Turkish Journal of Biology Turk J Biol (2017) 41: 98-104 http://journals.tubitak.gov.tr/biology/ © TÜBİTAK Research Article doi:10.3906/biy-1512-64

Expression of the ZmDEF1 gene and α-amylase inhibitory activity of recombinant against maize weevils

1 2 3 2 4, Thi Xuan Thuy VI , Hoang Duc LE , Vu Thanh Thanh NGUYEN , Van Son LE , Hoang Mau CHU * 1 Tay Bac University, Son La, Vietnam 2 Institute of Biotechnology, Vietnam Academy of Science and Technology, Hanoi, Vietnam 3 Thai Nguyen University of Sciences, Thai Nguyen, Vietnam 4 Department of Genetics and Modern Biology, Thai Nguyen University of Education, Thai Nguyen, Vietnam

Received: 24.12.2015 Accepted/Published Online: 21.07.2016 Final Version: 20.02.2017

Abstract: Plant are multifunctional small cysteine-rich proteins. They are active against fungi, bacteria, and many viruses, and they inhibit and α-amylase activities. In this study, we expressed the maize defensin gene (ZmDEF1) in tobacco seeds in order to establish the basis for generating transgenic maize plants resistant to weevils. The ZmDEF1 gene was isolated from Maison, a Vietnamese local maize cultivar, which is well known for having the highest resistance to weevils among other local cultivars. The ZmDEF1 gene was cloned into a binary vector, pBetaPhaso-dest, which carries phaseolin, a seed-specific promoter, to direct defensin expression in tobacco seeds. We obtained 13 transgenic tobacco lines from Agrobacterium-mediated transformation and regeneration, called the T0 generation. T0’s seeds (called the T1 generation) were collected and analyzed for ZmDEF1 gene expression. Reverse-transcription polymerase chain reaction (RT-PCR) results showed that 4 out of 13 lines (T1-1, T1-3, T1-10, and T1-17) expressed the ZmDEF1 gene at the transcriptional level. These lines were further analyzed by real-time RT-PCR until their transcript expression could be identified. The results showed that the line T1-17 expressed it at the highest expression level. The western blot method also showed that ZmDEF1 was expressed in all four lines above. These transgenic seeds can inhibit maize weevils’ α-amylase activity. Extracted protein from transgenic lines reduced weevil α-amylase activity by 67.9%–71.4% in comparison with protein extracted from nontransgenic plants. These results will hopefully provide a useful background to create transgenic maize plants with high resistance to weevils.

Key words: Defensin, cysteine, ZmDEF1 gene, Zea mays, Sitophilus zeamais Motsch.

1. Introduction Recently there has been increasing interest in searching Maize (Zea mays L.) is one of the major cereal crops with high for plants’ natural peptides that are able to inhibit harmful yield and economic value. Maize contributes to the steady agents. One group of natural peptides are plant defensins. production of cereals in the world and has an important role They are small cysteine-rich proteins containing about in economy and international trade as it is used for food, 45–54 amino acids. Plant defensins attract great interest animal feed, and materials for many industries. The demands as they are reported to be involved in different defense for food, animal feed, and materials as well as fuel in the pathways in plants (Selitrennikoff, 2001). Plant defensins world are growing rapidly, and it is estimated that about 200 are multifunctional, and, according to many studies, they million tons of maize would need to be produced annually to show and antibacterial activity (Wang et al., meet demands in 2017 (Edgertom, 2009). Similar to many 2011), inhibition of trypsin and α-amylase activities (Melo other crops, maize is less productive due to weevils. Though et al., 2002), inhibition of protein synthesis (Colilla et al., maize weevils (Sitophilus zeamais Motsch.) eat most types 1990), increased tolerance to heavy metals (Mirouze et al., of cereals, legumes, oil seeds, and other plant products, they 2006), and regulation of plant growth and development prefer corn. The adult weevils drill a hole into the grain, lay (Stotz et al., 2009). Based on their sequential characteristics eggs, and secrete a sticky mucus to block the hole. The larvae and functions (van der Weerden and Anderson, 2013), hatch in the seed and consume the embryo and other parts plant defensins are divided into 18 groups. The first until only the testa is left. Once large enough, the maggot group consists of defensins functioning as inhibitors of will drill holes to get out, grow wings, and infect other plants α-amylase or trypsin. Liu et al. (2003) isolated defensin (Gutierrez-Campos et al., 1999). from chickpea (VrD1) and demonstrated that VrD1 could * Correspondence: [email protected] 98 VI et al. / Turk J Biol inhibit insects, including weevils. VrD1 inhibits α-amylase a useful background to create transgenic maize plants with activities, and therefore it inhibits starch digestion in the high resistance to weevils. weevil gut. Pelegrini and Franco (2005) reported that defensin in cowpea (VuD1) functioned as an inhibitor of 2. Materials and methods α-amylase in weevil larvae, but not in mammals. These 2.1. Materials studies indicate the potential of using defensins to improve The local maize cultivar Maison was used as a source for weevil resistance in plants. ZmDEF1 isolation from RNA. Nicotiana tabacum C9-1 The three-dimensional structure of defensin is tobacco plants were used as transgenic targets to check composed of 3 β-sheets and one α-helix. Loop 3, which the activities of the recombinant vector, which contains is in the middle of the second and third β-sheets, plays ZmDEF1. The DH5α E. coli strain, A. tumefaciens CV58, an important role in the inhibition of weevil α-amylase and the vectors p201-SLHEP and pBetaPhaso-dest were activities. The interaction between loop 3 and the active used for cloning, vector design, and gene transformation. site of weevil α-amylase prevents starch from entering that The primers used for PCR and real-time RT-PCR are site (Lin et al., 2007). shown in Table 1. The hybrid maize grown in Vietnam today has high 2.2. Isolation and cloning of the ZmDEF1 gene yield, but its productivity and quality are greatly reduced Total RNA was isolated from maize by the use of TRIzol due to insects, especially weevils. The local maize cultivars reagent (Life Technologies). cDNA was synthesized by have low productivity and small grains, even though they using Maxima First Strand cDNA Synthesis Kit (Thermo have high weevil resistance. Current preserving methods Scientific). The ZmDEF1 gene was cloned from cDNA by such as drying, mixing with inert dust, or stirring are time- using the ZmDEF1-F and ZmDEF1-R primers. consuming and inefficient, while chemical treatment may 2.3. Agrobacterium-mediated transformation cause risks of environmental pollution. There are many Transformation of the transgenic structure containing studies on improving antifungal and antibacterial ability ZmDEF1 in tobacco plant C9-1 by A. tumefaciens was in maize (Murthy et al., 2009; Rahmaty and Khara, 2011; performed according to Topping’s protocol (1998). Wang et al., 2011); however, few studies about enhancing Transgenic samples regenerated in vitro then developed resistance to maize weevil have been conducted. Therefore, into whole plants in a natural environment and were called application of genetic engineering to improve weevil the T0 transgenic generation. The seeds collected from T0 resistance in maize could be a new and sustainable solution transgenic plants were the T1 transgenic generation and to maize preservation. In this study, the authors analyzed were used to analyze ZmDEF1 gene expression. ZmDEF1 gene expression isolated from maize in transgenic 2.4. Quantitative real-time RT-PCR for transcriptional tobacco at transcription and transition levels by real-time expression analysis reverse-transcription polymerase chain reaction (RT- ZmDEF1 transgene expression levels were determined PCR) and western blot and then examined its α-amylase by real-time RT-PCR with SYBR Green I fluorescent dye inhibitory function. Tobacco plants were used as models (Roche). The process consists of the following steps: 1) and defensin 1 was transformed into tobacco plants in extract total RNA with the TRIzol kit; 2) synthesize cDNA order to examine gene expression of recombinant proteins by using the First Strand cDNA Synthesis Kit (Maxima); 3) in seed defensins and to check whether the recombinant conduct real-time RT-PCR by using the Roche LightCycler defensin protein inhibited weevils’ α-amylase activities or 480; 4) calculate the expression level (R) by using the R = not. Hopefully these results in tobacco plants will provide 2–∆∆Ct method according to Livak and Schmittgen (2001).

Table 1. The primers used for PCR and real-time RT-PCR.

Primers Sequence (5’ - 3’) Product size (bp) ZmDEF1-F ACGCGTCGACATGGCGCCGTCTCGACGCA 243 (cDNA) ZmDEF1-R CCCAAGCTTGCAGATCTTCTTGCAGAAGCAC qDEF1-F TCCTCGTCCTCCTGCTC 148 qDEF1-R GAAGTTCTCGGTCTGGCA qAct-F GATCTTGCTGGTCGTGATCTT 152 qAct-R GTCTCCAACTCTTGCTCATAGTC

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2.5. Western blot analysis The purified RT-PCR product was simultaneously cut To extract total protein, 0.5 g of leaves was crushed in by SalI and HindIII then inserted into vector p201-SLHEP, liquid nitrogen and dissolved in 1.0 mL of PBS with 0.05% which contains two alternative positions, attL1 and attL2. Tween 20 (PBS-T), and then the mixture was centrifuged Vector p201-SLHEP-ZmDEF1 underwent the LR reaction at 13,000 rpm for 15 min. Proteins were denatured and with vector pBetaPhaso-dest to form a binary vector electrophoretic analysis was done on 10% SDS-PAGE, called pBetaPhaso–ZmDEF1 (Figure 1). pBetaPhaso-dest and then the samples were transferred to nitrocellulose contains a phaseolin promoter, 2S2 (a sequence to direct membranes by using the Pierce G2 Fast Blotter (25 V, 1.3 protein expression in the endosperms of seeds), and a mA for 20 min). Membranes were blocked in 5% skimmed c-myc tag. Inferentially, the c-myc that the recombinant milk in PBS-T for 1 h, then incubated with the first protein contains allows determining the expression of antibody attached to c-myc antigen (Santa Cruz Biotech) recombinant protein using c-myc antibody. for 3 h by shaking at room temperature, then washed three 3.2. Analysis of ZmDEF1 gene expression in T1 transgenic times with PBS, and then incubated with the secondary tobacco plants antibody (IgG) attached to horse radish peroxidase for To check the activity of the pBetaPhaso–ZmDEF1 structure, 2 h. Results were displayed using 3,3’,5,5’-tetramethyl the pBetaPhaso–ZmDEF1 structure was transformed into benzidine or 3,3’-diaminobenzidine tetrahydrochloride. tobacco C9-1 plants using A. tumefaciens. Eighteen lines 2.6. Alpha-amylase inhibitory assay of transgenic plants were obtained after the regenerating The α-amylase activity of weevils was determined process, and they grew and developed normally in the according to the Bernfeld method (Bernfeld, 1955). Maize greenhouse. Nontransgenic samples were also regenerated weevils (Sitophilus zeamais Motsch.) were crushed in ice without (Control-1) and 10 plants were with 0.2 M phosphate buffer (pH 6.8) and centrifuged at obtained in the greenhouse. 12,000 rpm for 10 min, and after that, the supernatant After 3–4 weeks in the greenhouse, these tobacco plants’ solution containing α-amylase was collected. Starch (1% leaves were collected for PCR to determine the presence of [w/v]) was used as a substrate for α-amylase activity assay. the transgene. Out of 18 lines, 13 were positive by PCR Solutions containing α-amylase from weevils and protein (Figure 2). These positive transgenic lines continued to be extracted from tobacco plants and starch were incubated analyzed. for 30 min at 30 °C, and then 3.5-dinitrosalicylic acid was To check ZmDEF1 expression in T1 transgenic plants, added. The suspension was heated at 100 °C for 10 min seeds were collected from the 13 positive T0 transgenic so that the reaction stopped; next, it was photolytically lines, because the pBetaPhaso–ZmDEF1 structure has the measured at 530 nm. Each assay was performed in triplicate. seed-specific phaseolin promoter (Mahesh et al., 2003), The α-amylase enzymatic activity is the amount (mg) of which directs ZmDEF1 expression only in seeds. RT-PCR degradable starch for 30 min at 30 °C (U/mg). results from collected seeds showed that 4 out of 13 lines expressed ZmDEF1 at the transcriptional level (Figure 3). 3. Results These lines were labeled as T1-1, T1-3, T1-10, and T1-17. 3.1. Construction of the transgenic pBetaPhaso– Next, the transcript expression levels of the transgene ZmDEF1 structure in the 4 positive T1 transgenic lines were quantified.

Based on previous evaluations of weevil resistance from Tobacco seeds from lines T1-1, T1-3, T1-10, T1-17, and different local Vietnamese maize cultivars, the researchers nontransgenic (negative control) were used to perform real- chose the cultivar Maison, which has the best resistance, as time RT-PCR with qDEF1-F/qDEF1-R primers (repeated 3 the source for ZmDEF1 gene isolation (Vi et al., 2015). The times). The cycle threshold (Ct) of the negative control was ZmDEF1 cDNA is 243 bp in length, encoding 80 amino after the 40th cycle, which means that the tested sample acids. The ZmDEF1 cDNA sequence from maize cultivar did not carry ZmDEF1. Because the nontransgenic plants Maison was deposited in GenBank (NCBI) with the code did not carry ZmDEF1, the Livak equation was applied to LN650982 (Vi et al., 2014). the lowest gene expression level (T1-3). The ΔCt, ΔΔCt,

Figure 1. pBetaPhaso-ZmDEF1 structure. Phaso Pro: Phaseolin promoter; attB1 and attB2: positions for the LR reaction; LB: left T-DNA border; RB: right T-DNA border; KanR: kanamycin resistance.

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Figure 2. ZmDEF1 gene presence in T0 transgenic tobacco lines. M: 1-kb DNA ladder; 1–18: transgenic plants; (-): nontransgenic plant; (+): PCR result from pBetaPhaso–ZmDEF1 vector.

Figure 3. ZmDEF1 transcript expression in T1 transgenic lines (RT-PCR). M: 1-kb DNA ladder; 1–18: transgenic plants; (-): nontransgenic plant; (+): PCR result from pBetaPhaso–ZmDEF1 vector. and R expression ratio values from the Livak method are 3.4. Analysis of α-amylase inhibitory ability of presented in Table 2. These transgenic lines have different recombinant ZmDEF1 protein ZmDEF1 transcript levels. The T1-17 transgenic line has To check whether the recombinant defensin protein can the highest expression (Figure 4). inhibit α-amylase enzymatic activity, the researchers extracted a solution containing α-amylase enzyme 3.3. Analysis of recombinant defensin 1 protein in T1 from maize weevil and protein from seeds of transgenic transgenic lines tobacco lines T1-1, T1-3, T1-10, and T1-17 as well as Proteins extracted from the seeds of tobacco lines T1-1, nontransgenic plants. Protein extracted from seeds was T1-3, T1-10, and T1-17 were used to perform western mixed with a solution containing α-amylase of weevils to blots with the c-myc antibody (Figure 5). Figure 5 shows perform the amylase enzymatic assay. Table 3 shows that that all four transgenic lines expressed recombinant samples containing the mixtures of protein extracted from defensin 1 protein. Thus, it could be concluded that transgenic seeds and solution containing α-amylase had the four transgenic tobacco lines obtained successfully significantly lower α-amylase enzymatic activity than the expressed ZmDEF1. samples containing either the mixture of nontransgenic

Table 2. Ct results and expression levels of ZmDEF1 gene from T1 transgenic tobacco lines.

T1 transgenic tobacco lines Ct_DEF1 Ct_Actin ΔCt(T) ΔCt(C) ΔΔCt R/T1-3 T1-3 21.98 23.61 –1.63 –1.63 0.00 1.00 T1-1 20.82 22.68 –1.86 –0.23 1.17 T1-10 21.60 23.52 –1.92 –0.29 1.22 T1-17 21.72 23.88 –2.16 –0.53 1.44

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Figure 4. Expression level of ZmDEF1 gene from T1 generation transgenic tobacco lines. Vertical bars represent standard errors.

Figure 5. Western blot for recombinant ZmDEF1 protein in T1 generation transgenic tobacco plants. M: Standard protein ladder (10–250 kDa); 1, 3, 10, 17: recombinant ZmDEF1 protein of T1 transgenic tobacco plant lines (T1-1, T1-3, T1-10, T1-17); (+): protein, ~35 kDa, with c-myc tag; (-): protein of nontransgenic plant.

Table 3. Enzyme assay of α-amylases of maize weevils in samples containing protein extracts from wild-type and transgenic tobacco plants.

Mixture of α-amylase and Mixture of α-amylase and protein of T1 transgenic plants Only Sample protein of nontransgenic α-amylases T1-1 T1-3 T1-10 T1-17 plant transgenic plant transgenic plant transgenic plant transgenic plant

Enzymatic activity (U/mg) 7.18 ± 0.29 7.13 ± 0.36 2.26 ± 0.46 2.01 ± 0.58 2.68 ± 0.73 2.15 ± 0.68 (mean ± standard error)

Enzymatic activity (%) 100 99.3 31.47 27.99 37.32 29.94 seed extract and α-amylase or α-amylase alone. Analytical 4. Discussion results of α-amylase activity are also shown in Figure 6. Plant defensins are divided into 18 groups, the first of These results indicated that α-amylase activity of maize which contains defensins, which can inhibit α-amylase or weevil (Sitophilus zeamais Motsch.) was inhibited by trypsin activities. There are many defensins with antifungal defensin recombinant protein extracted from seeds of T1 ability; among them, Group 15’s ZmESR6 has been proven transgenic plants. (Balandin et al., 2005). In this study, we wanted to improve

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A B Figure 6. A) Inhibitory effect of recombinant defensin on enzymatic activity of -α amylase from maize weevils (T1-1: solution containing α-amylases and protein extract of T1-1 transgenic plants; T1-3: solution containing α-amylases and protein extract of T1-3 transgenic plants; T1-10: solution containing α-amylases and protein extract of T1-10 transgenic plants; T1-17: solution containing α-amylases and protein extract of T1-17 transgenic plants; (-): solution containing α-amylases and protein extract of nontransgenic plants; E: solution containing only α-amylases). B) Recombinant protein defensin inhibits α-amylase activity in maize weevils (1: solution containing only α-amylases; 2: solution containing α-amylases and protein of T1-3 transgenic line; 3: solution containing α-amylases and protein of nontransgenic plant). resistance to weevils according to the α-amylase inhibition ZmDEF1 protein in the transgenic tobacco lines. Solutions mechanism, so we selected the ZmDEF1 gene to study. containing weevil α-amylase were incubated with protein The introduction of a foreign gene may trigger extracts from transgenic and nontransgenic seeds to posttranscriptional gene silencing (Baulcombe, 2004). examine the effect of ZmDEF1 on α-amylase enzymatic However, ZmDEF1 expression analyses in four T1 activity (Figure 6). The results were very promising: the generation transgenic tobacco lines (T1-1, T1-3, T1- average enzymatic activity when α-amylase was incubated 10, and T1-17) demonstrated that RNAi targeting of the with protein extract from the four transgenic lines ZmDEF1 transgene had not happened. (31.68%) was significantly lower than that of incubated To analyze the biological function of ZmDEF1 towards solution containing α-amylase with protein extract from the weevil’s digestion, transgenic tobacco seeds are used as nontransgenic seeds (99.3%) or α-amylase alone (100%) food for weevils. One gram of seed of each T1 transgenic (Table 3). These results showed that weevil α-amylase tobacco line was placed into a glass vase, ensuring activity was inhibited by recombinant ZmDEF1 protein. gas exchange conditions. Five pairs of maize weevils Research in tobacco plants has established the basis to (Sitophilus zeamais Motsch.) were released in the vase generate maize plants with resistance to weevils and will that contained transgenic tobacco seeds. After 6 days of contribute to improving maize preservation. the experiment, the weevils were dead. Tobacco seed mass remained the same as before releasing weevils. Repeated Acknowledgment experiments showed the same results. It was concluded The authors would like to express their gratefulness for the that the tobacco seeds were not appropriate food for help of the Key Laboratory of Gene Technology, Institute maize weevils. Therefore, a different approach had to be of Biotechnology, Vietnam Academy of Science and used to analyze the biological function of recombinant Technology.

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