Cloning And Functional Analysis Of Transcription Factor GmNAC3 Gene In Glycine Max

Soybean(Glycine Max)play a very important role in the source of protein and oil.Nowadays,many abiotic and biological stresses,such as drought,salt and metal,have seriously affected the growth and development of soybean.Transcription factors play an important role in plant regulation of drought stress.As one of the largest family of plant-specific transcription factors,the N-terminal of NAC protein is a highly conserved NAC domain that can specifically bind to the Ca MV 35 S promoter,and the C-terminal is a regulatory region controlling transcriptional activation.Many studies have shown that NAC transcription factors play an important role in the molecular process of plant resistance to plant stress.In this study,through proteomics analysis of drought-resistant soybean Jiyu 47 roots,some drought-related proteins were identified,and the transcription factor GmNAC3 gene was selected from the up-regulated proteins for cloning.Bioinformatics analysis and RT-q PCR were used to study the biological characteristics of GmNAC3 protein.Gene expression pattern and drought resistance analysis were studied to preliminarily clarify the regulatory mode and pathway of GmNAC3,and this study also provides candidate genes for soybean molecular breeding of stress resistance.The main results of the study are as follows:1.In this study,8687 proteins were extracted from the soybean jiyu 47 root system under drought stress for 24 h,and 7875 proteins were identified by mass spectrometry.Among the proteins quantified by protein difference analysis,the levels of 468 proteins were found to be changed,of which 324 were down-regulated and 144 were up-regulated.Including the transcription factor GmNAC3 protein.2.The full length of GmNAC3 gene was cloned by RT-PCR using soybean Jiyu 47 root system.Bioinformatics analysis showed that the complete ORF of GmNAC3 gene included1452 bp and encoded 483 amino acids.With a molecular weight of 53.6k Da and a theoretical isoelectric point of 4.81,it is an unstable hydrophilic protein with no signal peptide and locates on the nucleus.GmNAC3 contains a relatively conserved NAM domain and is most closely related to wild soybean with 99.59% consistency of amino acid sequence.3.Yeast self-activation experiment was conducted by constructing p Bridge-GmNAC3 yeast expression vector,and GmNAC3 protein was proved to have transcriptional activation activity.The transient expression vector p CAMBIA1302-GmNAC3 was constructed and transformed into Benji tobacco for subcellular localization experiment,which proved that GmNAC3 protein was located on the nucleus.The analysis of biological characteristics showed that GmNAC3 accords with the basic characteristics of transcription factor.4.In order to study the expression of GmNAC3 gene in flowering and pod stage,the whole soybean plant of Jilin Agricultural University was selected as experimental material,and the relative expression level of GmNAC3 gene in root,stem,leaf,flower and pod was determined.GmNAC3 gene was expressed in root,stem,leaf,flower and pod at flowering and pod setting stage,the lowest expression level in soybean leaves and the highest expression level in soybean roots,which was 8 times of that in leaves.In order to explore the expression pattern of GmNAC3 gene in soybean roots under drought conditions,the laboratory conducted a soybean hydroponic experiment,using a nutrient solution containing 20% PEG6000 to simulate drought treatment for 42 h,and taking soybean roots treated for 0h as the control,sampling every 6h.The results showed that drought stress could promote the expression of GmNAC3 gene.The expression of GmNAC3 gene showed a single-peak curve,and reached the highest level at 12 h,which was 16 times that of the control,and reached the lowest level at 42 h,which was 3 times that of the control.5.Plant expression vector p CAMBIA3301-GmNAC3-GFP was constructed,and transgenic soybean roots were obtained by agrobacterium-mediated method.Transgenic roots treated with20%PEG6000 simulated drought for 12 h were used as the experimental group,and those without drought were positive control and those treated with empty vector were negative control.Compared with the control group,SOD,POD,CAT activities and Pro content were increased in the experimental group,while MDA content was decreased.In order to further explore the molecular regulatory mechanism of GmNAC3 gene,the relative expression levels of some drought-resistance related genes were determined in this experiment.The results showed that the expression levels of LEA14,GSH-PX,6PGD and P5 CS were all up-regulated in the experimental group,while APX2 had no response to GmNAC3.These results suggest that GmNAC3 can up-regulate the expression of GSH-PX,P5 CS,LEA14 and 6PGD in plants.GmNAC3 can regulate the expression of some drought-related genes to cope with drought stress.6.In order to further clarify the drought resistance function of GmNAC3 gene,transgenic Arabidopsis thaliana with GmNAC3 gene was obtained by agrobacterium Agl0 infection.It was found that the germination rate and germination rate of transgenic Arabidopsis thaliana on MS0,3%PEG and 6%PEG medium were higher than those of wild type.Drought resistance analysis showed that transgenic Arabidopsis thaliana was more tolerant to drought and had improved resilience after rehydration.These results suggested that GmNAC3 overexpression increased germination rate,promoted rooting and improved recovery ability of arabidopsis seeds under drought conditions.In order to verify the antioxidant capacity of GmNAC3 gene under drought conditions,transgenic Arabidopsis thaliana under 6%PEG stress was used as experimental group,transgenic Arabidopsis thaliana without stress was used as positive control,and wild-type Arabidopsis thaliana under 6%PEG stress was used as negative control.Physiological and biochemical indexes related to drought resistance were determined.SOD,POD,CAT activities and Pro content of transgenic Arabidopsis were increased,while MDA content was lower in the control group,which was consistent with the results of soybean root experiment.It was further indicated that GmNAC3 overexpression could improve drought resistance of plants.