Functional Analysis Of Soybean Type ? Chalcone Isomerase (CHIs) Gene In Regulating Nodulation

The nodules formed by symbiosis of soybean and rhizobium can fix nitrogen in the air and transform it into biologically available nitrogen,which plays an important role in the sustainable development of agriculture.The results of previous studies showed that soybean isoflavones were secreted into the rhizosphere soil at the beginning of the establishment of the symbiotic system,which induced the attachment of rhizobia in the root and then symbiosis.Soybean isoflavones are synthesized by phenylpropane metabolism pathway.Type II chalcone isomerase(CHIs)is an important rate-limiting enzyme for its synthesis,including GmCHI1 A,GmCHI1B1,and GmCHI1B2,but the function of these genes for the establishment of soybean root nodule symbiosis system is not clear.This study identified major functional GmCHI1 A,GmCHI1B1 and GmCHI1B2 genes in the soybean-rhizobia symbiotic system based on the soybean hair rooting technology system.The results are as follows:1.The gene GmCHI1 A,GmCHI1B1 and GmCHI1B2 of type II soybean chalcone isomerase were cloned by RT-PCR using the cDNA of soybean Willams82 as template.Bioinformatics analysis showed that the GmCHI1 A,GmCHI1B1 and GmCHI1B2 is most recently related and belongs to the second subfamily.The subcellular localization predicted that GmCHI1 A,GmCHI1B1 and GmCHI1B2 were located in the nucleus.Amino acid sequence comparison analysis found that three genes retains all catalytic active site residues of CHI folding.2.The expression of GmCHI1 A,GmCHI1B1 and GmCHI1B2 gene in soybean tissues was detected by real-time quantitative RT-PCR,and the results showed that there were significant differences in the expression patterns of GmCHI1 A,GmCHI1B1,and GmCHI1B2.The expression of GmCHI1 A was highest in the root and the expression of GmCHI1B1 was highest in the embryo,and the highest expression of GmCHI1B2 was in flowers.3.Composite plants with overexpression expression of GmCHI1 A,GmCHI1B1 and GmCHI1B2 genes were obtained using soybean hair rooting technology system.The phenotype traits of compound plants such as nodule and hair root were investigated after 30 d of rhizobia infection treatment.The results showed that the number of nodule,the fresh weight of the nodule and the volume of the nodule was significantly increased in the roots of the overexpressed GmCHI1 A composite plants.At the same time,the content of chlorophyll,the content of nitrogen in the leaves,the length,the weight and the number of hairy roots also increased significantly.Overexpression of GmCHI1B1 composite plants was only significantly increased in nodule weight,nodule volume and the length,the weight,and the number of hairy roots.Overexpression of GmCHI1B2 composite plants did not significantly effect on all phenotypes.The content of isoflavones in the hairy roots of overexpression GmCHI1 A and GmCHI1B1 was determined.The results showed that the content of daidzein,glycitein,daidzein,genistein and total isoflavones was significantly increased in the roots of the overexpressed GmCHI1 A.The content of glycitein was significantly increased,while the content of other isoflavones did not significantly increase in the hairy roots of the overexpressed GmCHI1B1.4.The plant interference expression vector pFGC-GmCHI1 A was constructed to obtain the complex plants with low GmCHI1 A expression.The statistical analysis of root nodule and hair root phenotype data showed that,the number,weight and volume of nodule and the weight,number of hair roots of compound plants were significantly reduced,and other phenotypes were reduced,but the difference was not significant.The content of isoflavones was measured,and the results showed that the content of glycitein in hair roots was significantly reduced,while the content of daidzein,genistein,and total isoflavones decreased,but not significantly.5.Quantitative results showed that GmCHI1 A gene positively regulated the expression of enzyme genes GmCHS8,GmIFS1,GmHID in the isoflavone synthesis pathway,and the expression of related nodulation pathway genes ENOD40-1,GmNF1?,GmNF5?,pre-miR172 C,GmNSP1,and GmNSP2.In summary,it is speculated that among the type II soybean chalcone isomerase genes,GmCHI1 A is major functional gene for isoflavone synthesis in soybean roots and in the establishment of a soybean-rhizobia symbiosis system.The results of this study can provide references for the molecular mechanism of soybean nitrogen,and can also provide genetic resources for soybean varieties breeding with high nodulation ability.