Expression Analysis Of Ferritin Genes In Pear And Transgenic Micrografting Plant Of Apple

The yield of plant is severely affected by abiotic and biotic stresses,such as pathogeny, drought,low temperature,high light intensities,ozone,UV-B and salinity.All these adverse environmental conditions have a common consequence,i.e.,the formation of highly reactive oxygen species,which is potentially harmful to the cells.Ferritin is the only known mechanism to concentrate iron to the level required by the cells,to store iron in a soluble and biologically available form.Ferritin functions to protect cells against iron -mediated toxicity to protect the cells against the toxic effects such as hydroxyl radical produced,and to increase plant stress tolerance.Ferritin was widely distributed throughout the living kingdom for storing up to 4500 atoms of iron,which was necessary to achieve the sequestration of iron in a soluble and bioavailable form.So the expression of the ferritin gene is benefit for plants,not only increasing the stress tolerance,but also the iron content. The ferritin protein taking up iron could improve the plant tolerance to pathogeny and environmental stresses.In this study,the expression patterns of four ferritin genes(PpFer1, PpFer2,PpFer3,and PpFer4) in pear(Pyrus pyrifolia'Cuiguan') were investigated using quantitative real-time PCR.The success of in vitro micrografts of apple(Malus domestica) materials was presented.Micrografts were used to study on ferritin genes expression and expression changes induced by Fe(â…¢-citrate for 24 h at the transcriptional level by quantitative real-time PCR.The experiment research results were as follows:1.In this study,the quantitative real time RT-PCR method for analyzing the expression of ferritin genes was studied.The results showed that the primers were specific,and the method was reliable.Based on this method,the expression patterns of four ferritin genes (PpFer1,PpFer2,PpFer3,and PpFer4) in pear(Pyrus pyrifolia) were investigated. Analysis of tissue-specific expression revealed that these genes expressed higher in leaves than in other tested tissues.In stem,the expression of PpFer1 was higher than in fruit.In shoot tip,stem and fruit,there were no obvious differences on the expression of ferritin genes.The expression level of PpFer2 and PpFer4 was the lowest in stem.In fruit and shoot tip,the expression of PpFer2 and PpFer4 had no obvious difference.No significant changes in expression of PpFer2 were observed in stem,shoot tip and fruit.In leaves,the expression of PpFer2 was higher than PpFer2,PpFer3 and PpFer4.2.In this study,the expression patterns of four ferritin genes(PpFer1,PpFer2,PpFer3, and PpFer4) in pear(Pyrus pyrifolia) were investigated using quantitative real-time PCR. After Fe(â…¢)-citrate treatment,analysis of the expression revealed that these genes expressed higher in the early stage after treatments.Under different plant hormone treatments,Abscisic acid(ABA) significantly enhanced the expression of all four ferritin genes,especially PpFer2,followed by BA,GA₃,and IAA.These ferritin genes were differentially expressed in response to various abiotic stresses.The effect of oxidative stress (5 mM H₂O₂) was significant on the expression of four ferritin genes,followed by salt stress,heat stress and cold stress.The expression peaks of PpFer1 and PpFer3 in leaves appeared at 6h,6h,and 12h after pear plant was exposed to oxidative stress(5 mM H₂O₂), salt stress(200 mM NaCl).A significant increase in PpFer4 expression was detected at 6h after salt stress or heat stress.The expression of four ferritin genes was different under plant hormone and abiotic stress treatments.The sequences of promoters maybe contribute to the expression differences.Taken together these results suggested that ferritin genes might be functionally important in acclimation of pear(Pyrus pyrifolia) to abiotic stresses.This showed accumulation of ferritin genes could be operated by different transduction pathways under abiotic stresses and hormones treatments.3.In this study,transgenic apple(Malus robusta) plants expressing rolC in vitro were used as rootstocks.Shoot tips collected from the four lines of transformation of apple (Malus domestica)(G1,G2,G3,G4) with soybean ferrtin gene(PvFer) and non-transformed plant(G0) in vitro were the source of scions.This study researched on the way of micrografting,the effect of different concentration of hormone(6-BA) and different culture medium.The results showed that the survive rate was higher by wraping silver in graft union,however,the contamination rate was higher and the procedure was more complex than control.6-Benzyladenine(6-BA) could increase the micrografts with the transgenic stock survival rate and the concentration was 0.2 mg/L concentration.Moreover, the survive rate of micrografts was higher in rooting medium than in differentiation medium.Micrografts were used to study on ferritin genes expression and expression changes induced by Fe(â…¢)-citrate for 24h at the transcriptional level by quantitative real-time PCR.The data obtained shows the expression of endogenous ferritin gene(MdFer) in transgenic scion was higher than non-transformed scion,however,the expression of MdFer and PvFer was lower than control plant.Exposed to iron overload stress,the expression of these genes was strongly enhanced,indicating that ferritin genes in micrografts could be activated in response to iron treatment.