Functional Study Of NAC Transcription Factor GmSIN2 And Its Interactive Protein GmNIW In Soybean Stress Tolerance

Soybean is an important food and economic crop,an important source of vegetable oil and protein,and has an important position in people's lives and the national economy.In recent years,the dependence of soybean imports in China has remained high,and industrial safety is facing crises and challenges.Under this background,it is important to identify biotic and abiotic stress related genes,to reveal their genetic relationships and molecular regulatory networks,and to construct high-yield and stress tolerance soybean varieties with molecular design breedingNAC transcription factors are a family of plant-specific transcription factors.It has been found that many NAC transcription factors participate in plant stress response through direct transcriptional regulation of stress-related genes and regulate plant stress tolerance.They are potential gene modification targets to improve abiotic and biotic stress tolerance in crops.In former study,we have cloned a salt-inducible soybean NAC transcription factor gene GmSIN2 from the transcriptional profile analysis of soybean under salt stress and found the GmSIN2 OE soybean showed higher salinity tolerance and grew better than wild-type soybean in field.To reveal the mechanism that how it regulates the salt stress tolerance of soybean,we performed the yeast cDNA library screening and found the candidate interactive protein of GmSIN2 as GmNIW(NAC Interactive WD40 protein),which encodes a WD40 protein.Its function is unknown in soybean.On this basis,this thesis takes GmSIN2 and GmNIW as the research objects,and has conducted the following research and obtained the corresponding results:1.Validation of the protein-protein interaction between GmSIN2 and GmNIW in plantaBimolecular fluorescence complementation(BiFC)experiments were used to confirm the protein-protein interaction of GmSIN2 and GmNIW in plant cells,and it was preliminarily determined that the interaction site was in the nucleus2.Systematic comparison of GmNIW and GmSIN2 gene charactersThrough phylogenetic analysis,it was found that the GmNIW orthologous genes are widely present in all tested species from algae to angiosperm and are rarely duplicated,indicating that they originated very early and their function might be conserved,while the GmSIN2 orthologous genes are not found in algae but in mosses to angiosperms,and they suffers more duplication,indicating that their functions are prone to differentiation;through organ-specific expression pattern analysis,GmNIW and GmSIN2 showed different organ-specific expression patterns,and leaf and stem are their common expression sites;through stress response expression pattern analysis,it was found that GmSIN2 was strongly induced by short-term NaCl,H2O2 and MeJA treatment,while GmNIW was up-regulated in response to short-term NaCl stress,and down-regulated in response to H2O2 and MeJA treatment.3.The function of GmNIW and GmSIN2 in plant abiotic stress and biological stress responsesTo study the function of GmNIW and GmSIN2 in plant stress response,their plant overexpression vectors and CRISPR-CAS9 vectors were constructed respectively,and their ectopically expressed transgenic Arabidopsis homozygous lines,35Spro::GmSIN2-GFP transgenic soybean T2 lines,and 35Spro::GmNIW-GFP transgenic soybean T1 lines are obtained.The transgenic Arabidopsis/soybean were used to study GmNIW and GmSIN2's function in abiotic and biotic stress responses.It was found that GmNIW and GmIN2 ectopically expressed transgenic Arabidopsis showed increased resistance to Botrytis cinerea and salt stress;the GmSIN2 ectopically expressed transgenic Arabidopsis showed reduced sensitivity to ABA and H2O2,while the GmNIW ectopically expressed transgenic Arabidopsis showed no obvious change.This shows that salt tolerance and gray mold resistance may be the main biological pathways regulated by GmNIW and GmSIN2.Additionally,it was found that 35Spro::GmSIN2 transgenic soybean grow better than wild-type under drought,indicating that in addition to improving salt tolerance,GmSIN2 can also improve soybean drought tolerance.Whether GmNIW can also regulate soybean drought tolerance needs to be further studied.4.Preliminary study on the pathways coordinately regulated by GmNIW and GmSIN2To find out the pathways co-regulated by GmNIW and GmSIN2,RNA-seq experiments were conducted using GmNIW,GmSIN2 overexpression(OE)soybeans and wild-type soybeans.The differential expressed genes were enriched in GO terms,KEGG pathways,and the corresponding interaction networks were explored.The results showed that in GmNIW OE soybeans,the number of down-regulated genes was much greater than that of the up-regulated genes.The expression of genes involved in plant-pathogen interaction,stress response and other pathways is significantly increased in GmNIW OE soybeans,and that in plant hormone signaling pathways,signal transduction and transcriptional regulatory proteins were changed.The genes involved in the basic material and energy metabolism are significantly down-regulated.At the same time,some interaction networks in plant defense pathways,ABA,JA and other pathways are found in the differential expressed genes.In GmSIN2 OE soybeans,more genes are up-regulated instead of down-regulated.The terms or pathways as biological stress response,plant-pathogen interaction,hormone signal transduction,JA metabolism are significantly up-regulated,while the cell basic material and energy metabolism pathways are down-regulated.These results indicate that both GmNIW and GmSIN2 may be involved in the plant stress response process,especially the biotic stress response,and JA pathway may be the plant hormone pathway that they regulate together.In RNA-seq results,the expression of the key genes of JA biosynthesis and signal transduction is downregulated in GmNIW OE soybeans but up-regulated in GmSIN2 OE soybeans.The result of Real-time RT-PCR experiments further validate the conclusion:the JA synthesis key genes Glyma.18G280900.1(GmAOC1),Glyma.19G044900.1(GmAOC2),Glyma.13G047300.1(GmAOC4)and JA signaling key genes Glyma.13G112000.1(GmJAZ2)are up-regulated in GmSIN2 OE soybeans and down-regulated in GmNIW OE plants,indicating that GmNIW and GmSIN2 regulate the expression of JA biosynthesis and signal transduction genes in opposite way.The above results indicate that genes encoding GmSIN2 and its interaction protein GmNIW have their own characters in terms of evolution and organ-specific expression.They transcriptionally respond to NaCl,H2O2,and MeJA treatment in different patterns,participate in the regulation of salt tolerance and Botrytis cinerea resistance in plants,and affect the expression of genes involved in biotic stress and other stress related pathways.They may participate in the regulation of plant biotic and abiotic resistance by regulating JA pathway.