| Energy plant switchgr ass(Panicum virgatum L.)has strong toler ance to salinity and alkalinity.In order to better understand the mechanism of response of switchgr ass to alkali-s alt stress,we used alkali-sens itive genotype AM-314/MS-155(AM-314)and alkali-toler ant genotype Alamo as exper iment mater ials in this study.When the plants were grown to f ive leaves(E5)stage,the str ess treatment was carr ied out by soaking method(Na2 CO3 : Na HCO3=1: 9,C(Na+)=150 m M and p H=9.0),F ive process ing time points(0 h,3 h,6 h,12 h,24 h)were s et up.Relative water content,relative conductivity,osmotic adjustment substance content and antioxidant enzyme activity of root and leaf of switchgrass were measured.Then,according to the changing trend of phys iological and biochemic al indexes,thr ee small molecule RNA librar ies were constructed from root samples,and micro RNAs related to alkaline salt tolerance of switchgrass were excavated.To explore the pr imar y phys iological mechanism and the molecular regulation mechanism of micro RNAs,of switchgrass responding to alkali-str ess.the results are as follows:(1)Relative water content of roots and leaves of two cultivars increased w ith str ess time.After 24 hours of str ess,the r elative water content of r es istant cultivars could keep above 85%,while that of s ens itive cultivars decreas ed to 72%.Relative electr ical conductiv ity and malondialdehyde content of leaves increas ed with the increase of stress time.The content of MDA in roots increased first and then decr eased,reaching its maximum value in 6 hours,and increased by 40.12% and 32.06%(P<0.05)respectively in resistant and sensitive varieties compared with CK.(2)The contents of proline,soluble sugar and soluble protein in leaves increased with treatment time,while those in r oots f irst increased and then decr eased.The proline content in leaves and roots of r es istant var ieties was higher than sens itive var ieties.The content of proline,soluble sugar and soluble protein in res istant roots reached the maximum value after 6 hours of stress,which increas ed 113.99%,17.21% and 11.85 compared with the control respectively.Sens itive var ieties reached the maximum at 12 hours.The soluble protein content in sens itive leaves was significantly higher than in resistant varieties(P<0.05).(3)The activity of S OD,POD,CAT and glutathione content were bas ically the s ame.The activity of SOD and CAT incr eased f irst and then decreased w ith the increase of treatment time.SOD and CAT in leaves reached the maximum at 12 h,while POD reached the maximum at 6 h.The activities of three protective enzymes in r oots reached their maximum in advance(3 h,6 h).The activities of SOD and CAT in r es istant cultivars wer e higher than in sens itive cultivars,and the content of glutathione was lower than in sensitive cultivars.(4)Fresh root samples were taken after tr eatment for 0 h(CK),6 h,and 24 h,respectively,the differ entially expr essed micro RNA and its r egulatory network wer e analyzed.T he r esults shows that a total of 1049 known mi RNAs and 68 novel mi RNAs wer e identif ied.Among the res istant var ieties,ther e wer e 72 differ en tially expr essed mi RNAs,19 of which wer e sens itive,A total of 2595 target genes were pr edicted for all differ entially expressed micro RNAs in the two cultivars.41 of the differ entially expressed micro RNAs in the two cultivars were s ignif icantly down-regulated(P < 0.05).(5)GO analys is showed that diff erentially expr essed micro RNAs were s ignif icantly enr iched in metabolic processes,cellular processes and responses to stimuli(P<0.05).KEGG analys is showed that it was mainly concentr ated in metabolic pathways and genetic inf ormation process ing.Four diff erentially expressed micro RNAs(novel-mir-23,novel-mir-31,amg-mir 396,sbi-mir 5568c-5p)were f ound to be enr ic hed in the per oxisome metabolic pathway(ko04146)and involved in the scavenging of reactive oxygen spec ies(ROS)in the peroxisome,after 6 hours treatment in Alamo.Thr ee diff erentially expressed micro RNAs(novel-mir-6,zma-mi169i-3p and zma-mi167g-3p)were found to be enr iched in the glutathione metabolic pathway af ter 6 hours treatment in AM-314,m icro RNAs r educed glutathione catabolis m by inhibiting the express ion of glutathione transfer ase gene,which was consistent with the measur ed change tr end of glutathione content.T hrough analys is of diff erentially expr essed mi RNAs and their target genes,it was found that under alkaline-salt str ess,differ entially expressed mi RNAs in alkali-sens itive genotype were mainly involved in the regulation of cellular ROS c learanc e,ethylene s ignal tr ansduction,and root,leaf and f lower development.Compared with alkali-s ens itive genotype,mi RNAs in alkali-s alt respons e were also involved in water transpor t,DNA methylation,respons e to high osmotic pressure,activation of str ess-related genes and more complex responses to alkaline-salt str ess processes.alkali-salt response was s ignif icantly higher than alkali-sens itive genotype in the number of micro RNAs responding to alkaline-salt stress and in the functional divers ity of their regulatory target genes. |
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