Functional Analysis Of Foxtail Millet Autophagy Associated Gene SiATG8a In Regulated Plant Response To Low Nitrogen Stress

The supply of nitrogen affects the normal growth and development of plants by influencing flowering,aging,photosynthesis and the distribution of photosynthetic products.At present,in China,the rate of nitrogen fertilizer application is very high,but the use of nitrogen by plants is very low.On the one hand it has resulted in a waste of fertilizer,and on the other hand it has caused eutrophication and other serious environmental problems.It is very important to study plant nitrogen metabolism and signal transduction pathways regulating nitrogen transport to improve the efficiency of fertilizer use.Autophagy is a cellular degradation process that is highly evolutionarily conserved in yeast,plants,and animals.In plants,autophagy plays important roles in regulating the intracellular degradation and recycling of amino acids in response to nutrient starvation,senescence,and other environmental stresses.Foxtail millet(Setaria italica),which originated in China,has strong resistance to drought and nutrition deficiency stress and has been proposed as an ideal model system to study the physiological mechanisms of abiotic stress tolerance in monocotyledon plants.Although the genome sequence of foxtail millet(Setaria italica)is available,the characteristics and functions of abiotic stress-related genes remain largely unknown for this species.In this study,we firstly screened and identified the ATG family genes in foxtail millet.We analyzed the characteristic of the ATG family using the knowledge of bioinformatics.To study the function of SiATG genes,we cloned SiATG8 a gene.We analysed the sequence characteristics,expression patterns and transgenic plants under nitrogen starvation stress.With homologous proteins of SiATG8 a in Arabidopsis,we used AtATG8 a as a bait to screen interaction proteins in Arabidopsis by the split-ubiquitin system(hunter system),which provides a theoretical basis for elucidating autophagy pathways.The main experimental results are as follows:1.Base on the genome-wide analysis of foxtail millet genome,a total of 37 putative ATG genes in the foxtail millet genome were identified.Gene duplication analysis revealed that both segmental and tandem duplication events have played significant roles in the expansion of the ATG gene family in foxtail millet.Comparative syntenic mapping between the genomes of foxtail millet and rice suggested that the ATG genes in both species have common ancestors,as the ATG genes were primarily located in similar syntenic regions.2.Gene expression analysis revealed that the expression of 31 SiATG genes was induced by one or more phytohormone treatments,26 SiATG genes were induced by drought,salt and cold,24 SiATG genes were induced by darkness and 25 SiATG genes were induced by nitrogen starvation.qRT-PCR analysis revealed that among the 37 SiATG genes,the expression level of SiATG8 a was the highest after 24 h of nitrogen starvation,suggesting its potential role in tolerance to nutrient starvation.3.Subcellular localization experiments showed that SiATG8 a was localized in the membrane and cytoplasm of foxtail millet.Expression of SiATG8 a was also induced by nitrogen starvation.When foxtail millet seedlings were subjected to nitrogen starvation stress,the expression level of SiATG8 a gradually increased,and peaked at day 6 of the treatment.4.The plant expression vector pMWB014:SiATG8a was constructed and transformed into rice by Agrobacterium-mediated transformation.The stable inheritance and expression of SiATG8 a in transgenic plants was confirmed by PCR and qRT-PCR.The expression of 18 endogenous autophagy genes in transgenic rice was evaluated by qRT-PCR.We found that the insertion of the SiATG8 a gene did not affect the expression of these genes.Gene function analysis showed that the growth of SiATG8 a transgenic rice was better than that of wild type,survival rate,plant height,fresh weight and root surface area of SiATG8 a transgenic rice were significantly higher than wild type and the aboveground nitrogen content of transgenic rice was significantly higher than wild type.The total protein content was significantly decreased under nitrogen starvation conditions.These results indicated that heterologous expression of SiATG8 a in rice enhanced tolerance to nitrogen starvation and enhance nitrogen cycling.5.The plant expression vector pCAMBIA1302:SiATG8a was constructed and genetic transformation was used to obtain transgenic Arabidopsis.Gene function analysis showed that under nitrogen-starvation conditions,transgenic Arabidopsis plants expressing SiATG8 a were insensitive to nitrogen starvation,and growth was not inhibited.In addition,the lateral roots,total surface area,roots of transgenic plants was also higher than that of wild-type Arabidopsis.Transgenic plants accumulated more total nitrogen and had a less total protein content than wild-type plants.These results showed that the heterologous expression of SiATG8 a could improve the tolerance of transgenic Arabidopsis to nitrogen starvation.This result is consistent with the rice,which shows that SiATG8 a can significantly increase the tolerance of transgenic plants to low nitrogen stress in both dicotyledonous and dicotyledonous plants,these results had essential application values.6.We used the SiATG8 a homologous protein in Arabidopsis AtATG8 a as a bait to screen for AtATG8a-interacting proteins in Arabidopsis and identified a G protein α subunit by split-ubiquitin system.The interaction between GPA1 and AtATG8 a occurs in the cell membrane,which was verified by bimolecular fluorescence complementary assay(BiFC).The ultrastructure of Arabidopsis leaves were observed by transmission electron microscopy(TEM).Based on our results,we predict that ATG8 a could regulate the autophagy pathway by interacting with a G protein α subunit.In conclusion,we obtained the SiATG8 a gene base on the genome-wide analysis of autophagy associated gene in foxtail millet genome.Genetic function analysis showed that SiATG8 a could significantly improve the tolerance of rice and Arabidopsis to low nitrogen stress,and it had important application value.The mechanism of SiATG8 a homologous gene in Arabidopsis AtATG8 a regulation in plant autophagy was preliminarily analyzed.