| Drought is one of the main abiotic stress factors that limit crop yields.Drought frequently occurs in most of the wheat regions in China,which seriously affects food production safety.Analyzing the molecular mechanism of drought resistance in wheat and deeply digging drought-related genes has important theoretical and practical significance for improving wheat drought resistance and developing water-saving agriculture.Two wheat varieties(lines)with significant differences in drought resistance(Xinong 979(XN979)and drought-sensitive strain(LA-379))were selected for this experiment.Two treatments,drought and normal irrigation,were set up to determine the relative extent of the leaves.Water content,leaf electrical conductivity,leaf malondialdehyde(MDA)content and leaf proline content.The molecular mechanism of drought resistance was analyzed by proteomic analysis.Virus-induced gene silencing(VIGS)technology was used to silence the genes encoding the two differentially expressed proteins,W5DYH0 and W5ERN8,and their function was studied.The main test results are as follows.1.Drought resistance identification of drought-resistant wheat cultivar XN979 and drought-sensitive strain LA-379 showed that compared to normal irrigation treatment,the relative water content of wheat leaf in XN979 under drought treatment only decreased by 8.4%,which was not significant compared with the control.However,the leaf relative water content of LA-379 decreased by 40.4%,reaching a very significant level;compared with normal irrigation treatment,the leaf relative electrical conductivity of XN979 increased by only 20.1% under drought treatment,while LA-379 wheat varieties The relative conductivity of the drought-treated wheat leaves increased by 70.1%,reaching a very significant level;compared with normal irrigation treatments,the MDA content of wheat leaves under drought treatment increased by 74.0%,while LA-379 wheat.The MDA content in drought-treated wheat cultivars increased by 169.6%,which was significantly higher than that of XN979;the proline content of XN979 increased by 2.42 times under drought treatment,whereas the drought-treated wheat leaves in LA-379 wheat cultivars increased.Proline content increased by 1.04 times,indicating that XN979 is stronger than LA-379 wheat varieties in drought resistance.2.Comparative proteomic analysis methods were used to study the differentially expressed proteins in drought resistant wheat varieties XN979 and drought-sensitive strain LA-379 under normal irrigation and drought conditions.A total of two-stage mass spectra was obtained: 227076;the number of spectra matched to the identified peptides: 38070;the total number of identified peptides: 17416;the total number of unique peptides identified: 12126;the total number of identified proteins: 5369.482 differentially expressed proteins,199 up-regulated differentially expressed proteins,and 283 down-regulated expressed proteins were identified in XN979 wheat varieties;600 differentially expressed proteins were identified in LA-379 wheat varieties,301 of which were up-regulated and 299 were down-regulated.protein.335 unique differentially expressed proteins were identified in XN979 wheat varieties,453 unique differentially expressed proteins were identified in LA-379 wheat varieties,and differentially expressed proteins common to 147 two wheat varieties were identified.The differentially expressed proteins unique to the XN979 wheat variety are mainly involved in metabolic pathways,biosynthesis of secondary metabolites,carbon metabolism,photosynthesis,carbon fixation in photosynthetic organisms,glyoxylate and dicarboxylic acid metabolism,glycolysis/ Gluconeogenesis and other pathways.To evaluate the reliability of proteomics data,12 differential proteins were selected.Ta GAPDH gene and Ta Actin gene were used as internal reference genes respectively,and the expression levels at the transcriptional level were verified by Real time-PCR.The results showed that the results obtained using two different internal reference genes were basically the same.Most of the differentially expressed proteins have the same tendency to express at the transcription level and the other at the transcription level.This indicates that the expression of these genes may be regulated at the transcriptional,post-transcriptional,translational and post-translational levels..3.The function of genes selected by the two differentially expressed proteins was further studied using VIGS technology.The gene encoding the differential protein W5DYH0 was named Ta DSR1(Drought stress response 1),and the gene encoding the differential protein W5ERN8 was named Ta DSR2(Drought stress response 2).On the 10 th day after VIGS inoculation,the symptoms of BSMVPDS showed albinism,and the infection phenotype of spotted streak appeared on the third leaf of the silenced gene plant,indicating successful infection.The results of m RNA expression of differentially expressed proteins in the successfully inoculated plants showed that compared to normal irrigation(NS),Ta DSR1 and Ta DSR2 m RNA expression levels under drought stress(DS)and inoculation with ? The expression level of m RNA in the drought-treated plants of the empty vector negative control(BSMV0)was significantly increased,and the m RNA expression level of different strains was significantly down-regulated in the target gene-reduced expression plants.This indicated that drought stress could induce the up-regulated expression of the gene,and VIGS technology reduced the expression of the gene after it was silenced,and it was at a lower expression level under drought stress.The results of the physiological indicators of the differentially expressed protein-corresponding genes in the successfully inoculated plants showed that the drought-resistance(DS)and inoculation of the ?-null of the two wheat varieties XN979 and ZM9023 were compared with normal irrigation(NS)and drought treatment.The leaf relative water content of the drought-treated(BSMV0)and silencing Ta DSR1 and Ta DSR2 genes in the negative control of the vector were significantly reduced,but the target gene was reduced in the plants expressing the different genes compared to the drought treatment(DS).The relative water content of the leaves was significantly down-regulated;compared to normal irrigation(NS),the drought treatment(DS)of the two wheat varieties XN979 and ZM9023,the drought treatment(BSMV0)inoculated with the negative carrier of the ? empty vector,and the drought treatment.The relative conductance content and MDA content in leaves of different Ta DSR1 and Ta DSR2 gene silencing strains were significantly increased.Compared with drought stress(DS),the relative conductance content and MDA content in leaves of different strains were decreased in target gene-reduced expression plants.The contents also increased significantly;compared to normal irrigation(NS),drought treatment,drought stress(X)of two wheat varieties XN979 and ZM9023 The proline content of different strains of drought-treated(BSMV0)and Ta DSR1 and Ta DSR2 silenced vaccinated with ?-empty vector was significantly increased,compared with drought-treated(DS),the target gene was reduced expression.In the plants,the proline content in leaves of different strains was significantly reduced,and the leaves appeared sagging.The results showed that drought stress could induce the up-regulation of Ta DSR1 and Ta DSR2 genes and the changes of physiological indexes.VIGS silenced the two genes,which reduced the expression of these two genes and changed the physiological indexes,which in turn led to their drought resistance.Reduced sexuality.This shows that Ta DSR1 and Ta DSR2 play an important role in wheat drought resistance. |
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