Evolutionary Analysis Of SAMDC Gene In Crucifer Species And Research On YSAMDC Transformation In Tomato

Polyamine is a class of steroid hormones that plays an important role in plant growth and development. Polyamines can promote plant growth and enhance the resistance of plants to environmental stresses. The polyamine family usually included putrescine, spermidine and spermine. S-adenosylmethionine decarboxylase activity (SAMDC) is a key enzyme involved in the biosynthesis of the polyamines, viz. spermidine and spermine. The substrate for the SAMDC is S-adenosylmethionine (SAM) and the product is the decarboxylated SAM (dcSAM) which provides the aminopropyl groups for subsequent synthesis of spermidine and spermine. SAM is a precursor not only of polyamines, the anti-senescence plant growth substances, but also of ethylene biosynthesis. The purpose of this study is providing the helpful tools for understanding the physiological function of polyamines and the relationship between the expression of SAMDC gene on stress response and the biosynthesis of lycopene in tomato fruit.In this study, we constructed an efficient tomato genetic transformation system, and transformed the gene involving polyamines into tomato, which we hope to provide a strong basis for further investigating the function of SAMDC in regulating the level of polyamines and therefore developing strategy for crop improvement by regulation of SAMDC. Furthermore, SAMDC gene analogues in Cuciferae were obtained by PCR strategy of conserved regions and molecular evolution of the SAMDC gene were evalutidn. The main results are as follow:(1) An efficient method was developed for plant regeneration from tomato cotyledon and hypocotyl. The MS medium supplemented 1mg/L BAP, 0.15 mg/L 1AA 3% sucrose and 0.8% agar resulted high frequency of shoot regeneration in tomato. The frequency of shoot regeneration from cotyledon and hypocotyls was about 90% and 94.2% respectively.(2) An efficient genetic transformation system of tomato mediated by Agrobacterium tumefaciens was established, and the optimum conditions for the transformation of ySAMDC gene were as follow: The explants were infected by Agrobacterium tumefaciens strain LBA4404, which contains pBI35S-SAM and pBIE8-SAM for 5 -10 minutes, and cocultivated for 2 d after they were precultivated for 2 d, Then the explants were inoculated into the regeneration medium containing 7 mg/L Hyg to obtain the Hyg~R shoot, and the explants were transferred to fresh medium at intervals of 2-3 weeks. The Hyg~R shoots were cut off from the explants and cultured in rooting medium when they grew to 2-3 cm in hight. Roots apeared in shoots after 15 d culture, and then the cover of the container were opened gradually within the first week, and then the young transgenic plants were transferred to soil in larger pots and grown in a greenhouse.(3) More than 80 Hyg~R young plants were obtained by genetic transformation mediated byagrobacterium tumerfaciens with tomato cotyledon. PCR > RT-PCR results showed that 32 of 40 plants contain the 1100 bp (ySAMDC) amplification products and throuth GUS histochemical assay on transgenic plants were found to be positive..(4) In the present study, SAMDC gene analogues from 14 species of 6 genera in Cuciferae were obtained by PCR strategy using specific primers designed from conserved regions of SAMDC gene reported in GenBank. Constructed the the neighbor-joining (NJ) and minimum-evolution (ME) phylogenetic tree based on the alignment nucleotides sequences, phylogenetic tree showed that Brassica was closely related to Raphanus, and followed by Barbarea, Roripl Scop and Arabidopsis Heynh in orderly, but was remotely related to Capsella Medic.This research elucidated the relationship among plants at nucleotide level, which would provide some knowlegement of germplasms for improvement in Cuciferae crops.