Development Of Near-isogenic Lines Of Rice Blast Resistant Genes From Japonica Variety Yunyin And Its Proteomics Analysis

Rice blast, is one of the most devastating diseases all over the world. The recurrence and outbreak of Rice blast usually leads to sharp decline in rice production, and even no yield. In recent years, rice blast still prevails in some areas. In this study, resistant and susceptible materials were screened out from Near-Isogenic Line (NIL) of Yunyin resistant to rice blast using spray inoculation of Magnaporthe oryzae Sichuan-43in combination with marker-assisted selection. Proteomics and Mass Spectrometric Analysis of resistant and susceptible materials were described as follows:rice leaf total protein at three-leaf stage was extracted using TCA/Acetone Precipitation for2-dimensional electrophoresis (2-DE) with the first dimension isoelectrofocusing using17cm pH4-7IPG and12%SDS-PAGE in the second dimension.2-DE gel was subjected to Coomassie Brilliant Blue R250staining, and the difference in leaf protein expression levels between resistant and susceptible NILs was analyzed using PDQuest2-D Analysis Software. Protein spots with expressional difference in resistant and susceptible NILs during the process of infection were extracted and identified using MALDI-TOF-MS. A total of75different protein spots were extracted, in which67proteins with expressional difference were further identified.7proteins exhibited specific expression in resistant NILs,43proteins were up-regulated,7proteins were found down-regulated, and20proteins were identified as same families.Bioinformatics analysis showed that the identified proteins were involved in lots of biological and physiological processes. Among them,5proteins were related to photosynthesis, accounting for7.5%;25proteins, about37.3%, were involved in metabolism; in addition to12unclassified protein, the rest of proteins were found to be associated with other processes:6with anti-oxidant,10with synthesis and modification,5with signal transduction, and4with adverse stress.Amino acid sequences of selected proteins were aligned using BLAST.5 proteins in different physiological processes were predicted for their cDNA sequences and verified using qRT-PCR. The results showed that the genes encoding Peptidyl-prolyl cis-trans isomerase, cytochrome C peroxidases, and glutathione transferase expressed lower before inoculation, while improved the expression at12hours post inoculation. The gene encoding phospholipase D gene was in a relatively high expression in normal physiological process, while down-regulated from12to48h post inoculation, and its expression in resistant materials was notably higher than that in susceptible materials. Finally, the expression of Guanine nucleotide-binding protein beta subunit-like protein gene was up-regulated gradually after inoculation, followed by down-regulation, and finally returned to the level of pre-inoculation at96h post inoculation. The results indicate that resistance-associated genes are induced and expressed in rice defense against pathogen infection, which subsequently activate plant resistant responses.