Transcriptomic and Proteomic Research To Explore Bruchid-Resistant

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Transcriptomic and proteomic research to explore bruchid-resistant genes in mungbean isogenic lines Wu-Jui Lin, Chia-Yun Ko, Mao-Sen Liu, Chien-Yen Kuo, Dung-Chi Wu, ChienYu Chen, Roland Schafleitner, Long-Fang Oliver Chen, and Hsiao-Feng Lo J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b03015 • Publication Date (Web): 10 Aug 2016 Downloaded from http://pubs.acs.org on August 16, 2016

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Journal of Agricultural and Food Chemistry

Transcriptomic and proteomic research to explore bruchid-resistant 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,|| 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 ||

AVRDC- World Vegetable Center, Shanhua, Tainan, 74151, Taiwan

#These authors contributed equally to this work *CORRESPONDING AUTHORS

Dr. Hsiao-Feng Lo Department of Horticulture and Landscape Architecture, National Taiwan University, Taipei, 10617, Taiwan (Tel: +886-2-3366-4871; Email: [email protected])

1

ACS Paragon Plus Environment


1

ABSTRACT

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Mungbean (Vigna radiata (L.) Wilczek) is an important rotation legume crop for

3

human nutrition in Asia. Bruchids (Callosobruchus spp.) currently cause heavy damage as

4

pests of grain legumes during storage. We used omics-related technologies to study the

5

mechanisms of bruchid-resistance in seeds of the nearly isogenic lines VC1973A

6

(bruchid-susceptible) and VC6089A (bruchid-resistant). 399 differentially expressed genes

7

(DEGs) were identified between the two lines by transcriptome sequencing. Among these

8

DEGs, 251 DEGs exhibited high expression levels and 148 DEGs expressed low expression

9

levels in seeds of VC6089A. 45 differential proteins (DPs) were identified by isobaric tags

10

for relative and absolute quantification (iTRAQ). The 21 DPs had higher abundances in

11

VC6089A, and 24 DPs had higher abundances in VC1973A. According to transcriptome

12

and proteome data, only three DEGs/DPs, including resistant-specific protein (g39185),

13

gag/pol polyprotein (g34458), and aspartic proteinase (g5551) were identified and located

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on chromosome 5, 1 and 7, respectively. Both g39185 and g34458 genes encode a protein

15

containing a BURP domain. In previous research on bruchid molecular markers, the g39185

16

gene located closely to the molecular markers of major bruchid-resistant locus may be a

17

bruchid-resistant gene.

18 19

KEYWORDS: mungbean, bruchid, transcriptome, proteome, iTRAQ 2


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INTRODUCTION

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Mungbean, Vigna radiata (L.) Wilczek, is an important rotation leguminous crop in

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south and southeast Asia.1-4 For human nutrition, mungbean seeds constitute valuable protein

23

and carbohydrate sources, and sprouts are important vitamin and mineral sources.2 However,

24

two bruchids, the azuki bean weevil (Callosobruchus chinensis) and cowpea weevil (C.

25

maculatus), attack mungbean and cause severe losses during storage. To overcome this

26

challenge, bruchid-resistance has constituted an important breeding goal of mungbean.5 The wild mungbean accession TC1966 (Vigna radiata var. sublobata), which is

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completely resistant to C. chinensis, C. maculates, C. phaseoli and Z. subfasciatus, was used

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for bruchid-resistance breeding.6 In the first report on bruchid-resistance, a wild mungbean

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was controlled with bruchid-resistance by a single dominant locus.7 Based on segregation

31

populations of TC1966 and a mungbean cultivar (NM92) with different levels of

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bruchid-resistance and -susceptibility, Chen et al. first reported that the bruchid-resistant

33

genes (Br genes) were controlled by one major locus and two minor loci in wild mungbean.5,

34

8

35

random amplified polymorphic DNA (RAPD), and DMB-SSR 158 was mapped on linkage

36

group 9 by simple sequence repeat (SSR).5, 8 The two minor loci that were found to be tightly

37

linked with molecular markers mg7pgc325 and ma3pat361 through analysis by amplified

38

fragment length polymorphism (AFLP) were located at 114 and 132 cM on linkage group 7,

The major locus was tightly linked with molecular markers W02a4 through analysis by

3



39 40

respectively.8 Resistance mechanisms of plants against insect pests could be associated with antixenosis

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and antibiosis.9 Hence, bruchid-resistance in legumes relies on anti-nutritional compounds

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and secondary metabolites which are toxic to bruchids.10 A 4-week feeding study, comparing

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a commercial mungbean with a bruchid-resistant isogenic line, showed no negative effects on

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growth or any pathological effects on mice.11 Mungbean seeds contain 58.2~61.8%

45

carbohydrates, 22.9~23.6% protein, and 1.2% oil.12 Consequently, mungbean seeds can

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provide the major nutrients, starch and protein, for bruchid larvae. The specific alleles of the

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arcelin-phytohaemagglutinin-α-amylase (APA) locus from wild bean (Pbaseolus vugaris)

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provide some resistance to bruchids.13 An arcelin of common bean is a lectin-like protein

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bound to carbohydrates in the intestinal epithelium of insects and causes an alteration of

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insect gut structure.14, 15 The VrD1/VrCRP protein detected in the mungbean seed coat

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inhibited the development of C. maculates to adults in artificial seeds.16-18 VrD1 protein is a

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specific α-amylase inhibitor that inhibited α-amylase of insects, but not of animals.16, 19

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Moreover, although two novel cyclopeptide alkaloids, vignatic acid A and B, were isolated

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from mungbean,20 they were not the principal factors responsible for the bruchid-resistance.21

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Thus, the mechanisms of bruchid-resistance in TC1966 are still not clearly understood.

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The mungbean genome size (2n = 22 chromosomes) was estimated as 494-579 Mb by flow cytometry.22, 23 Recent mungbean genome-sequencing and gene annotation of 4


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bruchid-resistant mungbean, RIL59,22 and bruchid-susceptible mungbean, VC1973A,24

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provide an excellent foundation for omic-related analyses. To elucidate the mechanisms of

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bruchid-resistance, we used Illumina RNA-seq technology and the iTRAQ method to

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investigate the differentially expressed transcripts and proteins in two isogenic mungbean

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lines, VC6089A and VC1973A. By combining these omic-related technologies, our results

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reveal that three Br candidate genes/proteins are involved in bruchid-resistance mechanisms.

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The results will be applicable in mungbean breeding and in insect-resistance research of other

65

legume crops.

66 67

MATERIALS AND METHODS

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Plant Materials

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Mungbean bruchid-susceptible line (VC1973A, NM92) and bruchid-resistant line

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(VC6089A, TC1966, and recombinant inbred line 59 (RIL59)) were obtained from the World

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Vegetable Center - Asian Vegetable Research and Development Center (AVRDC). VC6089A

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is a nearly isogenic line (BC6F2) of VC1973A. It was derived from cross of VC1973A and a

73

wild bruchid-resistant mungbean TC1966 (Vigna radiate var. sublobata), and then

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bruchid-resistant hybrids were back-crossed for six times to VC1973A. The genome of

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VC6089A is only 1% different from VC1973A.17, 25 The contents of protein and soluble sugar

76

are shown in Table S1. RIL59, one of 200 F12 RIL, was generated from an inter-subspecific 5



77

cross between bruchid-susceptible mungbean variety NM92 and a bruchid-resistant accession

78

TC1966 (Vigna radiata var. sublobata).5,8,22 The bruchid-resistance analysis with 40 seeds

79

was performed following the Chen et al. method.5 Mungbean seeds with 0% damage were

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defined as bruchid-resistant and those with more than 80% damage as bruchid-susceptible.

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RNA Extraction

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Total RNA was extracted following the Pine Tree method26 with slight modification.

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Seeds were ground into powder with a mortar in a pestle filled with liquid nitrogen. The

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extraction buffer containing 2％ CTAB, 2％ PVP K30, 100 mM Tris-HCl pH 8.0, 25 mM

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EDTA, 2.0 M NaCl, 0.5 gL-1 spermidine, and 2％ beta-mercaptoethanol (added prior to use)

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was pre-heated at 65 °C in a water bath. One gram of seed powder was extracted with a 10

87

mL extraction buffer by vortexing in a 50 mL Falcon tube. Then, an equal volume of

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chloroform : isoamyl alcohol (24：1) was added and mixed well by vortexing. After

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centrifuging under 12 000 g at room temperature for 10 min, the supernatant was transferred

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to a new 50 mL tube. A half volume of phenol and 1 volume of chloroform：isoamyl alcohol

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(24：1) were added to the supernatant and mixed by vortexing. After centrifugation, the

92

supernatant was transferred to a new tube and mixed with 1/3 volume of 8 M LiCl. RNA was

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precipitated at 4 °C overnight. RNA was harvested by centrifuging under 12 000 g at 4 °C for

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30 min. This was followed by discarding the supernatant, air-drying for 5 to 10 min, and then

95

dissolution in 200 µL DEPC-H2O. Contaminated DNA was removed by the TURBO 6


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DNA-free kit (Ambion) following the manufacturer’s instructions. The DNA-free RNA

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sample was precipitated by adding 1/10 volume of sodium acetate, 2 volume of 100％

98

ethanol, and 15 µg/mL linear acrylamide (Ambion) at -70 °C overnight. After centrifugation

99

and discarding the supernatant, RNA was pelleted by 1 mL 75％ ethanol, air-dried for a few

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minutes, and then dissolved in 32 µL DEPC-H2O. The RNA quality was confirmed by a

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Bioanalyzer RNA 6000 NanoChip (Agilent Technologies, Santa Clara, CA, U.S.A.) coupled

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with an Agilent 2100 Bioanalyzer (Agilent Technologies) at the DNA Microarray Core

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Laboratory, Institute of Plant and Microbial Biology (IPMB), Academia Sinica, Taiwan.

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RNA Sequencing

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For transcriptomic analysis, two biological repeats of total RNA from VC1973A and

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VC6089A were performed. Four paired-end RNA libraries were constructed for sequencing

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by the Illumina Hiseq 2000 platform. The RNA-seq data of VC1973A and VC6089A are

108

available on the NCBI Sequence Read Archive under accession of SRP070726. The RNA-seq

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reads (1973A-seed-1: 19052484 reads, 2.88 Gb; 1973A-seed-2: 17591440 reads, 2.66 Gb;

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6089A-seed-1: 24883022 reads, 2.76 Gb; 6089A-seed-2: 19099398 reads, 2.88Gb) of all

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samples were trimmed for low quality bases and then individually aligned to the set of

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annotated transcripts using BWA MEM.27 For each data set, a quantification of transcript

113

expression was performed by using eXpress28 to calculate the transcripts per million (TPM)

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for each transcript. 7



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Computing Differentially Expressed Genes

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DESeq was used for differential expression analysis by calculating the total read counts

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of a gene in each sample. A transcript was denoted as differential expression genes (DEGs) if

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Padj> 0.1, P> 0.0529 and the fold change (FC) (resistant/susceptible) was greater than 2 or less

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than 0.5, respectively. Otherwise, a transcript was denoted as non-differentially expressed.

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Protein Extraction

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Total protein was extracted by TRIzol Reagent (Ambion) following the manufacturer’s

122

instructions. Seeds were ground into powder with a mortar in a pestle filled with liquid

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nitrogen. One hundred milligrams of seed powder was extracted with 1 mL TRIzol Reagent

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and 0.2 mL chloroform. After centrifugation with 1 200 g at 4 °C for 5 min, the proteins in

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the organic phase were transferred to a new tube, 0.3 mL 100％ ethanol was added, and then

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centrifuged with 2 000 g at 4 °C for 5 min. The supernatant was precipitated with 1.5 ml

127

isopropanol. After centrifugation with 1 200 g at 4 °C for 10 min, the protein pellets were

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washed for three times with 2 mL 0.3 M guanidine hydrochloride in 95％ ethanol. The

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protein pellets were harvested, air-dried, and dissolved in100~200 µL 9 M urea for usage.

130

Protease Digestion

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Protein treatment, protease digestion, and labeling prior to LC-MS analysis were

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performed as described previously30 with minor modifications. Protein concentration was

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measured with a Pierce 660 nm Protein Assay Kit (Thermo Scientific). Proteins of 100 µg 8


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extracted from each sample were reduced in 10 mM dithiothreitol (DTT) for 1 h at 37 °C.

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Cysteines (Cys) were alkylated in 50 mM iodoacetamide at room temperature for 30 min in

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the dark. Protein solutions were then diluted to 4 M urea with 50 mM Tris-Cl pH 8.5 and

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digested with 250 units/ml benzonase (Sigma-Aldrich) at room temperature for 2 h, followed

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by Lys-C (Wako, Japan) digestion [1:200 (w/w)] at room temperature for 4 h. Protein

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solutions were further diluted to < 2 M urea with 50 mM Tris-Cl pH 8.0, and incubated with

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2 µg of modified trypsin (w/w, 1:50, Promega) at 37 °C overnight. These protease-digested

141

solutions were acidified with 10% trifluoroacetic acid, desalted using an Oasis HLB cartridge

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(Waters, U.S.A.), and then dried with SpeedVac.

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Peptide Labeling with Isobaric Tags and SCX Fractionation

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The dissolution of dried peptides in dissolution buffer and labeling with iTRAQ reagents

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were performed according to the manufacturer’s instructions (Applied Biosystems). Digested

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peptides from mungbean VC6089A were labeled with iTRAQ 113, and those from VC1973A

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were labeled with iTRAQ 117 reagents. The labeling reactions with iTRAQ reagents were

148

incubated for 1 h at room temperature. Following the reactions, solutions from all of the

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different iTRAQ labels were combined and further fractionated on a strong cation-exchange

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(SCX, PolySulfoethyl A, 4.6×200 mm, 5 µm, 200 Å, PolyLC) high-performance liquid

151

chromatography (HPLC). SCX chromatography was performed with initial equilibrium

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buffer A containing 10 mM KH2PO4, 25% acetonitrile (ACN), pH 2.65, and followed by a 9



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0~15% buffer B (1 M KCl in buffer B, pH 2.65) gradient for 20 min, 15~30% buffer B

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gradient for 10 min, 30~50% buffer B gradient for 5 min, 50~100% buffer B gradient for 1

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min, and 100% buffer B for 5 min. The flow rate was 1 mL/min. Chromatography was

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recorded with Abs at 214 nm UV light. Fractions (0.5 min/fraction) were collected, pooled,

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and separated into 25 final fractions. Samples were desalted using an Oasis HLB cartridge

158

(Waters, U.S.A.) prior to LC-MS/MS analysis.

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LC-MS/MS Analysis

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Pooled and desalted fractions were re-dissolved in 0.1% formic acid and analyzed using

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LC-MS/MS (a DionexUltiMate 3000 RSLCnano LC system coupled to a Q Exactive hybrid

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quadrupole-Orbitrap mass spectrometer equipped with a nanospray Flex ion source, Thermo

163

Scientific). A C18 capillary column (Acclaim PepMap RSLC, 75 µm × 250 mm, Thermo

164

Scientific) was utilized to separate peptides with a 120 min linear gradient from 3% to 30%

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solvent B (0.1% formic acid in acetonitrile, ACN) at a flow rate of 300 ml/min. The Q

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Exactive MS was operated in the data-dependent mode, with the top 10 ions (charge states ≥2)

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for MS/MS analysis following the MS survey scan for each acquisition cycle. The selected

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ions were isolated in the quadrupole, and subsequently activated using higher-energy

169

collisional dissociation (HCD) and analyzed in an Orbitrap cell. The dynamic exclusion

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duration of ion selection was 15 s. The MS was set as follows: m/z 350~1,600 range,

171

resolving power of 70,000, automatic gain control (AGC) target of 3×106, and maximum IT 10


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of 30 ms. MS/MS was set as follows: resolving power of 17,500, AGC target of 1×105, and

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maximum IT of 150 ms. HCD was set at a collision energy of 30% normalized collision

174

energy (NCE).

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Protein Identification and Quantification

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Peptide identification was performed using Proteome Discoverer software (v1.4,

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Thermo Fisher Scientific) with SEQUEST and Mascot (v2.5, Matrix Sciences) search

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engines. MS data were searched against the mungbean protein sequence database with 49,951

179

entries. Search conditions were set as full trypsin digestion, two maximum missed cleavage

180

sites, precursor mass tolerance of 10 ppm, fragment mass tolerance of 50 mmu, dynamic

181

modifications

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carbamidomethyl (C), and iTRAQ8plex (N-terminus and K). Peptide spectrum matches

183

(PSM) were validated using the target decoy PSM validator algorithm, which automatically

184

conducted a decoy database search, and rescored PSMs using q-values and posterior error

185

probabilities (PEP). All PSMs were filtered with a q-value threshold of 0.01 (1% false

186

discovery rate), and proteins were filtered with a minimum of two distinct peptides identified

187

per protein. For comparative protein quantification, the ratios (113/117) of iTRAQ reporter

188

ion intensities in the MS/MS spectra of PSMs were utilized to calculate fold changes between

189

samples. Only unique peptides were used for protein quantification. All peptide ratios were

190

normalized by the median protein ratio.

of

oxidation

(M)

and

iTRAQ8plex

(Y),

11


static

modifications

of


191

Statistical Analysis

192

Proteins with significant changes in abundance between the two mungbean isogenic

193

lines were selected using a method described previously.30 The mean and S.D. from the log 2

194

ratios of the 1,271 proteins overlapping in two biological repeats were calculated. Next, 95%

195

confidence (Z score = 1.96) was used to select those proteins with distribution removed from

196

the main distribution. For down-regulated proteins, the confidence interval was 0.00318

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(mean ratio of the 1,271 proteins) – 1.96 × 0.189418 (S.D.), corresponding to a protein ratio

198

of 0.774814. Similarly, for up-regulated proteins, the mean confidence interval was

199

calculated (mean ratio + 1.96 × S.D.) to a protein ratio of 1.296338. Protein ratios outside of

200

this range were defined as being significantly different at P = 0.05. The cutoff value for

201

down-regulated proteins was 0.77-fold, and for up-regulated proteins was 1.30-fold.

202

Combining Transcriptomic and Proteomic Data

203

The lists of 399 DEGs and 45 DPs were saved as DEG.csv and DP.csv files, respectively. Two

204

files were analyzed, and then a Venn diagram was created by Partek Genomics Suite software

205

(Partek, Inc.). The overlap area of the Venn diagram represents the DEGs/DPs that belong

206

equally to both transcriptomic and proteomic data.

207

Chromosome Location Analysis

208 209

The sequences of genes that encode proteins with differential abundances were blasted on the mungbean genome database 12


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(http://plantgenomics.snu.ac.kr/mediawiki-1.21.3/index.php/Main_Page)24 to identify their

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locations on the chromosome.

212

Predicted protein domain and glycosylation sites

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The predicted protein domains were analyzed by NCBI BLAST

214

(http://blast.ncbi.nlm.nih.gov/Blast.cgi). The predicted N-glycosylation and O-glycosylation

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sites were analyzed by NetNGlyc and NetOGlyc software in the ExPASy website

216

(http://www.expasy.org/). Prediction of transmembrane helices in proteins was analyzed by

217

TMHMM server (http://www.cbs.dtu.dk/services/TMHMM/).

218 219

RESULTS AND DISCUSSION

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Transcriptomic Analyses of Bruchid-Resistant and -Susceptible Mungbeans

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Total RNA in VC1973A (bruchid-susceptible line) and VC6089A (bruchid-resistant line)

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were extracted for RNA sequencing (RNA-seq) by Illumina Hiseq 2000 to search for

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potential Br genes. Approximately 23,000 transcripts (54.8% of all transcripts in the

224

mungbean genome) were detected and annotated by BWA MEM in two biological repeats of

225

mungbean lines. The transcripts between VC1973A and VC6089A were analyzed by

226

DESeq.29 The transcripts with Padj lower than 0.1, P value lower than 0.05, and fold change

227

(FC) higher than 2 or lower than 0.5 were selected and defined as differential expression

228

genes (DEGs). A total of 399 DEGs were identified, among which 251 DEGs showed 13



229

up-expression and 148 DEGs showed down-expression in the seeds of bruchid-resistant line

230

(Table S2). The numbers of DEG exhibited a 1.73% difference in seed transcriptome

231

between VC1973A and VC6089A. Six DEGs (g11456, g4739, g34480, g21777, g27780, and

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g39181) were only expressed in the resistant line and one DEG (g13480) in the susceptible

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line. However, these seven DEGs showed lower expression levels (TPM 5.2 or < 0.35 in Table S2. Inf represented the genes expressed in VC6089A, but not in VC1973A.

b

35



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Table 2. Differential proteins between seeds of mungbean isogenic lines VC6089A and VC1973A ID

Description

Accession

Coverage

E-value

Ident

Fold change

Location

Functional category

g34458.t1 Gag/pol polyprotein

AAQ82037.1

76%

0

59%

4.460

Vr01

Other

g5551.t1

Aspartic proteinase

AAB03843.2

99%

0

94%

4.324

Vr07

Enzyme

g39185.t1 Resistant-specific protein-1(4)

BAC22498.1

99%

0

83%

3.772

Vr05

Other

BAA77691.1

99%

8.00E-96

94%

2.388

Vr09

Defense-related protein

g39724.t1 Small ubiquitin-related modifier 1

XP_008456407.1

98%

7.00E-59

91%

2.051

scaffold_335

g8174.t1

XP_003553937.1

99%

0

89%

1.584

Vr01

Enzyme

g21583.t1 40S ribosomal protein S4-1

KHN38535.1

99%

5.00E-161

98%

1.557

Vr06

Protein synthesis and digestion

g8171.t1

Alpha-dioxygenase 1

XP_002279884.1

98%

0

70%

1.542

Vr01

Enzyme

g1322.t1

Stem-specific protein TSJT1-like

XP_003545925.1

99%

4.00E-160

86%

1.537

Vr10

Development

g30519.t1 40S ribosomal protein S7-2

KHN47668.1

99%

1.00E-123

92%

1.512

Vr01


g3584.t1

KHN16793.1

53%

6.00E-64

99%

1.415

Vr07


g10115.t1 Early nodulin-like protein 1

KHN37083.1

96%

1.00E-25

66%

1.398

Vr06

Other

g6021.t1

Signal peptide peptidase-like isoform X1

XP_003526473.1

99%

0

96%

1.392

Vr05

Enzyme

g8483.t1

PITH domain-containing protein

KHN16193.1

99%

1.00E-118

95%

1.368

Vr08

Other

g19167.t1 TPR superfamily protein

KEH24003.1

75%

2.00E-139

74%

1.357

Vr03

Other

g38165.t1 Isoflavone reductase

KHN41959.1

99%

4.00E-178

82%

1.345

Vr11

Enzyme

g17708.t1 Cu/Zn superoxide dismutase

ADZ72850.1

99%

8.00E-92

100%

1.338

Vr10

Enzyme

g42017.t1 Lipid transfer protein I

AAQ74627.1

99%

2.00E-63

100%

1.331

scaffold_91

g17385.t1 tRNA (mo5U34)-methyltransferase

KHG13272.1

94%

5.00E-133

68%

1.329

Vr01

Enzyme

XP_003548805.1

99%

0

97%

1.312

scaffold_7

Enzyme

XP_004508989.1

99%

3.00E-144

96%

1.310

Vr11

Other

KHN12858.1

91%

3.00E-118

94%

0.767

Vr10

Chaperone

g27390.t1 Cowpea pathogenesis-related protein 3 (CpPR3)

Alpha-dioxygenase 1-like

40S ribosomal protein S10

DNA repair


g21722.t1 Glyceraldehyde-3-phosphate dehydrogenase A, chloroplastic-like g1820.t1

Ras-related protein RABH1b-like

g21463.t1 Putative prefoldin subunit 3

36


Page 37 of 44

g2325.t1


Sulfite reductase [ferredoxin], chloroplastic

XP_003537728.1

91%

0

93%

0.764

Vr02

Enzyme

g32355.t1 Actin-related protein 4-like

XP_003521054.1

99%

0

96%

0.762

Vr09

Development

g18315.t1 Ubiquitin receptor RAD23d

XP_008230399.1

99%

3.00E-177

71%

0.761

Vr08


g2293.t1

XP_003607456.1

54%

0

89%

0.759

Vr02

Other

g17312.t1 60S ribosomal protein L7-4

KHN24635.1

58%

9.00E-120

85%

0.754

Vr05


g9314.t1

XP_003546884.1

99%

3.00E-132

79%

0.754

Vr05

Enzyme

0.742

Vr06

Other

Prohibitin 1-like protein

ATP synthase subunit O, mitochondrial-like

g10116.t1 Uncharacterized protein g31165.t1 Glutamate 5-kinase

XP_003613945.1

97%

0

89%

0.738

scaffold_100

Enzyme

g36464.t1 Nucleic acid-binding, OB-fold-like protein

XP_007044641.1

88%

2.00E-73

90%

0.731

scaffold_293

DNA repair

g176.t1

KHN43543.1

99%

2.00E-175

93%

0.726

Vr03


g19307.t1 Non-specific lipid-transfer protein A

KHN12008.1

90%

5.00E-23

47%

0.725

Vr06


g8144.t1

Formate dehydrogenase

ACZ74696.1

99%

0

96%

0.724

Vr01

Enzyme

g5544.t1

L-ascorbate oxidase homolog

XP_003549771.1

99%

0

91%

0.722

Vr07

Enzyme

XP_003547730.1

94%

1.00E-50

46%

0.720

scaffold_2330

KF033519.1

100%

2.00E-171

89%

0.702

Vr10

Other

g26926.t1 Nuclear cap-binding protein subunit 1-like

XP_006599000.1

99%

0

91%

0.695

Vr04

Gene expression and regulation

g27806.t1 Pantothenate kinase

AES75860.2

99%

0

91%

0.684

Vr01

Enzyme

g32713.t1 Probable aldo-keto reductase 1-like

XP_006577284.1

99%

0

88%

0.682

Vr10

Enzyme

g5877.t1

AES88942.2

98%

0

77%

0.666

Vr02

Other

XP_003531365.1

99%

0

92%

0.657

Vr01

Enzyme

OTU domain-containing protein 6B

g18113.t1 Germin-like protein-like g30742.t1 Phaseolus vulgaris clone BE5d2113

Development

glutamate-rich protein mRNA

DUF1680 domain protein

g33674.t1 Bifunctional aspartate aminotransferase and glutamate/aspartate-prephenate aminotransferase-like g6005.t1

Glycine-rich RNA-binding protein GRP1A

XP_008236997.1

48%

7.00E-45

84%

0.636

Vr02

Gene expression and regulation

g851.t1

Pathogenesis-related protein 10

AAX19889.1

99%

4.00E-104

100%

0.557

Vr07


XP_006575386.1

99%

6.00E-159

98%

0.556

Vr06


g19819.t1 60S ribosomal protein L10

37



521

TOC Graphic: Exploring bruchid-resistant candidate genes in mungbean

522

isogenic lines by omics technologies

523

38


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Figure 1. The functional categories (A) and chromosome location (B) of 399 differential expressed genes (DEGs) analyzed in two isogenic lines. The up-expressed DEGs (dark gray) and down-expressed DEGs (light gray) in VC6089A were classified into 18 functional categories and chromosome locations. 122x177mm (300 x 300 DPI)



Figure 2. The functional categories (A) and chromosome location (B) of 45 differential proteins (DPs) analyzed in two isogenic lines. The DPs with higher abundance (dark gray) and lower abundance (light gray) in VC6089A were classified into eight functional categories and chromosome locations. 122x177mm (300 x 300 DPI)


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Figure 3. The bruchid-resistant candidate genes identified with transcriptomic and proteomic analyses. The 399 DEGs and 45 DPs were selected from RNA-seq and iTRAQ data between two isogenic lines. The three DEGs/DPs were identified from overlap of a Venn diagram and defined bruchid-resistant candidate genes. 62x46mm (300 x 300 DPI)



Figure 4. Expression levels of bruchid-resistant candidate genes in resistant and susceptible lines. Gene expressions of g39185 (A), g34458 (B), and g5551 (C) were measured by qRT-PCR in resistant lines (RIL59, TC1966, and VC6089A) and susceptible lines (NM92 and VC1973A). Data represent the mean ± S.D. in three independent experiments. 60x21mm (300 x 300 DPI)


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Figure 5. Map of the differential expressed genes (DEGs) and differential proteins (DPs) on chromosome 5. The DEGs located on chromosome 5 were represented on the left side. The DPs located on chromosome 5 were represented on the right side. The bruchid-resistant markers, DMB-SSR 158 and W02a4, were indicated by the letters with black color. The potential major Br gene was indicated in black color and with an asterisk. 122x177mm (300 x 300 DPI)



Exploring bruchid-resistant candidate genes in mungbean isogenic lines by omics technologies 47x26mm (600 x 600 DPI)


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Transcriptomic and Proteomic Research To Explore Bruchid-Resistant

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