Study On Abiotic Stress Resistance Of Transgenic Wheat With SiMYB1,SiATG8α

Wheat（Triticum aestivum L.）is one of three major food crops in the world and it’s a main food crop in China.However,wheat growth is often threatened by abiotic stresses such as low nutrition,high salt,drought and heat,which seriously affect its yield.Therefore,improving wheat resistance to various abiotic stresses is the key to ensure wheat yield and food security.At present,most researches on crop stress resistance focus on single abiotic stress resistance,but less on multiple abiotic stress synergistic resistance.Foxtail millet（Setaria italica L.）originated in China,which is drought resistant,barren tolerant and stress resistant.Its genome is small and its annotation information is perfect.It is a model C₄cereal plant rich in excellent stress resistance genes,which is of great significance for crop stress resistance molecular breeding.In the early stage,our laboratory identified SiMYB1according to the results of low nitrogen and drought transcriptome sequencing of foxtail millet,and transformed it into wheat variety Ji5265.SiATG8a was screened through the analysis of foxtail millet autophagy-related gene family,and transformed into wheat varieties Ji5265,Shi366 and Shi4056.On this basis,we analyzed the characteristics of these two genes,screened and identified the stress resistance of transgenic wheat lines,and provided candidate materials for improving the comprehensive stress resistance of main wheat varieties in China.The main results are as follows.1.Characteristics of SiMYB1 gene.SiMYB1 is located on Chromosome 6 of foxtail millet and belongs to R2R3-MYB transcription factor.The genomic sequence of SiMYB1 is1870 bp,with an open reading frame of 1632 bp,including 3 exons and 2 introns.The coding sequence is 915 bp,encoding 304 amino acids.The protein has a molecular weight of 33.45 k Da,an isoelectric point of 5.65 and two SANT domains.Phylogenetic analysis showed that SiMYB1 and At MYB20 had the closest homology.The results of expression characteristics analysis showed that SiMYB1 was mainly expressed in leaves and was induced by low nitrogen,Na Cl,PEG,heat and ABA stress.2.Resistance to abiotic stress in transgenic wheat with SiMYB1 gene.The results of molecular detection in T₃generation showed that the 3 overexpression lines（OE-40,OE-56,OE-69）had high positive rate and high expression of SiMYB1 gene.Compared with Ji5265,SiMYB1 could significantly improve the resistance of transgenic wheat to abiotic stresses such as low nitrogen（0.2 mmol/L N）,high salt（200 mmol/L Na Cl）,drought（25%PEG6000）and heat（38℃）.Under stress conditions,MDA and H₂O₂contents of transgenic lines were significantly lower than those of receptor,while proline content,catalase,peroxidase and superoxide dismutase activities were significantly higher than those of receptor.Under low nitrogen stress,plant height,shoot fresh weight,shoot dry weight and shoot nitrate content of transgenic lines were significantly higher than those of the receptor;Under high salt stress,the relative conductivity of transgenic lines was significantly lower than that of receptor,and the plant height,fresh weight and dry weight of shoot were significantly higher than that of receptor;Under drought stress,the survival rate of transgenic lines was significantly higher than that of receptor,and the water loss rate of detached leaves was significantly lower than that of receptor;Under heat stress,the survival rate,plant height,aboveground fresh weight and aboveground dry weight of transgenic lines were significantly higher than those of receptor,while the relative conductivity was significantly lower than that of receptor.The expression analysis of related stress genes showed that nitrate transport related genes（Ta NRT2.1,Ta NRT2.4 and Ta NRT2.5）,high salt stress related genes（Ta SOS1,Ta HKT1 and Ta NHX1）,drought stress related genes（Ta RD22,Ta RD29A and Ta COR47）and heat stress related genes（Ta HSP70,Ta HSP90 and Ta HSA32）were up-regulated in SiMYB1 transgenic wheat.3.Characteristics of SiATG8a gene.SiATG8a is located on Chromosome 6 of foxtail millet.Its genome sequence is 2743 bp and its open reading frame is 1233 bp,including 5exons and 4 introns.Its coding sequence is 360 bp and encodes 119 amino acids.The molecular weight of the protein was 13.73 k Da and the isoelectric point was 9.12.Evolutionary analysis showed that Os ATG8c and SiATG8a had the closest homology.The results of expression characteristic analysis showed that SiATG8a was mainly expressed in stems and was induced by low nitrogen stress.4.Resistance to low nitrogen stress in transgenic wheat with SiATG8a gene.The results of T₄generation molecular detection showed that the positive rate and SiATG8a gene expression of 3 Agrobacterium mediated overexpression lines（OE-32,OE-34 and OE-38）and 4 gene gun mediated overexpression lines（16SYJ67,16SYJ8,16SYJ9 and 16SYJ12）were higher.The results showed that overexpression of SiATG8a could significantly improve the resistance of transgenic wheat to low nitrogen stress.Under indoor low nitrogen condition,compared with Ji5265,the MDA and H₂O₂contents of OE-32,OE-34and OE-38 were significantly lower than those of Ji5265,while the proline content,catalase,peroxidase,superoxide dismutase activity,plant height,fresh weight,dry weight and nitrate nitrogen content of OE-32,OE-34 and OE-38 were significantly higher than those of Ji5265.The yield,panicle number,chlorophyll SPAD value and nitrogen utilization efficiency of transgenic lines 16SYJ67,16SYJ8,16SYJ9 and 16SYJ12 were significantly higher than those of the receptor under low nitrogen condition.The expression analysis of related stress genes showed that Ta NRT2.1,Ta NRT2.4 and Ta NRT2.5 were up-regulated in SiATG8a transgenic wheat.In conclusion,SiMYB1 can significantly improve the resistance of transgenic wheat to abiotic stresses such as low nitrogen,high salt,drought and heat;SiATG8a could significantly improve the resistance of transgenic wheat to low nitrogen stress.This not only provides new candidate genes and materials for comprehensively improving crop stress resistance,but also lays a foundation for the study of crop stress resistance regulatory network.