Screening,Cloning And Functional Characterization Of Key Genes For Chlorophyll Synthesis Of Apple Rootstock Malus Halliana In Response To Iron Deficiency

Iron(Fe)is an essential element for the chlorophyll synthesis.Fe deficiency will cause chlorosis in leaves of plants such as fruit tree,even whole plant died.Breeding Fe deficiency-tolerant rootstocks is an effective measure to prevent Fe deficiency.Malus halliana is a Fe deficiency-tolerant apple rootstock and did not show chlorotic symptoms.However,little is known about the mechanism of chlorophyll biosynthesis of Malus halliana in response to Fe deficiency.In this experiment,the Fe-efficient rootstock Malus halliana and the Fe-inefficient rootstock Malus hupehensis were employed as test materials.Under Fe deficiency,this report identified the key sites of chlorophyll biosynthesis of Malus halliana and screened differentially expressed genes such as PPOX1,PPOX2 and UROD via combining chlorophyll precursors contents and relative expression levels of related genes.The above three genes were cloned were transferred to tobacco(Nicotiana tabacum L.)to verify the function of the genes under Fe deficiency.The main results are as follows:1.Under Fe deficiency,the contents of chlorophyll precursors in Malus halliana were higher than that of Malus hupehensis,the regulatory site of chlorophyll synthesis in Malus halliana in response to Fe deficiency was the transformation from URO III to Proto IX.2.After application of exogenous sugar(Suc),the regulatory site of chlorophyll precursor synthesis in Malus halliana in response to Fe deficiency was conversion of ALA to PBG,suggesting that regulatory site was advanced.3.Quantitative real-time PCR(qRT-PCR)showed that the expression of PPOX1 and UROD was up-regulated and the expression of PPOX2 was down-regulated under Fe deficiency.4.PPOX1,PPOX2 and UROD genes were cloned from Malus halliana.The size fragments were 1644 bp,666 bp and 1143 bp,encoding 547,221 and 380 amino acids,respectively.Both of them had the highest homology with the amino acid sequence of apple.The amino acid sequence encoded by PPOX1 and UROD was closely related to the amino acid in white pear,and the amino acid sequence encoded by PPOX2 had the closest relationship with the amino acid in apple.5.A binary expression vector driven by constitutive promoter 35 S was constructed and transformed into tobacco by Agrobacterium tumefaciens.Transgenic tobacco plants were successfully obtained.6.Compared with the control,under Fe deficiency,transgenic tobacco with PPOX1 and UROD were more green,and transgenic tobacco with PPOX2 showed pale green.In addition,the qRT-PCR showed that the expression levels of PPOX1 and UROD genes in transgenic tobacco were up-regulated by 10.80 times and 3.65 times,respectively.The expression of PPOX2 was down-regulated by 0.23 times.