Expression of <Emphasis Type="Italic">GNA</Emphasis> and biting site-restricted <Emphasis Type="Italic">cry1Ac</Emphasis> in cotton; an efficient attribution to insect pest management strategies

Insect-resistant transgenic cotton has been commercialized for two decades. Most of the introduced cultivars express Bt gene(s) constitutively under the control of 35S promoter in whole-plant tissues. However, there have been other promoters considered by researchers to confine the toxin expression to targeted organ and tissues. We developed a triple-gene construct including GNA, cry1Ac and cp4 epsps genes. We attempted to confine cry1Ac expression to insect biting sites by cloning it to downstream of a wound-inducible promoter isolated from Asparagus officinalis (AoPR1). Moreover, to broaden the range of resistance, GNA was driven by the 35S promoter to target the sap-sucking insects like aphids which impose large losses in cotton production. To select the transformants in selection medium and for glyphosate tolerance, GNA and cry1Ac genes were accompanied with cp4 epsps gene. Two binary vectors harboring desired genes were constructed and utilized in the study (pGTGNAoC1AC and pGTGN35C1AC). Transformation of cultivar GSN-12 was carried out by employing Agrobacterium tumefaciens strain EHA105. Plantlets were primarily screened under glyphosate (N-phosphonomethyl glycine) selection pressure and subsequently subjected to molecular and biotoxicity assays. Introduction of cry1Ac and GNA to cotton plant conferred resistance to Spodoptera littoralis and Aphis gossypii Glover. Restriction of cry1Ac toxin protein to insect biting sites along with a plant lectin attributes significantly to insect pest management strategies.     

Promoters pro-SmAMP1 and pro-SmAMP2 from Wild Plant <Emphasis Type="Italic">Stellaria media</Emphasis> for the Biotechnology of Dicotyledons

Comparative results of the studied effectiveness of two new promoters, pro-SmAMP1 and pro- SmAMP2, from chickweed (Stellaria media L.) in various types of cultivated plants with transient expression and in stable transformants are given. The effectiveness of the promoters was evaluated through the expression of the reporter uidA gene by measuring the activity of its GUS protein product. It was found that the deletion variant (442 bp) of the pro-SmAMP1 promoter was significantly stronger in plants of Nicotiana benthamiana (Domin) with transient expression than the deletion variant (455 bp) of the pro-SmAMP2 promoter. The effectiveness of these short deletion variants of both promoters under transient expression in the plants of rapeseed (Brassica napus L.) and sugar beet (Beta vulgaris L.) was comparable with that of the viral CaMV35S promoter. The functionality of the pro-SmAMP2 promoter in the calluses of common flax plants (Linum usitatissimum L.) was shown. In the homozygous lines of transgenic tobacco plants (Nicotiana tabacum L.), all deletion variants of the pro-SmAMP1 promoter and the shortest version of pro-SmAMP2 were twice as strong as the CaMV35S viral promoter. The effectiveness of short variants of both promoters from the chickweed in controlling the gene encoding neomycin phosphotransferase II in the transgenic plants of tobacco and arabidopsis (Arabidopsis thaliana L.) growing on media supplemented with recommended concentrations of kanamycin are not inferior to the duplicated 2хCaMV35S viral promoter. The obtained experimental data show that short deletion variants of pro-SmAMP1 (442 bp) and pro-SmAMP2 (455 bp) promoters may be recommended as strong constitutive promoters for use in the biotechnology of crop plants.     

Towards better insect management strategy: restriction of insecticidal gene expression to biting sites in transgenic cotton

Most of the commercialized Bt crops express cry genes under 35S promoter that induces strong gene expression in all plant parts. However, targeted foreign gene expression in plants is esteemed more important as public may be likely to accept ‘less intrusive’ expression of transgene. We developed plant expression constructs harboring cry1Ac gene under control of wound-inducible promoter (AoPR1) to confine Bt gene expression in insect wounding parts of the plants in comparison with cry1Ac gene under the control of 35S promoter. The constructs were used to transform four Turkish cotton cultivars (GSN-12, STN-468, Ozbek-100 and Ayhan-107) through Agrobacterium tumefaciens strains GV2260 containing binary vectors p35SAcBAR.101 and AoPR1AcBAR.101 harboring cry1Ac gene under control of 35S and AoPR1, respectively. Phosphinothricin (PPT) was used at concentration of 5 mg L^?1 for selection of primary transformants. The primary transformants were analyzed for transgene presence and expression standard molecular techniques. The transformants exhibited appreciable mortality rates against larvae of Spodoptera exigua and S. littoralis. It was found that mechanical wounding of T ₁ transgenic plants was effective in inducing expression of cry1Ac protein as accumulated levels of cry1Ac protein increased during post-wounding period. We conclude that use of wound-inducible promoter to drive insecticidal gene(s) can be regarded as a valuable insect-resistant management strategy since the promoter activity is limited to insect biting sites of plant. There is no Bt toxin accumulation in unwounded plant organs, seed and crop residues, cotton products and by-products, thus minimizing food and environmental concerns.     

Transgenic Bt cotton driven by the green tissue-specific promoter shows strong toxicity to lepidopteran pests and lower Bt toxin accumulation in seeds

A promoter of the PNZIP(Pharbitis nil leucine zipper)gene(1.459 kb)was cloned from Pharbitis nil and fused to the GUS(b-glucuronidase)and Bacillus thuringiensis endotoxin(Cry9C)genes.Several transgenic PNZIP::GUS and PNZIP::Cry9C cotton lines were developed by Agrobacterium-mediated transformation.Strong GUS staining was detected in the green tissues of the transgenic PNZIP::GUS cotton plants.In contrast,GUS staining in the reproductive structures such as petals,anther,and immature seeds of PNZIP::GUS cotton was very faint.Two transgenic PNZIP::Cry9C lines and one transgenic cauliflower mosaic virus(Ca MV)35S::Cry9C line were selected for enzyme-linked immunosorbent assay(ELISA)and insect bioassays.Expression of the Cry9C protein in the 35S::Cry9C line maintained a high level in most tissues ranging from24.6 to 45.5μg g~(-1) fresh weight.In green tissues such as the leaves,boll rinds,and bracts of the PNZIP::Cry9C line,the Cry9C protein accumulated up to 50.2,39.7,and 48.3μg g~(-1) fresh weight respectively.In contrast,seeds of the PNZIP::Cry9C line(PZ1.3)accumulated only 0.26μg g~(-1) fresh weight of the Cry9C protein,which was 100 times lower than that recorded for the seeds of the Ca MV 35S::Cry9C line.The insect bioassay showed that the transgenic PNZIP::Cry9C cotton plant exhibited strong resistance to both the cotton bollworm and the pink bollworm.The PNZIP promoter could effectively drive Bt toxin expression in green tissues of cotton and lower accumulated levels of the Bt protein in seeds.These features should allay public concerns about the safety of transgenic foods.We propose the future utility of PNZIP as an economical,environmentally friendly promoter in cotton biotechnology.     

Different response of an elite <Emphasis Type="Italic">Bt</Emphasis> restorer line of hybrid rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) in adaptation to nitrogen deficiency

Transgenic Bacillus thuringiensis (Bt) rice have been reported to acquire effective resistance against the target pests; however, the insertion and expression of alien Bt genes may have some unintended effects on the growth characteristics of rice. A screen-house experiment was conducted and repeated twice to investigate the growth characteristics and Bt protein expressions in two Bt rice lines [MH63 (Cry2A*) and MH63 (Cry1Ab/Ac)], which had different Bt protein expression levels in leaves, under zero nitrogen (N0) and recommended nitrogen (NR) fertilizer applications. Compared to the counterpart MH63, MH63 (Cry2A*) under N0 experienced accelerated leaf senescence and a lower internal N use efficiency (IE_N), resulting in a 23.2% decrease in grain yield and a lower accumulated biomass. These variations were revealed to be correlated to the higher ratio of the Bt protein content to the soluble protein content (BTC/SPC) with a maximum value of 4.3‰ in MH63 (Cry2A*) leaves in the late growth stage. Under NR, no differences in growth characteristics between MH63 (Cry2A*) and MH63 were found. The growth characteristics of MH63 (Cry1Ab/Ac), with a lower BTC/SPC in the late growth stage compared to MH63 (Cry2A*), were identical to those of MH63 under the two N applications. Results show that the transgenic Bt rice MH63 (Cry2A*), with a relatively higher Bt protein expression in the late growth stage, had an inferior adaptation to nitrogen deficiency compared to its non-Bt counterpart. And this inferior adaptation was found to be correlated with the higher BTC/SPC in MH63 (Cry2A*) leaves in the late growth stage.     

Targeted expression of insecticidal hybrid SN19 gene in potato leads to enhanced resistance against Colorado potato beetle (<Emphasis Type="Italic">Leptinotarsa decemlineata</Emphasis> Say) and tomato leafminer (<Emphasis Type="Italic">Tuta absoluta</Emphasis> Meyrick)

The expression of insecticidal genes must be induced at appropriate time and in sufficient amount to confer protection against targeted pests. However, the increased scientific reports of resistance development in insect pest against insecticidal delta-endotoxins, produced by Bacillus thuringiensis, provide impetus for the development of alternative insect management strategies. The present study was conducted to investigate the importance of targeted expression of a hybrid insecticidal gene (SN19) in potatoes. For this purpose, two plant expression vectors were constructed by cloning hybrid SN19 gene (cry1Ba-domain I–III and cry1Ia-domain II) under the control of a wound-inducible promoter isolated from Asparagus officinalis (AoPR1) and CaMV 35S promoter, and were transferred to Agrobacterium tumefaciens strain EHA 105. Four potato genotypes (Marabel, Innovator, Tokat 10/1 and Tokat 6/24) were transformed with EHA 105 strain harboring pTF101.1 35S–SN19 and pTF101.1 AoPR1–SN19 constructs. Phosphinothricin (PPT) was used at concentration of 1 mg/l for selection of primary transformants. PCR results showed the presence of both introduced SN19 and bar genes in 43 plants out of total 154 putative transgenics. Expression of SN19 protein in primary transformants was confirmed by Western blot assays. The mechanical wounding of transgenic plants exhibited more accumulated levels of SN19 proteins during post wounding period. Leaf biotoxicity assays with Colorado potato beetle (Coleoptera) and tomato leafminer (Lepidoptera) exhibited 100% mortality of the pests in primary transformants. Based on our mortality results with both constructs, we concluded that the potato transgenic lines exhibited targeted expression of insecticidal gene under the control of AoPR1 promoter upon insect wounding with eliminated toxicity of Cry protein and hence can be further used effectively in potato breeding programme.     

Structural and functional analysis of new plant promoter pro-SmAMP1 from <Emphasis Type="Italic">Stellaria media</Emphasis>

The nucleotide sequence of a fragment of the promoter region of pro-SmAMP1 gene, having a length of 1257 bp and encoding antifungal peptides, was determined in chickweed (Stellaria media (L.) Vill.). Computer analysis of the nucleotide sequence revealed a number of cis-elements that are typical strong plant promoters. Five 5′-deletion variants were created taking into account the distribution of cis-elements:–1235,–771,–714,–603, and–481 bp of pro-SmAMP1 gene promoter, which were fused to the coding region of the uidA reporter gene in pCambia1381Z plant expression vector. The efficacy of pro-SmAMP1 promoter deletion variants was determined by transient expression in plants of Nicotiana benthamiana and using sequential generations of transgenic Nicotiana tabacum plants. It was found that the levels of GUS reporter protein activity in the extracts from transgenic and agroinfiltrated plants using all deletion variants of pro-SmAMP1 gene promoter were 3–5 times higher than those of 35S CaMV viral promoter. The highest activity of GUS protein was observed in the leaves of transgenic tobacco plants and closely correlated with the mRNA level of encoding gene. The levels of GUS activity did not differ significantly among 11 independent homozygous lines of T₂ generation of N. tabacum plants with different deletion variants of pro-SmAMP1 promoter. The results give reason to assume that all deletion variants of pro-SmAMP1 promoter provide stable and high level of expression of controlled genes. The shortest deletion variant–481 bp of pro-SmAMP1 promoter should be viewed as a potentially strong plant promoter for the genetic engineering of plants.     

Development of leaffolder resistant transgenic rice expressing <Emphasis Type="Italic">cry2AX1</Emphasis> gene driven by green tissue-specific <Emphasis Type="Italic">rbcS</Emphasis> promoter

The insecticidal cry genes of Bacillus thuringiensis (Bt) have been successfully used for development of insect resistant transgenic rice plants. In this study, a novel cry2AX1 gene consisting a sequence of cry2Aa and cry2Ac gene driven by rice rbcS promoter was introduced into a rice cultivar, ASD16. Among 27 putative rice transformants, 20 plants were found to be positive for cry2AX1 gene. The expression of Cry2AX1 protein in transgenic rice plants ranged from 5.95 to 122.40 ng/g of fresh leaf tissue. Stable integration of the transgene was confirmed in putative transformants of rice by Southern blot hybridization analysis. Insect bioassay on T₀ transgenic rice plants against rice leaffolder (Cnaphalocrosis medinalis) recorded larval mortality up to 83.33 %. Stable inheritance and expression of cry2AX1 gene in T₁ progenies was demonstrated using Southern and ELISA. The detached leaf bit bioassay with selected T₁ plants showed 83.33–90.00 % mortality against C. medinalis. The whole plant bioassay for T₁ plants with rice leaffolder showed significant level of resistance even at a lower level of Cry2AX1 expression varying from 131 to 158 ng/g fresh leaf tissue during tillering stage.     

Heterologous expression of two <Emphasis Type="Italic">Physcomitrella patens</Emphasis> group 3 late embryogenesis abundant protein (LEA3) genes confers salinity tolerance in arabidopsis

Salinity stress is a major limiting factor in agriculture and adversely affecting the whole plant. As a halophyte, the moss Physcomitrella patens, has been suggested to be an ideal model plant to study salinity tolerance and adaption. Two abiotic stress-responsive Group 3 Late Embryogenesis Abundant protein genes had been identified in P. patens and named as PpLEA3-1 and PpLEA3-2, respectively. Functions of these two genes were analyzed by heterologous expressions in Arabidopsis, driven either by their native P. patens promoters or by the 35S CaMV constitutive promoter. Phenotype analysis revealed that pLEA3::LEA3, pLEA3::LEA3::GFP and 35S::LEA3::GFP transgenic lines had stronger salinity resistance than that in the wild type and empty-vector control. Further analysis showed that the contents of proline and soluble sugar were increased and the malondialdehyde (MDA) were repressed in these transgenic plants after exposure to salinity stress. Our observations indicate that these two Group 3 PpLEA genes played a role in the adaption to salinity stress.     

Overexpression and oral immunogenicity of a dengue antigen transiently expressed in <Emphasis Type="Italic">Nicotiana benthamiana</Emphasis>

To understand the genetic and expression stability of transgenic insect-resistant poplar 741, this study compared the experimental plantations of transgenic insect-resistant poplar 741 lines (pb1, pb6, pb11, pb17, and pb29) with non-transgenic poplar 741, P. tomentosa Carr.f.yixianensis (poplar 84 K) and transgenic hybrid progeny lines cultured from immature embryos. The insect resistance and growth stability of transgenic poplar 741 were investigated by detecting exogenous genes by polymerase chain reaction (PCR), measuring the diameter at breast height (DBH) and volume growth, and performing insect-resistance tests against Clostera anachoreta and Hyphantria cunea. The inheritance and expression of the exogenous gene was also examined in transgenic hybrid progeny lines. The results revealed that the exogenous gene was stable, remaining stable in 8–10-year-old transgenic poplar 741 trees. No significant difference was found between the height of 10-year-old transgenic poplar 741 and non-transgenic poplar 741 in the experimental plantations in Baoding, China. The DBH and volume growth of pb17 was significantly greater than that of pb29 and pb11. The 8-year-old transgenic poplar 741 pb29 grown in Zhuozhou showed no significant difference from poplar 741 in terms of height growth, DBH, and volume. From 1999 to 2013, pb29-fed larvae (C. anachoreta larvae and H. cunea) exhibited stable mortality rates >79%. Likewise, pb11-fed larvae showed stable mortality rates (C. anachoreta larvae had mortality rates >75%, and H. cunea larvae exhibited rates >80%). pb17 conferred low insect-resistant stability, showing mortality rates that varied from 28.2 to 99.27% in C. anachoreta and H. cunea larvae. Among the hybrid progeny lines acquired by hybridization of pb1, pb29, and pb11 with 84 K poplar, the ratios of PCR-positive to PCR-negative lines for the BtCry1Ac gene were 1.31, 1.15, and 0.86, respectively. X ² tests showed that the ratio was consistent with the Mendelian law of 1:1 segregation controlled by an allele pair. The hybrid progeny of pb6?×?84 K had a segregation ratio of 3:1. The nptII gene followed the same segregation rule as Cry1Ac. The transgenic hybrid progeny that contained Cry1Ac gene exhibited the same insect resistance as the parent plants.     

Expression of plant antimicrobial peptide <Emphasis Type="Italic">pro-SmAMP2</Emphasis> gene increases resistance of transgenic potato plants to <Emphasis Type="Italic">Alternaria</Emphasis> and <Emphasis Type="Italic">Fusarium</Emphasis> pathogens

The chickweed (Stellaria media L.) pro-SmAMP2 gene encodes the hevein-like peptides that have in vitro antimicrobial activity against certain harmful microorganisms. These peptides play an important role in protecting the chickweed plants from infection, and the pro-SmAMP2 gene was previously used to protect transgenic tobacco and Arabidopsis plants from phytopathogens. In this study, the pro-SmAMP2 gene under control of viral CaMV35S promoter or under control of its own pro-SmAMP2 promoter was transformed into cultivated potato plants of two cultivars, differing in the resistance to Alternaria: Yubiley Zhukova (resistant) and Skoroplodny (susceptible). With the help of quantitative real-time PCR, it was demonstrated that transgenic potato plants expressed the pro-SmAMP2 gene under control of both promoters at the level comparable to or exceeding the level of the potato actin gene. Assessment of the immune status of the transformants demonstrated that expression of antimicrobial peptide pro-SmAMP2 gene was able to increase the resistance to a complex of Alternaria sp. and Fusarium sp. phytopathogens only in potato plants of the Yubiley Zhukova cultivar. The possible role of the pro-SmAMP2 products in protecting potatoes from Alternaria sp. and Fusarium sp. is discussed.