Physiological And Molecular Mechanisms Of Sorghum Seedlings Overexpressing AtVIP1 Gene In Response To Saline-alkali Stress

Sorghum(Sorghum bicolor(L.)moench)has strong tolerance to stress,and has attracted much attention as a "star" crop in China.With the continuous expansion of saline-alkali land area,it is important to elucidate the physiological and molecular mechanism of plant saline-alkali tolerance,and to cultivate new varieties of saline-alkali tolerant crops to adapt to agricultural environment and ensure food security.Transcription factors play an important role in the regulation of plant resistance to stress.VIP1(VIRE2-Interacting Protein 1)is a b ZIP transcription factor with various functions such as influencing Agrobacterium transformation efficiency,regulating osmotic stress signals and touch-induced root movement.Previous studies by the group suggested that VIP1 may be involved in plant response to soda salinity stress,but no related studies have been reported.In this experiment,transgenic sorghum lines overexpressing AtVIP1 gene were treated with salt alkali stress(Na HCO3:Na2CO3: 5:1,75 mm,p H 9.63)at the three leaves and one shoot.The key differentially expressed genes were screened by measuring the growth morphological indexes,stress resistance physiological indexes and endogenous hormone content,and the MAPK and ABA signal pathways and the expression patterns of key genes in flavonoid anthocyanin anabolism were analyzed,Finally,the physiological and molecular mechanisms of sorghum seedlings overexpressing AtVIP1 gene in response to saline alkali stress were clarified.The main conclusions are as follows:(1)Sorghum overexpressing the AtVIP1 gene had higher salinity tolerance,with higher whole plant fresh and dry weight,plant height,lateral root number,root to crown ratio and root vigor than the wild type.The wild type started browning of the main roots at the beginning of the stress,with new leaves curled and wilted at 72 h and severe root rot at the base of the roots,while the transgenic sorghum showed later onset of main root browning and mild symptoms,with normal extension of new leaves at 72 h.The chlorophyll content and soluble protein content were significantly higher than those of the wild type.(2)Overexpression of AtVIP1 gene increased the activity of reactive oxygen scavenging enzyme system in sorghum under saline stress,and the MDA content of transgenic sorghum was significantly reduced by DAB and NBT staining,indicating less accumulation of H2O2 and O2-and enhanced antioxidant enzyme activity.At the early stage of stress(4-12 h),SOD,CAT and GR acted significantly;at the mid-stress stage(24-72 h),all enzymes were functional;at the late stage of stress(120 h),CAT and GSH-Px played an important role,and sorghum overexpressing AtVIP1 gene had higher O2-resistance and stronger reactive oxygen species scavenging ability,thus reducing the damage of reactive oxygen species on membrane lipids and showing The sorghum overexpressing AtVIP1 gene has higher O2-activity,better reactive oxygen species scavenging ability,thus reducing reactive oxygen species damage to membrane lipids and showing better salinity tolerance.(3)Overexpression of the AtVIP1 gene significantly affected the content of growth-and defense-related hormones in sorghum roots.After stress,IAA and GA3 contents were significantly higher in transgenic sorghum than in the wild type;ABA contents were significantly higher than in the wild type except at 72 h of stress;while SA and JA contents were significantly higher than in the wild type at 72 h of stress.(4)A total of 1471 differentially expressed genes were identified in the differential transcriptome analysis of salinity stress.GO analysis showed that biological processes were mainly enriched in metabolic processes and adversity response processes,and the KEGG pathway was mainly involved in phenylalanine biosynthesis,phytohormone signaling and glutathione metabolism pathways.COG analysis of differentially expressed genes showed that defense mechanisms were more predominant in different time periods,and 42 differentially expressed genes related to antioxidant enzymes were obtained.(5)After screening for differential genes based on transcriptome data,q RT-PCR was used to further validate their expression patterns after saline stress.Of the 21 genes related to MAPK signaling pathway,transgenic sorghum Sb MPK3 and Sb MPK13 both showed significantly higher expression after saline stress,while Sb MPK6 and Sb MPK14 showed significantly higher expression at the beginning of stress(24 h).Eight genes related to ABA synthesis and signaling pathway,Sb NCED1 and Sb Sn RK2 were significantly up-regulated in transgenic sorghum while Sb PP2C5 was significantly down-regulated in expression.19 genes related to flavonoid synthesis and metabolism.When transgenic sorghum was stressed for 72 h and 120 h,the expression of flavonoid synthesis related enzyme genes such as Sb C4 H,Sb CHS,Sb CYP,Sb F3 H,Sb ANR and Sb ANS were significantly up-regulated,and the total flavonoid content at 0 h and 72 h was significantly higher than that in WT group,and the anthocyanin content at 120 h was significantly higher than that in WT group.In summary,the AtVIP1 gene plays a very important role in sorghum salinity stress response by promoting plant growth and root genesis,enhancing reactive oxygen species scavenging,promoting flavonoid synthesis to enhance antioxidant processes,regulating ABA stress hormone synthesis and its signaling,and participating in the MAPK signaling cascade pathway to enhance transgenic sorghum resistance to salinity stress.The results of this study provide a basis for further elucidation of the molecular mechanism of transcription factor VIP1 that regulates salinity tolerance in sorghum.