Identification,cloning And Expression Characteristics Analysis Of Grape Fe-S Cluster Assembly Genes

Iron is an essential trace mineral element for plant growth.Fe-S clusters,as the active part of ferrithiin,play an important role in life activities such as respiration,photosynthetic,sulfur and nitrogen assimilation,amino acids and purine metabolism,plant hormones and coenzyme synthesis.The Fe-S cluster assembly genes were screened and identified in grape genome by homologous cloning.The detailed characteristics of Fe-S cluster assembly genes and their encoded proteins in grape were analyzed by using a variety of bioinformatics software.The expression patterns of Fe-S cluster assembly genes in different tissues of grape and their responses to iron deficiency stress were analyzed by using real-time fluorescent quantitative PCR.The findings are as follows:1、In total,46 Fe-S cluster assembly genes were retrieved and cloned from grape,which were mainly located in plastids,mitochondria and cytoplasm,containing 14,21 and 11 genes,respectively.Bioinformatics analysis showed that grape Fe-S cluster assembly genes were distributed on a total of 16 chromosomes,containing 1-21 introns with different lengths.Grape Fe-S cluster assembly proteins were found in a variety of subcellular structures.Moreover,the subcellular localization of Fe-S cluster assembly proteins with different assembly mechanisms in plastids,mitochondria and cytoplasm is quite different.2、 Real-time fluorescence quantitative PCR analysis showed that the expression levels of 46 grape Fe-S cluster assembly genes in different tissue parts of the annual tree and tissue culture seedlings were significantly different in three years of Maseran.Overall,Its expression in fruit is higher than that in leaves,The expression of leaf part of seedling was higher than that of root and stem;Gene DRE2 expression levels were relatively high and stable at each site.Presumably,Fe-S clusters in grape mainly rely on DRE2 to transfer electrons;Gene ISU1 expression was highest in all parts,HSCA1 and ISA1 followed.Andthese three genes all belong to the mitochondrial ISC mechanism,suggesting that mitochondrial ISC mechanisms require more functional scaffold proteins,chaperones,and electron metastases;Some typical scaffold protein ISU2、ISU3 and NFU5 loss.3、Real-time fluorescence quantitative PCR analysis showed that the response of grape Fe-S cluster assembly gene to iron deficiency stress transcription level was significantly different in different parts of Maseran tissue culture seedlings.The expression levels of 16,14,21 genes in the roots,stems,and leaves of tissue culture seedlings were regulated by iron deficiency treatment.The expression levels of root Fe-S cluster assembly genes were easily induced and down-regulated by iron deficiency stress,while the expression levels of above ground(stem and leaf)Fe-S cluster assembly genes were easily inhibited and up-regulated by iron deficiency stress.4、Fe-S cluster assembly genes are slightly distinct among different species.The sequence identity of ISU1 homologous proteins from 10 plant species is as high as77%,and there are many highly conserved domain regions.Phylogenetic tree analysis indicates that ISU1 members belonging to the same genus,such as Arabidopsis and thellungiella of Cruciferae,rice and Brachypodium of Gramineae,and peach and apple of Rosaceae,are tended to be closely clustered,while grape ISU1 and tomato ISU1 were closely clustered together.