 gene into tomato, and performed molecular detection. Eventually, we had obtain transgenic tomato plants, which provide technical and theoretical basis for using plasmid-specific expression of exogenous protein provides and lay the material foundation for the oral treatment of diabetes melites. Specific research results are as follows:(1) The establishment of expression vector:five fruit specific promoters of recombinant expression vector were established: pCA- PG-IGF-1、pCA-TGS-IGF-1、pCA-ACO-IGF-1、pCA-AGPL1-IGF-1、pCA-2A11-IGF-1. And five expression vectors were introduced into agrobacterium strain named LBA4404.(2) The establishment of efficient agrobacterium transformation system of tomato: Two kinds of tomato materials were cited in the experiment: AC++and1448, and respectively conduct the dregeneration system test. We introduced orthogonal test design method to study the effects of different hormone levels( IAA, ZT, AgNO3) on the tomato regeneration system. Determine the ptimize medium for callus and adventitious buds induction: MS+ ZT 2.0mg/L+ IAA 0.5mg/L+ AgNO3 3.0mg/l; the optimize medium of inducing rooting: 1/2 MS +IAA 0.3mg/L. We introduced orthogonal test design method to optimize the transformed factors( Dip solution, acetosyringone, infection time, co-cultured days) which affects the transformation efficiency, and the transformation system with high efficiency was established as follows: Liquid medium to infect the solution for MS, no acetosyringone concentration, the immersion time was about 10 min, co-culture days for 2 d.(3) The identification of the resistant plants by PCR amplification: After the transformation of the system, the final 41 strains were obtained,and including 12 plantlets with TGS, 6 plantlets with ACO, 6 plantlets with PG, 15 plantlets with AGPL1,2plantlets with AP.The foreign gene IGF-1 was proved to be integrated into the genome of Lycopersicon esculentum Mill.by PCR test. Some of positive plantlets were obtained, 21 of plantlets were postive and positive rate of 51.21%, including 6 plantlets with TGS, 3 plantlets with ACO, 3 plantlets with PG, 8 plantlets with AGPL1, 1plantlets with AP.