| As an important economic crop,an enhanced production of legumes is necessary to provide sufficient food and renewable energy for the increasing human population,as well as basic compounds for industrial processes.Legumes often interact with other types of living organisms.The most common are pathogenic organisms and symbiotic microbes.Because of great impact on legume production,more and more studies are focusing on the interaction between legumes and parasitic or symbiotic organisms.Previously,a variety of legumes are used for the studying plant disease resistance.Taken soybean as example,the most common soybean diseases include soybean cyst nematode,soybean mosaic virus,soybean rust and so on.Among them,Soybean cyst nematode(SCN)is a kind of strict endoparasitic pest.As a SCN-resistant gene,Rhg4 has been identified on Chr.8,which encodes a serine hydroxymethyltransferase.In other green plants,a high number of SHMT genes have been indentified.Studies on these SHMT proteins,on one hand,confirmed their cellular locations;on the other hand,also revealed their intriguing roles of SHMTs in influencing plant defense abilities.Therefore,in order to understand why plants evolved multiple SHMT genes and elucidate the relationship between them,we further analyzed the phylogeny of these SHMT genes and combined with the sequence analysis of both wild soybean and cultivated soybean to understand the mechanism of SCN resistance.Additionally,model legume M.trunctula was uesd for investgating the root nodule symbiosis because of its smaller genome and rich genetic variation in present research.We mainly studied the roles of genes invloved in the bacterial release from infection thread and cargo-exchange between plants and symbionts.The main results were as follows:1.Soybean cyst nematode resistance emerged via artificial selection of duplicated serine hydroxymethyltransferase genesThe present study conducted sequence analysis of 117 SHMT genes of 18 representative plants and reconstructed its phylogeny.Two groups and four subgroups of the SHMT gene family were identified.In subgroup ?a,rhg4 and its homolog rhg4h,were detected,which were derived from a whole genome duplication event in the Glycine lineage.The two duplicated genes underwent differential degrees of functional constraint in the soybean populations,with the rhg4 gene accumulating more non-synonymous substitutions.The SCN-resistant allele,Rhg4,was not detected in wild soybeans;Instead,a very similar allele was observed in wild soybeans at a frequency of 7.4%,although it lacked the two critical non-synonymous substitutions.Taken together,these findings support that the SCN-resistant Rhg4 allele likely emerged via artificial selection during the soybean domestication process,based on a SCN-sensitive allele inherited from wild soybeans.2.Genetic mapping of retrotransposon insertional mutants in the model legume Medicago truncatulaThe Tnt1 mutant population displays a variety of phenotypes.Tnt1 is a tobacco retrotransposon adapted for establishing insertional mutation lines of Medicago truncatula.Although a powerful resource for functional genomics,the large number of insertion sites often present in a line sometimes pose a challenge for determining the causal insertion of a particular phenotype.In order to accelerate forward genetic screening using the Tnt1 mutant collection,additional genetic tools are needed.In this study,we established a mapping population between the Tnt1 background ecotype R108 and A17,the type accession of M.truncatula with a well-assembled genome sequence.By comparing the two genomes for homologous sequences and two types of polymorphisms(SNPs and indels)we designed a set of CAPS markers and a large collection of SSR markers.Our collection of CAPS and SSR markers can be of use to researchers interested in cloning mutants that reveal multiple aspects of legume biology.It could also be used to identify the genetic basis of natural variations between M.truncatula accessions.Using these resources,we were able to map the causal mutation in a Tnt1 line with a nodulation defect.The genetic tools we describe here provide valuable additions to the toolkit of M.truncatula genetics.3.A symbiotic SYP132 protein produced by alternative terminationThe key to establish symbiosis is how legumes effectively communicate with rhizobia,that is to say,transporting secretory protein or other important cargo from plant to bacteria,through vesicle trafficking,which requires syntaxin proteins.Previous studies revealed that during symbiosis,a syntaxin protein,named SYP132,was co-expressed with DNF1(Defective in Nitrogen Fixation 1),which encoded a nodule-specific signal peptidase,so it was hypothesized that SYP132 plays an important role in root nodule symbiosis.In our study,we found that the MtSYP132 could produce two transcripts,SYP132A and SYP132C,through alternative cleavage and APA.Of which,SYP132A is located on symbiosome membrane and involved in the establishment of symbiosis.In order to study how the SYP132 alternative cleavage and APA mechanism evolved in plants,we systematically analyzed the SYP13 gene of 37 representative plants and found that the SYP13 gene in plants mainly consists of two groups,namely,Group ? and Group ?,while the Group ? is further divided into Group ?a and Group ?b.Group ? contains SYP132 of both monocots and dicots plants,while alternative splicing and APA only took place in dicots in this clade.Through the sequence analysis and cellular sub-localization experiments,we found that the first three amino acids of TM domain of SYP132 in M truncatula accounts for the differences in their localization and function. |
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