Gene Mapping Of Two Disease-like Mutants And Functioninal Study Of GmHPL In Soybean

In nature,plants are inevitably subjected to a variety of abiotic and biotic stresses,and plants will be damaged even to death.Among them,pathogens can seriously harm the growth,development,yield and quality of plants.Disease-like mutant is an effective tool to study the mechanism of plant hypersensitive response,which is of great significance to reveal the mechanism of plant disease resistance.NT302 was derived from Sudou-1 and NT301 was derived from Yuyu through physical and chemical mutagenesis and screening.The NT302 mutant was mainly studied for its disease-like phenotype,chloroplast development,photosynthetic physiological characteristics,seed traits and active oxygen accumulation.Furthermore,the crosses were built with NT302 and wild type for genetic analysis,gene mapping and cloning candidate genes.The candidate genes were verified by cotton VIGS(Virus-Induced Gene Silencing).Biotic resistance identification were carried out using Bacterial Leaf Pustule pathogens and Common Cutworm.What's more,transcriptome analysis was also performed.At the same time,we carried out phenotype identification,observation of transverse section of leaf structure,genetic analysis,gene mapping and candidate genes analysis of mutant NT301.The main results are as follows:1.The chloroplast degenerated and photosynthesis activity was decreased in NT302 mutant,at the same time,the 100-seed weight of NT302 decreased and the content of ROS increased.In the vegetative growth stage,the leaves of NT302 were normal,but brown lesion spots appeared on the chlorotic leaves in the reproductive growth stage in absence of pathogens and continuously expanded to the whole plant until the end of the whole growth period.The ultrastructures of chloroplast were observed by transmission electron microscopy in different stages.It was found that the chloroplast structure of NT302 was normal,with complete thylakoid membrane,starch granules and osmophilic granules.In the reproductive growth stage,the chloroplast membrane system2.gradually degenerated and disappeared,leaving only a few starch granules with larger and irregular osmophilic granules,indicating that the chloroplast structure was seriously destructed.The photosynthetic pigment content of mutant was normal in the vegetative growth stage but decreased in the reproductive growth stage compared with wild type.The analysis of photosynthetic activity showed that net photosynthetic rate,transpiration rate and stomatal conductance decreased,but intercellular carbon dioxide concentration increased in the mutant.The length,width and thickness of mutant seeds significantly decreased by 34%,35%and 27%respectively,the 100-seed weight and the oil content decreased by 54%and 6.5%,while the protein content of increased by 9.5%,with the ratio of oil to protein decreasing by 24.6%.DAB staining was carried out at different stages.It was found that red brown precipitates increased with the number and size of brown lesion spots increasing,indicating that H2O2 excessively accumulated.3.The target gene of the disease-like mutant NT302 encodes hydroperoxide lyase(GmHPL),which belongs to CYP74B subfamily.The genetic analysis of three crosses showed that NT302 disease-like phenotype was controlled by a pair of recessive nuclear genes.Two F2 populations were used to map the target gene to a 220kb region on chromosome 12 with 23 candidate genes.Sequencing showed that there was a single base substitution(CAG-TAG)of 61st nucleotide in the coding region of Glyma.12g191400,which lead to a premature stop codon and resulted in a truncated protein with only 20 amino acids.The same mutation occurred in the mutants of F2 population.According to the annotation,Glyma.12g191400 gene encodes the hydroperoxide lyase of cytochrome P450 super family,which was named as GmHPL.The cotton orthologous gene silenced(VIGS)plants had similar phenotype as NT302,which showed brown lesion spots,decreased chlorophyll content and excessive H2O2 accumulation.According to amino acid sequence alignment,GmHPL was divided into CYP74B subfamily by phylogenetic tree analysis,cleaving only 13-hydroperoxides.It was found that the GmHPL enzyme was located on the chloroplast membrane through the GmHPL-GFP fusion protein expressing in tobacco epidermal cells.The results of expression level in different tissues showed that GmHPL gene was highly expressed in leaves and pods.The expression level of GmHPL gene in the mutant was significantly lower than that in wild type.The content of lipid peroxide in the top three leaves of mutant increased significantly,indicating that the deficiency of HPL in the mutant resulted in the accumulation of lipoxygenase metabolites,which led to the significant increase of total lipid peroxide content in the leaves,thus inducing the occurrence of disease-like necrosis of the leaves.3.GmHPL is involved in LOX pathway and competes with AOS branch,playing an important role in defense response.The expression level of JA(Jasmonic acid)biosynthesis genes like GmLOX1,GmAOS and GmOPR3 in NT302 was significantly higher than those in wild type.In HPL gene silenced cotton plants,JA biosynthesis genes like GhLOX1,GhLOX3,GhAOS,GhAOC1 and responce gene like GhJAmyb were also significantly triggerd.Accordingly,the JA content of mutant NT302 was higher than that of wild type with development of soybean,while the SA(Salicylic acid)content showed no significant difference between wild type and mutant.The above results showed that HPL and AOS compete to catalyze the common substrates,thus affecting JA biosynthesis.The GmHPL promoter contains the stress motif wbox(TTGAC)and ABA(Abscisic acid)response elements(ABRES;ACGTG),which were at 386bp and 260bp upstream of the promoter,respectively.Transcriptome analysis showed that plant hormone signaling pathway like JA pathway,ABA pathway,AUX(auxin)pathway and BR(brassinolide)pathway co-expressed with fatty acid metabolism pathway in NT302,and calcium signaling pathway,resistance protein PTI1(pto-interacting protein 1)and RPM1 were activated in plant and pathogen interaction pathway.qRT-PCR showed that GmHPL was induced by mechanical injury,MeJA and Common Cutworm.The mutant NT302 increased the resistance to the masticatory insect Common Cutworm and decreased the resistance to Bacterial Leaf Pustule.These results showed that NT302 was involved in abiotic and biotic stress of soybean.4.rll and rl2 are new genes controlling the character of disease-like rugose leaf in soybean.The first trifoliolate of mutant NT301 exhibited the rugose leaf phenotype,like infected by Soybean mosaic virus.It was a new mutation treated by 60Co ray irradiation,which was different from the phenotype of soybean disease-like mutants previously reported.The genetic analysis of F2 and F2:3 families in two crosses showed that NT301 disease-like phenotype was controlled by two pairs of recessive nuclear genes,named rl1 and rl2.rl1 was mapped to a 1.86Mb interval on chromosome 18,with BARCSOYSSR₁8₀419 and BARCSOYSSR₁8₀485 as flanking markers,containing 139 candidate genes.rl2 was mapped to a 130kb interval on chromosome 8,between SNP2(44937110)and SNP3(45064149),containing 15 candidate genes.In order to identify candidate genes of rl2,semi quantitative and quantitative RT-PCR analysis were carried out.The results showed that the expression level of Glyma.08g332500 of the mutant was four times as much as that of the wild type,the difference of which was the most significant among the candidate genes.Therefore,Glyma.08g332500 is the most likely candidate gene of rl2.