Legume Research, 38 (2) 2015 : 178-181 AGRICULTURAL RESEARCH COMMUNICATION CENTRE

Print ISSN:0250-5371 / Online ISSN:0976-0571 www.arccjournals.com/www.legumeresearch.in

Cloning and characterization of protease inhibitor genes from some legumes

Manoj Saini, P . Nandeesha, Mayank Kaashyap, Prasoonpal Gupta, Mukesh Mohan1 and Subhojit Datta*
Biotechnology Unit,
Indian Institute of Pulses Research, Kanpur 208 024, India.
Received: 21-04-2014 Accepted: 06-11-2014 DOI: 10.5958/0976-0571.2015.00079.X
ABSTRACT
Protease inhibitors are natural defense proteins that inhibit the proteolytic activity of proteases. Legume protease inhibitors
are diverse in their molecular mechanism and many of them have successfully been deployed in crop plants to confer
resistance against insect pests. Nine combinations of degenerated primers,designed, based on multiple aligned gene
sequences from NCBI Gene bank database were used to amplify the protease inhibitor genes in of ten different legume
crops (two varieties each)viz.fieldpea,chickpea, lentil, lathyrus, pigeonpea , frenchbean,mungbean ,clusterbean ,
horsegramand cowpea .The primer combination BBF 2/BBR2 amplified the expectedamplicon of 800 bp invarities
ofmungbean,horsegramand cowpea. These amplicons obtained from each genotype were further ligated to the pJET1.2/
blunt vector and transformed into XL1 blue strain of E.coli. Out of 41 clones sequenced, two clone sequences obtained
from cowpea (DCS 6) showed homology with substilisin-like serine protease inhibitor of Desulfovibria vulgaris and
serine/threonine protein kinase of Epichlocfastucae.These genes can be transformed into Agrobacterium based binary
vectors and used for transformation of crops. Therefore these findings offer new genes for transgenic development against
lepidopteron pests.

Key words: Blast, Cloning, Insect resistance, Protease inhibitor, Pulse crops.

INTRODUCTION on analysis of the complete sequence of several genomes, it

Pulse crops occupy a pivotal position in ensuring
nutritional security to world population and especially to
the undernourished masses in arid and semi-arid tropical
regions.They are rich in proteins and found to be main source
of protein to vegetarian people of India. It is second important
constituent of Indian diet after cereals.Pulses, being legumes,
fix atmospheric nitrogen into the soil and play important
role in sustainable agriculture by maintaining the soil
fertility.Besides increasing the inherent productivity of crop
plants, it is important to minimize the losses caused to the
production.Despite the use of insecticides, the worldwide
pre-harvest losses due to insect pests vary between 15%-
35% of total production in most of major field crops
(Krattiger 1997).The natural defense mechanism enables
plant to protect themselves against pests by synthesizing
specific macromolecules such as protease inhibitors, a-
amylase inhibitor, lectins and phenolic compounds. Protease
inhibitors are protein that inhibits the proteolytic activity of
protease.Protease inhibitors are natural defense-related
proteins often present in seeds and induced in certain plant
tissues by herbivory or wounding (Koiwaet al., 1997). Based
*Corresponding authors e-mail: subhojit@email.com.
1
Department of Biotechnology, C S Azad University of Agriculture and Technology, Kanpur 208 002, India.
is estimated that about 2% of all gene products are protease
(Barrett et al., 1998). Large number of protease inhibitors
have been isolated and characterized from plants, animals
and microorganisms and extensively studied in legumes like
pea, pigeon pea, cowpea and soybean and implicated in
defense mechanism against microbial and insect attack
(Richardson,1977, Valueva and Mosolov, 2004, Christeller,
2005).
During the last decade, with the commercial
introduction of insect resistant transgenicplants having
Btgenes, the major concern with these crops has been the
development of resistanceby pests and poor public
acceptance. Therefore, there is a need to discover new
effective plant genes, which would offer resistance/protection
against pests. Protease inhibitor is one of the prime
candidates with highly proven inhibitory activity against
insect pest and also knows to improve the nutritional quality
of food. The protease inhibitors actsspecifically against
protease of particular group of insects and therefore, it is
very important to identify the right candidate gene before
the crop plant is transformed with protease inhibitor gene
 Volume 38, Issue 2, 2015
(Johnson et al.,1989). Trypsininhibitor gene when transferred
from cowpea to tobacco was shown to confer resistance to
wide range of insect pests including Lepidoptera such as
Heliothis and Spodoptera, coleopterans such as Diabrotica,
Anthonomonus and Orthoptera such asLocusts (Hilderet
al.,1987). Recently protease inhibitors have been implicated
in defense mechanism against microbial and insect pest
control. Therefore this study was undertaken to assess the
presence of protease inhibitor gene in major pulse crops and
to clone and characterize legume protease inhibitor genes.
MATERIALS AND METHODS
Plant material and DNA isolation: Seeds of two varieties
each of ten different legume crops viz.fieldpea (IPF 99-25,
HUDP 15),chickpea(C 235, BG 256), lentil (DPL 15, IPL
81),lathyrus(Pusa 24 and Bio L 212), pigeonpea (Bahar,
Type 7),frenchbean (EC 406072, HUR 15),mungbean
(Pantmung 3, TAP 7),clusterbean (HGH 365, RGC 986),
horsegram(AK 42, HG 25)and cowpea(DCS 6, IC 39890)
were selected for the present analysis. Seeds were germinated
on paper towel soaked in sterilized water under etiolated
conditions to minimize the contamination of chloroplast
DNA. One-week old seedlings were ground in preheated
CTAB buffer and incubated at 60 C for one hour. The
aqueous phase containing DNA was separated using
Chloroform: Isoamyl alcohol (24:1) (Abdelnooret al., 1995).
The DNA was precipitated with chilled iso-propanol and
the pellet was dissolved in 100 l of T10E1 buffer. The RNA
was eliminated by adding 0.5 Units of RNase. The
concentrations of DNA were quantified by measuring
absorbance using Hoefer Dyna Quant 200 DNA
fluorometer (Amersham Biosciences, Piscataway, NJ,
U.S.A.) and stocks were maintained at 25 ng/l.
Primer designing: For primer design, NCBI Genebank
database were used and sequences of Bowman Birk type
protease inhibitor gene of pea and cowpea were downloaded
and aligned using default parameters of ClustalW software
(Larkin et. al. 2007). Three forward (BBF1, BBF2 and BBF3)
and three reverse (BBR 1, BBR2 and BBR3) primers
(Table 1) were designed using the Primer 3.0 software and
were custom synthesized from Operon Technologies, USA.
TABLE 1: Primers used for amplification of legume protease
inhibitor genes
Primer Primer Sequence (5-3) Annealing
(s) temperature (C)
BBF1 ATGGWGTTGAWGAAYAAGAAAG 55.7
BBR1 GTGRCATGYTTYATAGCAGAAST 60.1
BBF2 TTGRGCTTCRCCGCAAMT 58.8
BBR2 TCAGTTCTTRATKACCTCCTC 58.7
BBF3 GGTGATGATGTCAAATCAG 55.8
BBR3 TACATAGTTATACTTGCTCA 52.2
179
Amplification of protease inhibitor gene by PCR: Nine
different combinations of these three pairs of degenerate
primers were used for amplification of protease inhibitor
gene with annealing temperature ranging from 37C -
50C.These were BBF 1/BBR1,BBF 2/BBR2,BBF 3/BBR3,
BBF 1/BBR2, BBF 1/BBR3,BBF 2/BBR1,BBF 2/BBR3,BBF 3/
BBR1, and BBF3/BBR2.The PCR reactions were performed
in 20 l volume containing 25 ng of template DNA, 45 pM
each forward and reverse primers, 25 M of each dNTPs
(MBI Fermentas, La Jolla, USA) and 0.6 Units of Taq
polymerase (Bangalore Genei, Bengaluru, India). PCR
program was run on PTC-200 (MJ Research, U.S.A.)
thermal-cycler consisting of initial denaturation at 94C for
3 min followed by 39 cycles each consisting of denaturation
at 94C for 1 min, annealing and elongation at 72C for 2
min. The Tm used for annealing was as per the primer
specifications (Table 1). Final extension step included 72C
incubation for 7 min. The amplified DNA fragments were
resolved on ethidium bromide stained agarose gel (2%) in
1X TBE buffer at 50V. The gels were visualized on trans-
UV and photographed in BioRad Gel Doc XR 2.0.
Cloning and sequence analysis: The amplicons of expected
size were eluted from the gel using QIAGEN kit as per
manufactures instructions. Thefragments were ligated into
pJET1.2/blunt using Genejet kit (Fermentas). The ligated
product was transformed into E.coli XL 1 blue competent
cells by following standard molecular biology procedures
(Sambrook et al. 1989). Recombinant plasmids were isolated
using QIAprep spin miniprep kit (QIAgen) according to
manufactures instruction protocol. Recombinant clones were
identified by restriction digestion analysis using Xba1 and
Xho1 restriction enzyme (Fermentas).The positive clones
were sequenced using forward primer of pJET1.2
(5CGACTCACTATAGGGAGA GCGGC3) (Fermentas),
using a Big dye terminator method by an ABI 3100 genetic
analyzer apparatus (Applied Biosystem) based on Sangers
method at Bangalore Genei, Benagaluru. Sequence analyses
were performed using the BLASTx and BLASTnprogramme
of NCBI site (http:// www.ncbi.nim.nih.gov).
RESULTS AND DISCUSSION
The primer combinations BBF2 and BBR2 amplified
product of 800 bp in mungbean (Pantmung 3 and TAP 7),
horsegram (AK 42) and cowpea (DCS 6 and IC 39890)
(Fig 1). The size of amplicons was in correlation to the earlier
reported full length size of protease inhibitor gene (Datta
and Tiwari, 2006).The primer combination BBF 1/BBR1
amplified amplicons of 350 bp in fieldpea (IPF 99-25 and
HUDP 15), lentil (IPL 81 and DPL 15) and lathyrus (Pusa
24 and BioL 212). The primer combination BBF 3/BBR1
180 LEGUME RESEARCH
M 1 2 3 4 5 6 7 8 9 10 11 12 13

FIG 1: PCR amplification by primer combination BBF2/BBR2

at 37C annealing temperature. Lane M: 100 bp DNA ladder
plus, Lane 1: MungbeanPantmung 3, Lane 2: Mungbean TAP 7,
Lane 3: Horsegram AK 42, Lane 4: Cowpea DCS 6,
Lane5:Cowpea IC 39890.
amplified amplicons of 200 bp in all selected legume
genotypes. The primer combinations (BBF3/BBR3), (BBF1/
BBR2), (BBF 1/BBR3) (BBF 2/BBR3) and (BBF 3/BBR2)
produced multiple bands. The appearance of multiple bands
has been reported earlier as the result of amplification of
more than onelocusby each primer combination (Holton et
al., 2002). The primers combination BBF1/BBR2, BBF2/
BBR2, BBF2/BBR1, BBF2/BBR3 and BBF3/BBR2 did not
produce any bands.Of the total 41 clones sequenced, 16
clones from mungbean(TAP 7), 14 clones from cowpea (DCS
6) and 11 clones from cowpea (IC 39890)showed sequence
read length between 324-866 bp (Fig 2).
Sequence analysis: TheBLASTx results of nucleotide
sequence of clone number SD24 showed strong homology
of 50% query coveragewith subtilisin-like serine protease
of Desulfovibria vulgaris.More than 200 subtilisinlike
enzymes are presently known, Subtilisin inhibitors were
found in Vignaunguiculata (Vartak et al. 1980) and
Vignaradiata (Kapuret al., 1989),Subtilisinlike proteases
have been identified and the genes cloned in only few plant
species, including Arabidopsis (Ribeiro et al., 1995).Incase
of cowpea (DCS 6), the nucleotide sequence of SD38
showed homology with Serine/Threonine protein kinase of
Epichloefastucae.The nucleotide sequences of clone
number SD24 showed four frames -1, +2, +3 and -3. The
frame -1 started at position 30 to 314 base pairs and
contained a 285 bp open reading frame encoding a predicted
protein of 94 amino acids (Fig. 3). These results are in
FIG 2: Restricted recombinant plasmids showing released
inserts. Lane M: 1 kb DNA ladder. Lane 1: control (pJET1.2/
blunt vector, 2974 bp). Lane 2-5:cowpea DCS 6. Lane 6-9:
cowpea IC 39890, Lane 10-13: mungbeanTAP-7.
314 atgtgcggagggagagaacggtgcaattgccccagaccaagtctc
M C G G R E R C N C P R P S L
269 atccccctacccctactccaaggggcttcacactctgggagttcc
I P L P L L Q G A S H S G S S
224 ttctacttgaccagcttattcggatttcaattaccagccccctat
F Y L T S L F G F Q L P A P Y
179 aggtattgtcttccaatcagtgggttcatgccgcacagaaagcga
R Y C L P I S G F M P H R K R
134 gacctacttctttctatctcggataactatgaaatttggcctagt
D L L L S I S D N Y E I W P S
89 aataagcttagagtggaagagtcggtgaggaggaggtcatcaaga
N K L R V E E S V R R R S S R
44 actgaatcttgctga 30
T E S C *
FIG 3: Open reading frame (285 bp) and deduced amino acid
(94) sequence of protease inhibitor gene from cowpea IC 39890.
accorda nce t o t he evidence tha t inh ibi tors fr om
dicotyledonous plants consist of a single polypeptide chain
with a molecular mass of 8 kDa (Habib and Khalid, 2007).
The frames +3 and -3 were showing length of 37 and 34
amino acidsrespectively. The frames -1, +3 and -3 were
showing initiation codon (ATG) and termination codon
(T GA). Th ese genes can be t ran sform ed in to
Agrobacterium based binary vectors and used for
transformation of crops. Therefore these findings offer
new gen es for t ran sgeni c development again st
lepidopteronpests.

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