| The growth and development of crop is affected by low temperature stressleads to the decrease of yield and quality.Improving crop resistance to low temperature stress is an effective way to solve the above problems.At present,the non-aba-dependent signaling pathway dominated by ice1-cbf is mainly used for the regulation of plant response to low temperature stress,while the signaling pathway of other hormones regulating plant response to low temperature stress is still unknown.In addition,most of the studies on plant resistance to low temperature stress are focused on seedling stage,and little is known about the regulatory pathways of plant resistance to low temperature stress at maturity stage.Setaria italica Lis a multigrain crop widely cultivated in northern China.It is characterized by strong stress resistance and is a model crop for the study under stress resistance of gramineae crops.At present,the research on the functional genome of millet resistance has just started,and the molecular mechanism of its resistance is still unknown.Digging out the key anti-stress genes of millet and analyzing their anti-stress function has important practical and theoretical significance for revealing the anti-stress molecular mechanism of millet and improving the anti-stress of gramineous crops.In this study,SiTGA5,a bZIP transcription factor gene involved in plant response to low temperature stress,was cloned from millet by means of reverse genetics,to elucidate its biological function in improving plant resistance to low temperature stress,and to reveal its signaling pathway in regulating plant response to low temperature stress.The main findings are as follows:1.Analyzing the transcriptome data of millet under low temperature stress,it was found that the transcription factor gene SiTGA5 was bZIP.The phylogenetic tree was constructed by amino acid multi-sequence alignment,the evolutionary relationship of SiTGA5 was analyzed,and the conserved structural domain and evolutionary relationship of SiTGA5 were determined.It was found that SiTGA5 belongs to the second subgroup of TGA transcription factors of bZIP type,and SiTGA5 has the highest homology with rice OsbZIP08.2.The results of gene expression analysis showed that under salicylic acid treatment,the expression level of SiTGA5 gene was significantly up-regulated and reached its peak at 2.5 hours,4times of the normal level.Under low temperature stress,the transcriptional abundance of SiTGA5 gene increased significantly,reaching about 3 times the maximum normal level at 2 h,and then decreased.The results of tissue specific expression analysis showed that SiTGA5 was the most expressed in roots.Analysis of protein subcellular localization in the protoplast of arabidopsis thaliana revealed SiTGA5 protein localization in the nucleus.The results of transcriptional activation experiments showed that SiTGA5 had no transcriptional activation activity.3.Transgenic rice was detected by PCR,and the copy number of exogenous genes in 5 lines was detected by digital PCR.The results showed that the transgenic lines were all single copies.In the seedling stage and booting stage,SiTGA5 transgenic rice lines 370,372,373,376 and 378 were treated at low temperature.The results showed that after treatment,the biomass of all the five lines in the seedling stage were higher than that of the recipient rice Kitaake,and the biomass of 370,372 and 373 increased significantly.After booting treatment,the seed setting rate of all 5 lines were higher than that of Kitaake.The results showed that the overexpression of millet transcription factor SiTGA5 could improve the resistance of transgenic rice to low temperature stress.4.OsNPR1 is an important member of the disease-related SA signaling pathway.The interaction between SiTGA5 and OsNPR1 was identified by yeast two-hybridization,pull-down,BiFC,and tobacco luciferase complementation.The results showed that SiTGA5 could interact with OsNPR1.5.Promoter structure analysis revealed that the promoter region of the disease resistance related protein OsPR1 a gene contained 4 elements of bZIP transcription factor binding.DNA binding experiments showed that SiTGA5 could bind to specific sites in the promoter region of OsPR1 a in vitro.Meanwhile,in the presence of OsNPR1,the binding ability of SiTGA5 to OsPR1 a promoter was enhanced,indicating that the interaction between OsNPR1 and SiTGA5 could enhance its transcriptional activation activity.In the DNA binding experiment,the content of SA was increased by gradient.The presence of OsNPR1 can enhance the DNA binding ability of SiTGA5,and this effect is regulated by SA.LUC experimental results showed that in the presence of OsNPR1,the activation ability of SiTGA5 to PR1 a was also enhanced,and the promoter activity of OsPR1 a was significantly enhanced in the presence of SiTGA5.The above experiments demonstrated that SiTGA5 can activate the expression of the disease-resistant gene OsPR1 a through transcription,and the realization of this function is regulated by OsNPR1,a receptor protein in the salicylic acid signaling pathway. |
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