The Study Of The Affect On Ascorbate Content In Tomato By Transgene Pyramiding

Ascorbic acid(L-ascorbate acid,AsA)is an important antioxidant that can remove reactive oxygen species in plants.It is also involved in a variety of metabolic processes in plants.In the human body,AsA can remove the free radicals in the body,and it is an indispensable nutrient,but the human lose the ability to synthesize AsA,so we have to absorb necessary ascorbic acid from fresh vegetables and fruits.Tomato(Solanum lycopersicum)is an important model plant in botanical research and it is rich in ascorbic acid.Therefore,the study of ascorbic acid content in tomato plays an important role in nutrient quality and has become the focus of the research.Now AsA synthesis pathway is elucidated,and the key genes of the pathways are also studied clearly.Most of the traits are controlled by multi-gene in plants,and AsA content is also controlled by ploygene.Previously it is reported that transgenic pyramiding improves the plant's salt tolerance,disease resistance and amino acid content and so on,but there is no gene polymerization in ascorbic acid research,so in this study,the overexpression transgenic lines of the key genes of the As A pathway are studied by pollination and pyramiding,the effects of transgenic polymerization on AsA in tomato were analyzed by physiological and biochemical experiments.The main results of this topic are as follows:1.Hybridizing the transgenic lines overexpressing GMP,GME,GGP,GGP,GalUR,MIOX,MDHAR and DHAR by artificial pollination,then the GMP×GME,GGP×GPP,GalUR×MIOX,MDHAR×DHAR,GMP×GME×GGP ×GPP and GMP×GME×GGP×GPP×MDHAR×DHAR have been obtained,and the target genes of pyramiding lines were screened by positive detection.2.By using the method of TCA to measure AsA of Ailsa Criag(AC),single gene and pyramiding lines,the results show that compared with the AC,the As A content of leaf and fruit in pyramiding lines are significantly increased,except that the fruits of GGP×GPP line did not change significantly.The qPCR is used to analyze the relative expression of As A synthesis and metabolism related genes in the plant,and we found that except for the AsA metabolic and synthetic related genes in leaves of GMP×GME×GGP×GPP lines,gene expression in leaves and fruits of other pyramding lines were enhanced.It is indicated that the AsA content is controlled and regulated by multi-gene at multi-point.3.We measured the AsA content of one-month seedling at 16 h Light/8 h Dark,the result showed the light-induction on AsA in leaves of pyramiding lines only affected the AsA content and the fluctuation amplitude,and did not alter the original trend of AsA accumulation along circadian cycle.And by the results of AsA synthetic precursor feeding showed that gene pyramiding alter the AsA biosynthesis pathway contribution in leaves and fruits,and subsequently affect ascorbate content.It is assumed that the main cause of increasing AsA content was the contribution of D-mannose/L-galactose(D-Man/L-Gal)pathway in leaf,and for the GMP×GME,GMP×GME×GGP×GPP lines AsA content level was increased compared with wild type,for which three pathways were attributive to inceased ascorbate content,however,GGP×GPP lines mainly use the D-Man/L-Gal pathway.4.The enzyme activity of the AsA synthetic enzyme such as GMP,GalUR were measured,the result showed that the two enzymes in pyramiding lines were significantly increased,which is possibly the cause of increased AsA content.The methyl viologen simulated oxidation adversity,the chlorophyll,malondialdehyde and H2O2 were measured,and it showed that transgenic pyramiding also improved the resistance to oxidative stress.5.In oder to study the source and the sink regarding AsA,we measured the AsA content in fruit and leaf petioles and their exudates.We found that the AsA content of the fruit petiole and its exudate in the pyramiding lines was higher compared with AC,and combining the result of the AsA content after feeding AsA in the green ripe stage of AC and pyramiding lines,we suggested transgenic pyramiding enhanced the transportation capacity.In the addition,a lot of agronomic traits in the pyramiding lines were also altered,such as the shape and weight of the fruit,stem diameter,and the soluble solids and so on.