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Multimeric Association of Purified Novel Bowman-Birk Inhibitor From the Medicinal Forage Legume Mucuna pruriens (L.) DC.

ABSTRACT: A Bowman-Birk protease, i.e., Mucuna pruriens trypsin inhibitor (MPTI), was purified from the seeds by 55.702-fold and revealed a single trypsin inhibitor on a zymogram with a specific activity of 202.31 TIU/mg of protein. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under non-reducing conditions, the protease trypsin inhibitor fraction [i.e., trypsin inhibitor non-reducing (TINR)] exhibited molecular weights of 74 and 37 kDa, and under reducing conditions [i.e., trypsin inhibitor reducing (TIR)], 37 and 18 kDa. TINR-37 revealed protease inhibitor activity on native PAGE and 37 and 18 kDa protein bands on SDS-PAGE. TINR-74 showed peaks corresponding to 18.695, 37.39, 56.085, and 74.78 kDa on ultra-performance liquid chromatography (UPLC) coupled with electrospray ionization/quadrupole time-of-flight-mass spectrometry (ESI/QTOF-MS). Similarly, TINR-37 displayed 18.695 and 37.39 kDa peaks. Furthermore, TIR-37 and TIR-18 exhibited peaks corresponding to 37.39 and 18.695 kDa. Multiple peaks observed by the UPLC-ESI/QTOF analysis revealed the multimeric association, confirming the characteristic and functional features of Bowman-Birk inhibitors (BBIs). The multimeric association helps to achieve more stability, thus enhancing their functional efficiency. MPTI was found to be a competitive inhibitor which again suggested that it belongs to the BBI family of inhibitors, displayed an inhibitor constant of 1.3 × 10-6 M, and further demonstrates potent anti-inflammatory activity. The study provided a comprehensive basis for the identification of multimeric associates and their therapeutic potential, which could elaborate the stability and functional efficiency of the MPTI in the native state from M. pruriens.

SUBMITTER: Lone JK

PROVIDER: S-EPMC8655843 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundThe tropical legume Mucuna pruriens (L.) DC. can meet three agricultural needs: low-cost protein, high-value medicines, and green manure or cover crops. But like other underutilized crops, it needs more modern breeding resources. Identifying marker-trait associations (MTAs) can facilitate marker-assisted breeding and crop improvement. Recent studies have demonstrated the feasibility of identifying MTAs using a small number of accessions (< 100). We have characterized a panel of 70 M. pruriens accessions across two consecutive years and performed association analysis for 16 phenotypic traits related to seed (seed length, seed width, seed thickness, seed yield per plant, hundred seed weight); pod (pod length, pod width, number of pods per cluster, number of pods per plant); inflorescence (inflorescence length, flower buds per inflorescence, flower length, pedicel length), and biochemical attributes (L-DOPA, total protein, total carbohydrate), using 66 genic-microsatellite markers following mixed linear model.ResultsThe results showed significant phenotypic (P < 0.05) and genetic diversity (Shannon's information index, I = 0.62) in our germplasm collection. Many tested traits were highly heritable (broad-sense heritability ranging from 42.86 to 99.93%). A total of 15 MTAs was detected at an adjusted significance level of P < 5.55 × 10- 3 for nine traits (seed length, seed thickness, seed width, hundred seed weight, seed yield per plant, inflorescence length, flower buds per inflorescence, flower length, and petiole length), contributed by 10 SSR markers (MPU_19, MPU_42, MPU_54, MPU_57, MPU_58, MPU_83, MPU_89, MPU_108, MPU_111, and MPU_122.) with phenotypic variance explained (PVE) ranging from 14.7 to 31.1%. Out of the ten trait-associated markers, the BLAST analysis revealed putative functions of seven markers, except MPU_57, MPU_58, and MPU_83.ConclusionFifteen MTAs identified for important traits with phenotypic variance explained > 10% from mixed linear model offer a solid resource base for improving this crop. This is the first report on association mapping in M. pruriens and our results are expected to assist with marker-assisted breeding and identifying candidate genes in this promising legume.

Project description:BackgroundThe medicinal legume Mucuna pruriens (L.) DC. has attracted attention worldwide as a source of the anti-Parkinson's drug L-Dopa. It is also a popular green manure cover crop that offers many agronomic benefits including high protein content, nitrogen fixation and soil nutrients. The plant currently lacks genomic resources and there is limited knowledge on gene expression, metabolic pathways, and genetics of secondary metabolite production. Here, we present transcriptomic resources for M. pruriens, including a de novo transcriptome assembly and annotation, as well as differential transcript expression analyses between root, leaf, and pod tissues. We also develop microsatellite markers and analyze genetic diversity and population structure within a set of Indian germplasm accessions.ResultsOne-hundred ninety-one million two hundred thirty-three thousand two hundred forty-two bp cleaned reads were assembled into 67,561 transcripts with mean length of 626 bp and N50 of 987 bp. Assembled sequences were annotated using BLASTX against public databases with over 80% of transcripts annotated. We identified 7,493 simple sequence repeat (SSR) motifs, including 787 polymorphic repeats between the parents of a mapping population. 134 SSRs from expressed sequenced tags (ESTs) were screened against 23?M. pruriens accessions from India, with 52 EST-SSRs retained after quality control. Population structure analysis using a Bayesian framework implemented in fastSTRUCTURE showed nearly similar groupings as with distance-based (neighbor-joining) and principal component analyses, with most of the accessions clustering per geographical origins. Pair-wise comparison of transcript expression in leaves, roots and pods identified 4,387 differentially expressed transcripts with the highest number occurring between roots and leaves. Differentially expressed transcripts were enriched with transcription factors and transcripts annotated as belonging to secondary metabolite pathways.ConclusionsThe M. pruriens transcriptomic resources generated in this study provide foundational resources for gene discovery and development of molecular markers. Polymorphic SSRs identified can be used for genetic diversity, marker-trait analyses, and development of functional markers for crop improvement. The results of differential expression studies can be used to investigate genes involved in L-Dopa synthesis and other key metabolic pathways in M. pruriens.

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Multimeric Association of Purified Novel Bowman-Birk Inhibitor From the Medicinal Forage Legume Mucuna pruriens (L.) DC.

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