Expression of an Insecticidal Protein under Phloem Specific Promoter in Cotton Plants to Control Whitefly

doi:10.21203/rs.3.rs-1992089/v1

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Expression of an Insecticidal Protein under Phloem Specific Promoter in Cotton Plants to Control Whitefly

https://doi.org/10.21203/rs.3.rs-1992089/v1

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Whitefly the Bemisia tabaci is a phloem sap-sucking hemipteran pest that causes enormous damage to many crop plants, particularly cotton. Bt endotoxins which successfully controlled the chewing pest proved to be mostly ineffective against the whiteflies and many other hemipteran pests. In the past few years, some insecticidal proteins showed significant toxicity against the whiteflies such as Hvt, lectin, and TMA12. In this study, transgenic cotton lines expressing plant-derived insecticidal protein TMA12 under constitutive promoter (2X35S derived from Cauliflower mosaic virus-CaMV) and a phloem-specific promoter (Nuclear shuttle protein-NSP derived from Banana bunchy top virus-BBTV) were developed to compare their efficacy against the whiteflies. Before developing cotton transgenic lines, tobacco transgenic plants were also developed to get preliminary results about the efficacy of the transgene. After molecular characterization, cotton transgenic lines were evaluated for resistance against whitefly in the contained environment. Whitefly survival and fecundity were calculated for each transgenic line. Bioassay data proved the effectiveness of insecticidal protein against whiteflies. These results also concluded that the NSP promoter is potentially an effective strategy against B. tabaci but still its efficiency is not equal to the 2X35S promoter. The use of a phloem-specific promoter has advantages over the constitutive promoter like tissue-specific expression saves the molecular resources of the plant cells and is less prone to biosafety concerns. In the future, more phloem-specific promoters can be explored for enhanced effectivity against B. tabaci.

There are several genetic engineering approaches to developing crop plants’ resistance to insects [1–6]. GM crops expressing proteins and toxins such as chitinases [7], biotin-binding protein [8], alpha-amylase inhibitors [9], protease inhibitors [10], and Bt endotoxins [11–13] have successfully protected from chewing pests and largely reduced the consumption of insecticides. Specifically, the GM crops expressing Cry proteins (Bt δ-endotoxins) to control coleopteran and lepidopteran insects provided successful examples of the safe use of GM crops without significant effect on beneficial insects [14].

Mostly, Bt insecticides provided resistance against chewing pests but were effective to control sap-sucking pests specifically whiteflies. To overcome this limitation, researchers developed some modified versions of Bt which provided significant resistance against the sap-sucking pest. A Bt crystal protein Cry51Aa2 was isolated and characterized with insecticidal activity against the Lygus species when provided in an artificial diet but its efficacy was not commercially acceptable when expressed in the cotton plants [15]. Later a study reported the improved efficiency of the modified version of Cry51Aa2 in cotton plants against the Lygus species [13]. Similarly, Chougule et al. reported that the fusion of a Bt cytolytic toxin, Cyt2Aa with an aphid gut-binding peptide (12 amino acid) enhanced the toxicity against the aphids [12]. However, there is no Bt toxin is effective against the Bemisia tabaci [16].

In recent years, some insecticidal proteins have been identified, which showed significant toxicity to the B. tabaci. Javaid et al., [17] reported that the expression of two insecticidal proteins, onion leaf lectin and Hadronyche versuta neurotoxin (Hvt) under the phloem-specific promoter provided resistance against B. tabaci, Myzus persicae (aphid), and Phenacoccus solenopsis (mealybug). Waheed et al. [7] reported an insecticidal protein endochitinase (Chit1) derived from an entomopathogenic fungus (Metarhizium anisopliae) also effective against the B. tabaci. Shukla et al. [18] reported the expression of TMA12 under constitutive promoter in cotton which resulted in significant resistance against whiteflies.

Expressing insecticidal protein in the crop plant is a sustainable and one-time investment technique that can contribute well to the development of whiteflies resistance. However, biosafety concerns limit their application for commercial cultivation. In this study, transgenic plants including both N. tabacum and G. hirsutum (var. Coker 312) expressing TMA12 protein under constitutive (2X35S) and phloem-specific promoter (Nuclear shuttle protein-NSP) were developed and their effectiveness against the whiteflies was tested. The tma12 gene is derived from a fern plant (Tectaria macrodonta) that naturally resists pests. A study found that it is a lytic polysaccharide monooxygenase and potentially binds to chitin, which is a major component of insect exoskeleton [19]. Shukla et al. [18] reported the activity of this protein against whiteflies by expressing it in cotton plants under a 2X35S promoter. NSP is a phloem-specific promoter derived from the Banana bunchy top virus (BBTV) [20] and the 2X35S promoter is a tandem repeat derived from the Cauliflower mosaic virus (CaMV) [21]. The study aims to develop transgenic cotton plants expressing TMA12 protein under NSP promoter and 35S promoter to compare their effectiveness against the B. tabaci. As the whitefly is a sap-sucking pest, therefore it was assumed that a phloem-specific promoter could provide a more targeted approach and some previous studies show the better effectivity of the gene expressed under phloem-specific promoter against sap-sucking pests such as aphids [22].

Synthetic tma12 Gene and Codon Optimization

The coding sequence of the tma12 gene was retrieved from the National Center for Biotechnology Information (GenBank accession code: JQ438776.1). To get enhanced expression in the transgenic cotton, the sequence was codon optimized by using the Gossypium spp. codon usage table and Geneious V8.0 software [23]. After codon optimization, the protein sequence remained the same but the DNA sequence has 70% identity of the original gene sequence. This codon-optimized gene sequence of tma12 was commercially synthesized by ATUM, Newark, CA, USA. The supplier provided the synthetic gene cloned in the plasmid pJIT201 (Figure S1 in Additional file 1).

Cloning of tma12 Gene under CaMV-2X35S Promoter and NSP Promoter

Two different promoters were used for the expression of TMA12 protein: 1) constitutive promoter CaMV-2X35S and 2) phloem-specific promoter-NSP). Plasmid pJ201 with the gene of interest was restricted with HindIII and EcoRI and the fragments were checked on the agarose gel. The desired band of the gene of interest (651bp) was eluted from the gel by Wizard SV Gel and PCR Clean-Up System (Promega). Then the gene of interest was cloned in the pJIT163 at HindIII and EcoRI sites under the 35S promoter. Then the expression cassette (CaMV-2X35S promoter, GOI, and CaMV terminator) was restricted from the pJIT163 with SacI and EcoRV and then ligated in the pCambia 2300 at SacI and SmaI sites. To clone the gene under the NSP promoter, the pJIT163 (with the gene of interest) was restricted with SacI and HindIII to remove the CaMV-2X35S promoter and at the same sites, NSP promoter was ligated. The ligation mixture was transformed into E. coli top 10. The recombinant plasmids having tma12 under CaMV-2X35S promoter and NSP promoter are shown in Figure S2 (Additional file 1).

Transformation into Agrobacterium LBA14404

To transform the recombinant plasmid DNA pCambia 2300 (tma12 constructs under CaMV-2X35S promoter and NSP promoter) into Agrobacterium tumefaciens with electroporation method, DNA was diluted to 15-20 ng/µL concentration. About 5 µL of DNA was mixed with 100 µL of agrobacterium cells in a 1.5 mL tube and incubated on ice for 30 min. The mixture was transferred to the cuvette (1mm gap) and the electroporator was adjusted at voltage: 1440 volts, resistance: 200 W, capacitance: 250 μFD. After passing an electric pulse through the cuvette, 1 mL LB medium was poured into the electroporation cuvette and after mixing well was transferred to a 2 mL tube. Then the tube was incubated at 28 ºC and 180 rpm for 2-3 hours. Then 150 µL of cells were spread on the LB agar plate supplemented with kanamycin and rifampicin. The LB agar plates were incubated at 28ºC to get the single colonies. These single colonies were cultured into the LB broth. The transformation was confirmed with colony PCR. Agrobacterium LBA4404 50 mL cultures (one transformed with tma12 construct under CaMV-2X35S promoter and the other under NSP promoter) were prepared to transform the N. tabacum.

Transformation into N. tabacum

To get the stable expression of tma12 gene, N. tabacum was transformed with the agrobacterium transformation method described by Horsch et al. [24]. N. tabacum seeds were sterilized by immersing in the 10 % bleach for 10 min and followed by rinsing (2-3 times) with autoclaved distilled water. After completely drying, the seeds were sprinkled over MS medium plates. MS0 medium contained 4.34 g/L of Murashige and Skoog salt, 3 % sucrose, and 2 % agar; and pH was adjusted to 5.7. Then for seed germination, MS agar plates were placed in a growth chamber with controlled conditions (28 + 2 ºC temperature and 16:8 daylight cycle). After germination, each seedling was shifted to an isolated container cast with MS agar medium and allowed to grow for at least 4 weeks to get the explants. After cutting the explants, these were pre-cultured on the solidified MS1 medium supplemented with hormones BAP (1 mg/L) and NAA (1 mg/L) under dark conditions for 2-3 days.

Overnight cultures of LBA4404 transformed with pCambia 2300 plasmids having GOI cassettes (tma12 under CaMV-2X35S promoter and NSP promoter) (Figure S2, Additional file 1) were diluted by 10 times in liquid MS1 medium. For inoculation, pre-cultured explants were dipped in these Agrobacterium diluted cultures for 20-30 min. Explants were removed from the cultures and dried in the aseptic condition. To co-cultivate, these explants were grown over solidified MS1 medium for 2-3 days in growth chamber under dark conditions. After co-cultivation these explants were rinsed with cefotaxime (250 mg/mL) to kill the excessive Agrobacterium cells. After drying these explants were shifted to the shooting medium (MS1 medium supplemented with kanamycin 50 mg/mL). Shoots initiation started after 3-4 weeks. These expected transgenic shoots were cut off and shifted to the rooting medium (recipe described in Table S1-Additional file 1). After the development of roots, plantlets were shifted to the soil in pots for further growth in a controlled environment. These transgenic lines (T₀) were subjected to molecular characterization and bioassays. Seeds from the T₀ lines were also collected to get the T₁ lines. T₁ lines were also subjected to the molecular characterization and whitefly bioassays to check the efficacy of tma12.

Molecular Characterization of Tobacco Transgenic Lines

The T₀ and T₁ lines of tobacco plants expressing tma12 under 35S promoter and NSP promoter were molecularly characterized. For preliminary screening of the transgenic lines, samples were collected from the plants and subjected to the direct PCR without extracting the DNA using the commercial kit Phire Plant Direct PCR (Thermo Scientific). In this protocol, a small piece of the young leaves about 2 mm in size was taken and crushed in the 20 µL of the dilution buffer with a pipette tip. Then this sample mixture was briefly centrifuged and 0.5 µL of the supernatant was taken as the template for the 20 µL reaction. The reaction mixture contained 10 µL of the 2X Phire plant PCR buffer, 0.5 µM of each of the forward and reverse primers (tm12F: 5’-ATGCATGGCTCTATGGAAGATCCT-3’/tm12R: 5’-TCATGTAGTAGAATGAAGTGACAA-3’), 0.5 µL of the template sample, 0.4 µL of the Phire Hot Start II DNA Polymerase and water to make up volume 20 µL. Thermocycling profile was set as 1 cycle of initial denaturation at 94 ºC followed by 35 cycles of 94 ºC for 30 sec, 55 ºC for 30 sec, 72 ºC for 30 sec, and then 1 cycle of final extension at 72 ºC for 5 min. After completing the polymerase chain reaction, samples were electrophoresed through the agarose gel for the confirmation of required band size (651 bp).

Bioassays: Evaluation of Tobacco Transgenic Lines

The confirmed lines were subjected to bioassays to find the efficacy of the transgenic lines against the whitefly. For the bioassays, whiteflies were reared in a controlled environment on the tobacco wild type plants for at least 2 months. Transgenic plants (when reached the 4-6 leaves stage) were exposed to the whiteflies in the random block design along with the control plants (wild-type Coker312) of the same stage in the closed containment with the controlled condition described above. The mortality data of the whiteflies were collected after 14 days of exposure to the whiteflies.

Transformation into Cotton

After checking the efficacy (against the whitefly) of the tma12 gene expressed under both promoters in model plant N. tabacum, cotton transgenic lines expressing TMA12 under 2X35S promoter and under NSP promoter were also developed in this study. To develop transgenic cotton lines Agrobacterium transformation method was used as described by Umbeck et al. [25]. G. hirsutum var. Coker 312 was used to transform with LBA4404 having tma12 construct under 2X35S promoter and NSP promoter.

Seeds of Coker 312 were delineated with H₂SO₄ and then sterilized by immersing the seeds in the mixture of HgCl₂ (0.2%) and SDS (1%) for 15-20 min. After washing with distilled water 4-5 times, seeds were dried and then grown in the glass jars in the growth chamber with controlled conditions (described in the previous section). After about a week, seeds germinated into the hypocotyls which were dissected to prepare the explants. These explants were immersed in the Agrobacterium LBA4404 culture for 20-30 min. After inoculation, these explants were dried and grown over the cotton callus induction (CCI) medium in the petri plates incubated in the dark at 28 ºC for 2 days. Then explants were sub-cultured to the CCI medium supplemented with kanamycin (50 mg/mL) and cefotaxime (250 mg/mL). Sub-culturing had been done to fresh medium after every 2 weeks until the callus emerged from the explants (hypocotyls). These calli were removed and sub-cultured further in the medium as described above.

After 8-10 weeks, the matured calli were sub-cultured to the embryo maturation medium (Table S1). Then after 4-6 weeks, embryos were separated from the calli under aseptic conditions and grown in the embryo germination selection medium until development of complete plantlets. These plantlets were grown in the same medium for root development in the glass jars. After development of roots these putative transgenic plants were shifted to the soil in the pots in the growth chamber.

Molecular Characterization of Transgenic Cotton Lines

PCR Confirmation

DNA was extracted from all the putative transgenic lines of cotton by the CTAB method described above. Integration of the tma12 sequence in the putative transgenic lines was confirmed by the PCR using specific primers. For PCR, DreamTaq Green PCR Master Mix 2X was used, which is ready to use solution, contained optimized DreamTaq DNA Green buffer, DreamTaq Polymerase, MgCl2, and dNTPs. This master mix is optimized to amplify up to 6 kb of the DNA in length. The 20 µL of the total reaction mixture contained the 10 µL of DreamTaq Green PCR Master Mix, 0.5 µM of each of the forward and reverse primers (Ptm12F: 5’-CATGGCTCTATGGAAGATCCT-3’/Ptm12R: 5’-TCATGTAGTAGAATGAAGTGACAA-3’), 25-50 ng of DNA and water to make volume 20 µL. The PCR profile was 98 °C for 3 mins, followed by 35 cycles of 95 °C for 30 sec, 60 °C for 30 sec, and 72 °C for 1 min, with a final step of 72 °C for 5 mins. After completing the PCR, the product was analyzed by running the agarose gel.

Expression Analysis

To estimate the relative expression of the TMA12 in the cotton transgenic plants, real-time quantitative PCR (qPCR) from the cDNA was performed. In the qPCR, sadI gene was used as a reference gene to normalize the C_t value. Each reaction mixture contained 5 µL PowerUpTM SYBRTM Green PCR master mix (Applied Bioscience, USA), 1 µL (10 ng) cDNA, 0.25 µL of each of the forward and reverse primer (qtm12F: 5’-ATGGCAGTATGGAGGATCCTATAAG-3’/qtm12R: 5’-GCGTTTGGGATATTAACTTCGTTCC-3’) and 3.75 µL of water. PCR reactions were initiated with an incubation at 95 °C for 30 sec, followed by a 40 times cycle of 95 °C for 5 sec, 60 °C for 1 min followed by a melting curve was collected analysis. The Ct values were used to quantify and analyze gene expression according to the 2^-^DD^CT method [26]. The RT-qPCR was performed including all positive and negative controls in triplicates. In these experiments, QuantStudioTM 6 Flex System (ThermoScientific, USA) was used with 384 well plates. Changes in the expression level of the gene of interest was calculated by comparing the DDCT values of samples of transgenic plants to those collected from wild type plants.

Copy Number Estimation

To estimate the number of copies of the tma12 that was integrated into the genome of each of the transgenic lines of cotton plants qPCR was performed using DNA as template and sad1 as a reference gene. Each reaction mixture contained 5 µL PowerUpTM SYBRTM Green PCR master mix (Applied Bioscience, USA), 1 µL (25 ng) DNA, 0.25 µL of each of the forward and reverse primer (qtm12F: 5’-ATGGCAGTATGGAGGATCCTATAAG-3’/qtm12R: 5’-GCGTTTGGGATATTAACTTCGTTCC-3’) and 3.75 µL of water. PCR reactions were initiated with an incubation at 95 °C for 30 sec, followed by a 40 times cycle of 95 °C for 5 sec, 60 °C for 1 min, followed by melt curve analysis. The 2^-^DD^CT method [27] was used to estimate the copy number of tma12 with relation to the internal control in each of the transgenic plant.

Bioassays: Evaluation of Cotton Transgenic Lines

Whiteflies (Asia II 1) populations were reared on the cotton plants (wild type: Coker 312) in the closed containment with conditions described above. After estimating expression level of tma12 by qPCR, two plants of each transgenic event were exposed to whiteflies in the closed containment with controlled condition. The eggs and nymphs were counted from 1-inch square area of the three upper leaves and adults were counted from the upper 3 leaves of each plant (transgenic lines and as well as the controlled plants). Thus, each transgenic line had six data points. Then data was tested using ANOVA followed by the Tukey’s HSD test [28].

Cloning of tma12 under 35S and NSP Promoter in pCambia2300

The synthetic gene tma12 was codon optimized and commercially synthesized by the ATUM, Newark, CA, USA. Supplier provided the synthetic gene cloned in the plasmid pJIT201 (plasmid map is shown in the Figure S1 in Additional file1). The confirmation of the gene in plasmid was done by the restriction analysis using HindIII and EcoR1 enzymes and restricted fragments were resolved in the agarose gel (Figure S3 in Additional file1). Then gene was cloned in the pCambia 2300 under 2X35S promoter and NSP promoter. Figure S4 (Additional file 1) shows the restriction confirmation of the gene in the pCambia2300 and Figure S5 (Additional file 1) shows the PCR confirmation of the gene in the binary vector pCambia2300 (under both the promoter CaMV-2X35S and NSP) transformed in the Agrobacterium LBA4404.

Transformation into N. tabacum

Both the construct tma12 under CaMV-2X35S promoter and NSP promoter in the pCambia 2300 vector were transformed to N. tabacum by agrobacterium transformation method in different experiments. Transformation stages of transgenics plants are shown in the Figure S6 (Additional file 1). A total of 16 transgenic lines of tma12 under CaMV-2X35S promoter were developed and 5 lines of tma12 under NSP promoter were developed. All the transgenic lines were confirmed by PCR using specific primers (Figure 1 panel A). These transgenic lines were grown to get the seeds for T₁ generation. Seeds of T₁ generations were grown on the selection medium MS supplemented with kanamycin (50 mg/mL). Then kanamycin resistant plants were grown in pots and confirmed by the PCR as shown in the Figure 1 panel B, C and D. Two plants (T₁ generation) of every line were used as biological replicates in the bioassay’s experiments (Figure S7 in Additional file 1).

Bioassays: Evaluation of Tobacco Transgenic Lines

The transgenic tobacco plants (T₁) when reached to 4-6 leaves stage were exposed whiteflies infestation after confirmation through PCR. Whiteflies were let to acclimatize for a week on the tobacco plants and after 2^nd week of exposure to the whiteflies, mortality data was taken. The mean mortality rate of whiteflies for each of the transgenic lines and the control plants is shown in the graph (Figure 2). Two to three plants from the same line were used as biological replicates. The mortality rate of whiteflies on all the transgenic lines (except 35S-5) expressing TMA12 under CaMV-2X35S promoter was observed significantly higher as compared to control plant (wild type). The mortality rate ranged from 32 to 88 percent on the 7^th day of the exposure to whiteflies. There were 5 transgenic lines expressing TMA12 under the NSP promoter. The mortality rate of the whiteflies on 4 transgenic lines was significantly higher as compared to control plants. The mortality rate of whiteflies on the line NSP-1 was not significantly different from the control plants. Mortality rates ranged from 57 to 80 percent on the 7^th day of the exposure to whiteflies.

Transformation into Cotton

There transgenic events expressing TMA12 under CaMV-2X35S promoter (35S-E66, 35S-E68, and 35S-81) and one transgenic event expressing TMA12 under NSP promoter (NSP-E5) was developed. There were 3-6 transgenic plants of each transgenic event, which were used as biological replicates in the bioassay experiments (Figure S8 in additional file 1). The confirmation of transgenic plants was done through PCR (Figure 3).

Transgene Copy Number

To estimate the number of copies of the tma12 that integrated into the genome of each of the transgenic line of cotton plants, absolute quantification was determined by real time quantitative PCR using sad1 as an endogenous control gene. The 2^-^DD^CT method [27] was used to estimate the copy number of tma12 in each of the transgenic plant. The amplification of serial dilution of the transgene and standard curve is shown in the Figure S9 (Additional file 1). By this method, it was estimated that there was 1 copy of the tma12 in all three the transgenic events having CaMV-2X35S promoter and 3 copies in the transgenic event with NSP promoter. Table 1 shows the calculated values for 2^-(∆∆Ct).

Relative Expression of TMA12

The relative expression level of each of transgenic line as compared to wild type is shown in Figure 4. The highest expression level was observed in the 35S-E68 that is about 2-fold higher as compared to the control. Although all the transgenic events (with 35S promoter) have the same copy number integration but have different expression levels of the protein, might be due to different sites of integration. The expression level was higher in the transgenic line NSP-E5 as compared to transgenic lines 35S-66 and 35S-81 because NSP-E5 has three copy numbers while the others have 1 copy number of the transgene.

Bioassays: Evaluation of Cotton Transgenic Lines

The adult whiteflies on the upper three leaves were counted from T₀ transgenic plants. Similarly eggs, and nymphs were also counted in a 1-inch square area of each of the upper three leaves of transgenic plants. There were two transgenic plants of each transgenic event in the bioassays so there were six data points of each transgenic event. In Figure 5, graph A shows the count of adult whiteflies per leaf of transgenic plants. Graphs (B and C) show the eggs and nymphs per square inch area of the upper three leaves, respectively. Data were analyzed by the ANOVA followed by the post-hoc Tukey’s HSD test. Transgenic plants were significantly different from the control plants in terms of adult whiteflies count, eggs, and nymphs data (Figure 5 and Figure 6). Based on this it was concluded that transgenic plants expressing TMA12 under the 35S promoter showed more resistance than the transgenic plants expressing TMA12 under the NSP promoter.

A transgenic approach is a sustainable approach with a one-time investment and it can largely contribute to integrated pest management as proved by the successful example of Bt cotton [29, 30]. In this study, transgenic cotton lines expressing TMA12 under CaMV-2X35S promoter and NSP promoter were developed to compare their efficacy against the whiteflies. Before developing cotton transgenic lines, tobacco transgenic plants were also developed to get preliminary results about the efficacy of the transgene as described in the results. Transgene tma12 was codon optimized for the improved expression in the cotton plant. Along with the codon optimization, adjustment of the AT/GC bias was also considered to get maximum expression in the cotton. The sequence of the protein and function is not affected by the codon optimization.

The phloem specific promoter was previously used effectively against the sap sucking pests. In a study, NSP promoter was used to express the insecticidal proteins lectin and a neurotoxin (Hvt) in tobacco and resulted in the 100% mortality of whiteflies [17]. In another study, a GroEL gene expressed under Arabidopsis phloem-specific promoter in the tomato effectively controlled the virus transmission by the B. tabaci [31]. Phloem-specific promoters have also been used to express protein to control other sap sucking pests e.g. aphid [32]. The purpose of using the phloem-specific promoter is to contain the gene expressing on targeted location phloem sap cells because the whitefly is a sap sucking pest and moreover it saves the resources of the plant cell. In this study, it is evident from the data that cotton transgenic line (NSP-E5) expressing TMA12 under phloem-specific promoter is significantly resistant to the whiteflies as compared to the control plants. But the cotton transgenic lines expressing TMA12 under constitutive promoter still performed better as compared to the lines with NSP promoter. Some previous studies in which constitutive and phloem-specific promoters were compared, observed similar results and some other reported contrasting results. A study described similar results according to which ASAL gene is more effective under 2X35S promoter than that of rolC promoter but Umer et al. [22] observed the contrasting results which described enhanced effectivity of the agglutinin (insecticidal lectin) gene (pta) under phloem specific promoter (rolC) as compared to the 35S promoter against the aphid (Myzus persicae). Therefore, it can be concluded from this study and some previous reports that using the phloem-specific promoter is effective strategy against phloem sap-sucking pest but still their efficiency is not equal to the CaMV-2X35S promoter but in the future, more phloem-specific promoters can be explored for enhanced effectivity.

The expression analysis shows that although there is a single copy number integration of transgene in all transgenic lines (35S-E66, 35S-68, and 35S-81) that are expressing TMA12 under CaMV-2X35S promoter these showed different expression levels of the protein which is probably due to the difference in the site of integration in the genome. The effect of the site of the integration on the expression level is quite evident from the past research studies [33]. Bioassays data showed, the least count of whitefly infestation and the lowest fecundity was observed on the 35S-E68. These results were also justified by the expression analysis which described the most expression level of 35S-E68 as compared to the other transgenic lines. The cotton transgenic line (NSP-E5) expressing TMA12 under NSP promoter, integrated three copy number of the transgene. The expression level of the NSP-E5 was lower than the 35S-E68 but more than the 35S-E81 and 35S-E66. As the NSP-E5 has three copy numbers but the expression level is not always directly related to the copy number [22] because different other parameters are also involved. If there would be a single copy of transgene in the NSP-E5 like the 35S transgenic lines, then the comparison of the expression analysis would be possible. Probably the greater number of transgenic lines with the NSP promoter might give more resolved results about the comparison of the promoter effectiveness.

This study provides the transgenic resource with optimized codon transgene and compared the effectiveness of the constitutive and phloem-specific promoters to control the whiteflies. From data it can be concluded that using NSP promoter is potentially an effective strategy against phloem sap-sucking pests but still its efficiency is not equal to the 35S promoter. But using a phloem-specific promoter has advantages over the constitutive promoter like tissue-specific expression saves the molecular resources of the plant cells and is less prone to biosafety concerns. In the future, more phloem specific promoters can be explored for enhanced effectivity. Moreover, using the gene tma12 which is plant derived is not liable to biosafety concerns.

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Table 1: Copy number of tma12 in the cotton transgenic lines

Transgenic lines (cotton)	∆∆Ct	2^-(∆∆Ct)	Estimated Gene Copy Number
35S-E66	0.93	0.52	1
35S-E68	0.69	0.62	1
35S-E81	0.91	0.53	1
NSP-E5	-1.34	2.53	3

No competing interests reported.

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Expression of an Insecticidal Protein under Phloem Specific Promoter in Cotton Plants to Control Whitefly

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Abstract

Figures

Introduction

Methodology

Synthetic tma12 Gene and Codon Optimization

Cloning of tma12 Gene under CaMV-2X35S Promoter and NSP Promoter

Transformation into Agrobacterium LBA14404

Transformation into N. tabacum

Molecular Characterization of Tobacco Transgenic Lines

Bioassays: Evaluation of Tobacco Transgenic Lines

Transformation into Cotton

Molecular Characterization of Transgenic Cotton Lines

PCR Confirmation

Expression Analysis

Copy Number Estimation

Bioassays: Evaluation of Cotton Transgenic Lines

Results

Cloning of tma12 under 35S and NSP Promoter in pCambia2300

Transformation into N. tabacum

Bioassays: Evaluation of Tobacco Transgenic Lines

Transformation into Cotton

Transgene Copy Number

Relative Expression of TMA12

Bioassays: Evaluation of Cotton Transgenic Lines

Discussion

Conclusion

References

Table 1

Additional Declarations

Supplementary Files

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