Screening And Functional Investigation On BZIP Transcription Factors In Broomcorn Millet (Panicum Miliaceum L.) Against Salt

The research on broomcorn millet in China mainly focuses on cultivation techniques,breeding new varieties,excellent agronomic traits,development of agricultural production and commercial value,collection and preservation of germplasm resources and so on.However,the mining and utilization of gene resource is far from sufficient.Investigating the germination mechanism of broomcorn millet under salt stress is beneficial to improve the germination power and potential of seeds,so as to improve the crop yield.It is of great significance to study and utilize the millet germplast with high salt tolerance and excavate the favorable genes,improve crop varieties,increase farmers’ income in millet producing areas and maintain the regional balance of crops of the nationa.In this study,the seeds of 17 local species of Broomcorn millet(Panicum miliaceum L.)were used as test materials,treated with water(RO water)and Na Cl with different concentrates to simulate salt stress and evaluate the salt tolerance by analyzing their whitening time and germination rate.As a result,a salt-tolerant variety and a salt-sensitive variety were selected and the seeds of them under both treatments at three germination stages were sampled for RNA-Seq.Through RNA-seq analysis,significant differences of the transcriptional abundance of a class of bZIP transcription factors were detected,suggesting that these genes may be involved in the salt stress response in broomcorn millet.Therefore,effective screening of related genes and in-depth understanding of the functions and mechanisms of these genes are necessary to analyze the salt tolerance of broomcorn millet.Bioinformatics analysis was conducted on bZIP related transcription factors screened,and screened out the genes that were involved in regulating plant hormone signal transduction pathway during seed germination under salt stress,so as to enable normal seed germination.To verify this conjecture,we carried out relevant studies and explored the functions and related mechanisms of bZIP transcription factor.The specific results are as follows:144 bZIP transcription factors were identified by gene family analysis,which were named PmbZIP1-PmbZIP144 and divided into 10 subfamilies(A to E,G,H,I,S and U,respectively).The molecular weight ranges from 9.19 k Da(PmbZIP59)to 68.3 k Da(PmbZIP51).The isoelectric point ranges from 4.52(PmbZIP102)to 11.57(PmbZIP43).Twelve different motifs(Motifs)were identified in PmbZIPs.The number of introns in PmbZIP ranged from 0 to 12.Promoter analysis revealed that PmbZIP promoter regions contained multiple cis-acting elements,such as plant hormone response element,MYB binding site,light response element,abiotic stress response element,seed specific regulatory element and root specific regulatory element.Gene family analysis will lay the foundation for further study of bZIP family gene in Broomcorn millet.Transcriptome sequencing revealed that 36 PmbZIP transcription factors were involved in seed germination under salt stress.The differential expression analysis of these genes showed that six bZIP transcription factors,PmbZIP131,PmbZIP125,PmbZIP33,Pm ABI5,PmbZIP118 and PmbZIP97,were differentially expressed in this period.Further expression analysis indicated that the expression Pm ABI5 was induced by the treatments of salt(200m M Na Cl),drought(20% W/V PEG6000)and low temperature(4℃)could induce the expression of in the seedlings of Shanbei japonica millet(Y9),and by ABA(100 μM ABA),6h in Yumi1(Y1,salt-sensitive material),and PmbZIP131 was induced by ABA,low temperature and high temperature(42℃)in Y1.There was no significant alteration in the expression of Pm ABI5 in the Shanbei japonica millet(Y9).The expression of PmbZIP97,PmbZIP125 and PmbZIP118 did not change in most treatments,and two U subgroup genes(PmbZIP97 and PmbZIP89)were strongly induced by ABA,salt,low temperature,high temperature and drought stress in Y1.The abovementioned six genes were overexpressed in Arabidopsis thaliana and the phenotypes of the transgenic lines suggested that PmbZIP33,Pm ABI5,PmbZIP118 and PmbZIP97,the overexpression of which significantly increased the germination rate of transgenic lines,were involved in regulating seed germination under salt stress.Pm ABI5,PmbZIP118 and PmbZIP97 were also involved in root growth regulation after germination,and the root length,root area and lateral root number of overexpression lines were significantly increased by comparison to those of wild type.To further explore the functions of Pm ABI5 in monocots,it was subsequently overexpressed in rice.The results showed that the root growth of rice overexpression transgenic lines was significantly enhaced,and the grain length was significantly increased compared to the wild type(Nipponbare).Bi FC showed that Pm ABI5 formed heterodimer with PmbZIP33 and PmbZIP131,and also homologous dimer with Pm ABI5.Further yeast single hybridization experiments showed that Pm ABI5 and PmbZIP131 regulated the expression of Pm NAC1 by binding to the G-box in the promoter.These results indicate that Pm ABI5 can directly regulate seed germination and seedling growth,and indirectly improve salt tolerance of plants by regulating the expression of Pm NAC1 gene through the formation of heterodimer with PmbZIP131.