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Aug 10, 2016 - ABSTRACT: Mungbean (Vigna radiata (L.) Wilczek) is an important rotation legume crop for human nutrition in Asia. Bruchids (Callosobruchus ...
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Transcriptomic and Proteomic Research To Explore BruchidResistant Genes in Mungbean Isogenic Lines Wu-Jui Lin,†,§,∥ Chia-Yun Ko,§,∥ Mao-Sen Liu,§ Chien-Yen Kuo,‡ Dung-Chi Wu,‡ Chien-Yu Chen,‡ Roland Schafleitner,Δ Long-Fang O. Chen,§ and Hsiao-Feng Lo*,† †

Department of Horticulture and Landscape Architecture, National Taiwan University, Taipei 10617, Taiwan Institute of Plant and Microbial Biology, Academia Sinica, Taipei 11529, Taiwan ‡ Department of Bio-industrial Mechatronics Engineering, National Taiwan University, Taipei 10617, Taiwan Δ AVRDC−World Vegetable Center, Shanhua, Tainan 74151, Taiwan §

S Supporting Information *

ABSTRACT: Mungbean (Vigna radiata (L.) Wilczek) is an important rotation legume crop for human nutrition in Asia. Bruchids (Callosobruchus spp.) currently cause heavy damage as pests of grain legumes during storage. We used omics-related technologies to study the mechanisms of bruchid resistance in seeds of the nearly isogenic lines VC1973A (bruchid-susceptible) and VC6089A (bruchid-resistant). A total of 399 differentially expressed genes (DEGs) were identified between the two lines by transcriptome sequencing. Among these DEGs, 251 exhibited high expression levels and 148 expressed low expression levels in seeds of VC6089A. Forty-five differential proteins (DPs) were identified by isobaric tags for relative and absolute quantification (iTRAQ); 21 DPs had higher abundances in VC6089A, and 24 DPs had higher abundances in VC1973A. According to transcriptome and proteome data, only three DEGs/DPs, including resistant-specific protein (g39185), gag/pol polyprotein (g34458), and aspartic proteinase (g5551), were identified and located on chromosomes 5, 1, and 7, respectively. Both g39185 and g34458 genes encode a protein containing a BURP domain. In previous research on bruchid molecular markers, the g39185 gene located close to the molecular markers of major bruchid-resistant locus may be a bruchid-resistant gene. KEYWORDS: mungbean, bruchid, transcriptome, proteome, iTRAQ



INTRODUCTION Mungbean, Vigna radiata (L.) Wilczek, is an important rotation leguminous crop in South and Southeast Asia.1−4 For human nutrition, mungbean seeds constitute valuable protein and carbohydrate sources, and sprouts are important vitamin and mineral sources.2 However, two bruchids, the azuki bean weevil (Callosobruchus chinensis) and the cowpea weevil (Callosobruchus maculatus), attack mungbean and cause severe losses during storage. To overcome this challenge, bruchid resistance has constituted an important breeding goal for mungbean.5 The wild mungbean accession TC1966 (V. radiata var. sublobata), which is completely resistant to C. chinensis, C. maculatus, C. phaseoli, and Z. subfasciatus, was used for bruchidresistance breeding.6 In the first report on bruchid resistance, a wild mungbean was controlled with bruchid resistance by a single dominant locus.7 On the basis of segregation populations of TC1966 and a mungbean cultivar (NM92) with different levels of bruchid resistance and susceptibility, Chen et al. first reported that the bruchid-resistant genes (Br genes) were controlled by one major locus and two minor loci in wild mungbean.5,8 The major locus was tightly linked with molecular markers W02a4 through analysis by random amplified polymorphic DNA (RAPD), and DMB-SSR 158 was mapped on linkage group 9 by simple sequence repeat (SSR).5,8 The two minor loci that were found to be tightly linked with molecular markers mg7pgc325 and ma3pat361 through analysis by amplified fragment length polymorphism (AFLP) were located at 114 and 132 cM on linkage group 7, respectively.8 © 2016 American Chemical Society

Resistance mechanisms of plants against insect pests could be associated with antixenosis and antibiosis.9 Hence, bruchid resistance in legumes relies on antinutritional compounds and secondary metabolites that are toxic to bruchids.10 A 4-week feeding study, comparing a commercial mungbean with a bruchid-resistant isogenic line, showed no negative effects on growth or any pathological effects on mice.11 Mungbean seeds contain 58.2−61.8% carbohydrates, 22.9−23.6% protein, and 1.2% oil.12 Consequently, mungbean seeds can provide the major nutrients, starch and protein, for bruchid larvae. The specific alleles of the arcelin−phytohemeagglutinin−α-amylase (APA) locus from wild bean (Phaseolus vugaris) provide some resistance to bruchids.13 An arcelin of common bean is a lectin-like protein bound to carbohydrates in the intestinal epithelium of insects and causes an alteration of insect gut structure.14,15 The VrD1/ VrCRP protein detected in the mungbean seed coat inhibited the development of C. maculatus to adults in artificial seeds.16−18 VrD1 protein is a specific α-amylase inhibitor that inhibited αamylase of insects, but not of animals.16,19 Moreover, although two novel cyclopeptide alkaloids, vignatic acids A and B, were isolated from mungbean,20 they were not the principal factors responsible for the bruchid resistance.21 Thus, the mechanisms of bruchid resistance in TC1966 are still not clearly understood. Received: July 5, 2016 Accepted: August 10, 2016 Published: August 10, 2016 6648

DOI: 10.1021/acs.jafc.6b03015 J. Agric. Food Chem. 2016, 64, 6648−6658

Article

Journal of Agricultural and Food Chemistry

19052484 reads, 2.88 Gb; 1973A-seed-2, 17591440 reads, 2.66 Gb; 6089A-seed-1, 24883022 reads, 2.76 Gb; 6089A-seed-2, 19099398 reads, 2.88Gb) of all samples were trimmed for low-quality bases and then individually aligned to the set of annotated transcripts using BWA MEM.27 For each data set, a quantification of transcript expression was performed by using eXpress28 to calculate the transcripts per million (TPM) for each transcript. Computing Differentially Expressed Genes. DESeq was used for differential expression analysis by calculating the total read counts of a gene in each sample. A transcript was denoted as differential expression genes (DEGs) if Padj > 0.1, P > 0.0529, and the fold change (FC) (resistant/susceptible) was >2 or