Cloning and Expression of Cadherin Fragment from Helicoverpa armigera

Helicoverpa armigera is an important pest of agricultural crops which causes severe damage to the fruits and pods of crop plants, larval stage which causes this kind of damage. Helicoverpa armigera cadherin fragment contains its toxin binding region which enhance cry1Ac activity against Helicoverpa armigera larvae because this kind of interaction between HaCad1 receptors and cry1Ac induces toxin oligomerization. The aim of current investigation was to clone and expression the cadherin fragment. Cadherin gene was isolated from mid gut portion of Helicoverpa armigera and cloned into protein expression vector pMALc2x for expression study. The recombinant clone was transformed into protein expression host and protein was isolated and analysed the expected kDa by SDSPAGE and Western blotting. The protein band was 70 kDa that confirmed 26.9 kDa cadherin protein was fused with 42.5 kDa Maltose binding protein. Present study successfully cloned and expressed cadherin fragment HaCad1 and Cry1Ac protein in Helicoverpa armigera. Our result provided a tactic for cadherin gene cloning and transformation into cry1Ac transgenic plants that can significantly enhance the insecticidal activity of Cry1Ac against Helicoverpa armigera.


I. Introduction
Pest management has always been an important challenge to agricultural community.Crop yields are substantially reduced by insect pests that attack them.Bt crystal proteins are the first insecticidal protein for introduction into plants because of their environmental safety.A variety of genes encoding for different classes of insecticidal proteins such as protease inhibitors, lectins, amylase inhibitors, scorpion-venom toxins, cytokinin biosynthesis enzymes, cholesterol oxidase, chitinase and δendotoxins of Bacillus thuringiensis are being tested for insect control.Among these, the insecticidal crystal proteins of Bt assume major significance due to their potency, insect specificity and lack of activity in mammals and other organisms (Kumar et al., 1996).Bacillus thuringiensis is a ubiquitous gram-positive, spore forming bacterium.It produces parasporal insecticidal crystal proteins (ICPs) also known as Cry proteins which exhibit toxicity to certain insect species.These insecticidal crystal proteins (ICPs) display insect specific toxicity and are harmless to humans and non-target animals.Consequently, they have been widely used in biopesticides and transgenic plants to control agricultural pests (Schnepf et al., 1998;Pigott and Ellar, 2007).Bacillus thuringiensis is already a useful alternative or supplement to synthetic chemical pesticide.Cadherin belongs to one of the families of animal glycoproteins responsible for calcium-dependent cell-cell adhesion, Cadherin (named for calcium dependent adhesion).The interaction between Bacillus thuringiensis insecticidal crystal protein Cry1A and Cadherin receptors in lepidopteran insects induces toxin oligomerization, which is essential for membrane insertion and mediates Cry1A toxicity.Published with open access at http://www.journalbinet.com/calcium (Ca 2+ ) ions to function, hence their name.Recent literature showed that the Cadherin-like protein in midgut of several insects served as the receptor of Bt toxin Cry1A and variation of Cadherin-like protein is related to insect's resistance to cry1A.cDNA sequence of a cadherin gene in Helicoverpa armigera (HaCad) and the cry1Ac toxin binding region (TBR) to residues 1217-1461 (Wang et al., 2005).Now a days cloning of the cadherin fragment is a novel strategy to enhance insecticidal activity or to overcome the resistance of insects.As Helicoverpa armigera is a major crop pest, cloning of cadherin gene fragment into the protein expression vector can help to manage this pest.The view of the present study was to clone the receptor region of HaCad1 into E .coliexpression vector pMALc2X and analyze the protein by SDS-PAGE and Western blotting.

II. Material and Methods
An experiment was conducted in National Research Center on Plant Biotechnology, Indian Agricultural Research Institute, New Delhi, India with a view for designing of primers for amplification of HaCad1 from Helicoverpa armigera: Based on published HaCad1 (Genebank no.DQ973295 - Peng et al., 2009) gene sequences, a set of specific primers were used to amplify the receptor binding portion of cadherin from the cDNA sample of Helicoverpa armigera midgut portion.
Total RNA isolation from H. armigera mid gut: Total RNA was isolated from the mid gut portion of the 4 th instar larvae of the Helicoverpa armigera.RNA isolation was done by using TRizol reagent (Invitrogen).Quality and quantity of the total RNA was checked by gel electrophoresis and Nanodrop 1000 spectrophotometer respectively (Figure 01).cDNA synthesis from the total RNA: Single stranded cDNA was prepared by using AffinityScript QPCR cDNA Synthesis Kit.(Stratagene) (Figure 02).The following reaction components were added in microcentrifuge tube in order: RNase-free H 2 O 5.0 μl Total RNA (1 μg) 1.0 μl First strand master mix (2×) 10.0 μl Oligo (dT) primer (0.1 μg/μl) 3.0 μl AffinityScript RT/RNase Block enzyme mixture 1.0 μl The above reaction was kept in thermal cycler and followed the condition according to the manufacturer's instruction.After cDNA synthesis the quality and quantity of the cDNA was checked on 1.2% agarose gel and Nanodrop respectively.
Amplification of the cadherin gene fragment: Gradient PCR was performed to find out the annealing temperature for amplification of cadherin receptor region at 50-60ºC by using Taq DNA polymerase enzyme (Bangalore Genei).Pfu DNA polymerase was used to amplify the final product and avoid the mismatch of the nucleotide sequences.Amplification was performed in an eppendorf thermal cycler (Eppendorf AG, Germany) (Figure 03).

Analysis and Purification of PCR products:
After amplification of cDNA, it was checked in 1% agarose gel electrophoresis and purified the cDNA using QIAquick PCR purification kit (QIAGEN, Germany).

Cloning of cadherin gene (HaCad1) fragment into pMALc2x expression vector:
For the expression of cadherin gene, 732 bp of PCR purified cadherin gene was restricted with BamHI and SacI site and cloned into correspondent site of pET29 (a+) vector.The cadherin fragment was further cloned into pMALc2X expression vector by BamHI -HindIII restriction sites for improving the protein expression.Routine lab protocols were followed for ligation, transformation and screening of transformed colonies (Figure 04).

Overexpression of cadherin protein:
The clone was transformed into the TB1 host cells and the transformants were used for screening.Protein was isolated according to manufacture instruction (New England BioLabs) from single colony and induced by 1mM IPTG.The overexpressed protein was checked by SDS-PAGE and Western blotting.
Screening for overexpression colony by SDS-PAGE analysis and Western Blotting: Isolated protein was checked on 12% SDS-PAGE (BIO-RAD Mini-Protein-3 Electrophoresis system).
Because of the small fragment of the cadherin gene the expected band size was not clearly seen under the SDS-PAGE.For further confirmation, western blotting was carried out.SDS-PAGE separated proteins were transferred to nitro cellular membrane using mini trans-blot electrophoretic transfer cell apparatus (BIO-RAD, USA).After adding primary and secondary antibody to the membrane, it was further washed with PBST for three times and air dried on what man paper and immediately photograph was taken (Figure 05a and 05b).

Experimental setup and cloning of cadherin gene
Based on published HaCad1 (Gene Bank no.DQ973295- Peng et al., 2009) gene sequence, set of primers were designed for amplification of receptor binding portion of cadherin fragment.Total RNA was isolated from the larval mid gut (Figure 01) and converted into single stranded cDNA (Figure 02) and used as template for PCR amplification (Figure 03).732bp PCR amplified cDNA fragment (toxin binding region) was cloned into the protein expression vector.PCR amplified 732bp gene fragment was restricted with BamH1 and SacI and cloned into the corresponding sites of protein expression vector pET29a (+).Cadherin fragment was inserting out from pET29a (+) vector using BamHI-HindIII sites and further cloned into pMALc2x expression vector (Figure 04) for improving the expression.The clone was further confirmed by multiple restriction digestion analysis.

Analysis of protein expression by SDS-PAGE and Western Blotting
PMALc2X-cadherin vector were transformed into TB1 E. coli host to express the protein.Since the gene fragment was extremely small consisting of 732bp and the proteins molecular weight being only 26.9 kDa it which was giving a very faint band on the SDS-PAGE so it was decided that this protein would be cloned into pMALC2X vector which contains a maltose binding fusion protein which is 42.5kDa.After cloning both proteins fuse together and yielded 70kDa fusion protein was found in SDS PAGE (Figure 05a).For further confirmation western blotting was carried out and a 70kDa fusion protein was obtained which contains 26.9kDa of cadherin protein along with 42.5kDa of maltose binding protein (Figure 05b).

IV. Discussion
Genetically engineered crops to produce Bt toxins for insect control have reduced the use of conventional insecticides.Bacillus thuringiensis produces insecticidal crystal protein.Previously, toxin binding region of Helicoverpa armigera cadherin fragment (HaCad1) cloning into binary vector and retransformation into the cry1Ac plants through Agrobacterium mediated plant transformation was done by Momena et al. (2014).The cry1Ac toxin binding sites of HaCad have been mapped to residues 1217 to 1461 by Wang et al. (2005) and found that HaCad1 enhanced the insecticidal activity of Bacillus thuringiensis cry1Ac toxins to Helicoverpa armigera.A critical step in the mode of action of cry proteins is binding of toxins to mid guts membrane receptors (Schnepf et al., 1998;Whalon and Wingerd, 2003).It was proposed that midgut epithelium receptors were involved in insect toxicity by interacting sequentially with the cry1A toxin (Bravo et al., 2004 and2007;Jurat-Fuentes and Adang, 2006;Gomez et al., 2007).Cadherin belongs to one of the families of animal glycoproteins responsible for calcium-dependent cell-cell adhesion hence, named cadherin.The interaction between Bacillus thuringiensis insecticidal crystal protein Cry1A and cadherin receptors in lepidopteran insects induces toxin oligomerization, which is essential for membrane insertion and mediates Cry1A toxicity.Based on these previous reports the present study has done to demonstrate the amplification of receptor binding portion of cadherin fragment from H. armigera, cloning to protein expression vector and over express the protein, analysed by SDS-PAGE and western blotting.
In SDS-PAGE analysis it shown very faint band due to low molecular weight of protein, so that it was further confirmed by western blot analysis.The result shows that 70 kDa protein band on the SDS-PAGE and western blot, which indicates 26.9 kDa cadherin proteins, was fused with 42.5 kDa Maltose binding protein.

Figure
Figure 01.Analysis of total RNA from mid gut portion of H. armigera Figure 02.Synthesis of cDNA from midgut total RNA of H. armigera Figure 03.PCR amplification of cadherin gene fragment using cDNA sample