AtDREB2A Gene Expression Under Control of the Inducible Promoter and Virus 5’-untranslated Regions Improves Tolerance to Salinity in Nicotiana Tabacum

Transcriptional factor DEHYDRATION-RESPONSIVE ELEMENT BINDING PROTEIN 2A (DREB2A) induces the expression of many genes in dehydration, heat shock, and salinity in Arabidopsis. Deletion of sequence coding the 30 amino acid central region transforms full length (FL) protein DREB2A FL into a more stable and constitutively active form known as DREB2A CA. Here, using agrobacteria, a leaf disc transformation of Nicotiana tabacum v. Samsun NN was carried out by transgenes AtDREB2A-FL and AtDREB2A-CA coding the proteins with His-tag on the С-end. The effects of combinations of constitutive 35S CaMV promoter or inducible rd29A promoter with different viral or artificial 5’-untranslated regions (UTR), 5’TMV, 5’PVY, 5’AMV or 5’ARC1, known as translational enhancers were evaluated on the both transgenes’ expression. Using an antibody to His-tag, recombinant protein synthesis was detected in transgenic plants in normal and heat shock conditions. After comparative analysis, it was shown that the properties of different 5’-UTRs vary greatly and depended on separate conjunction of promoter and transgene. The integration of AtDREB2A CA under control of the rd29A promoter and 5’TMV or 5’AMV in genome effectively improved tolerance of tobacco transgenic plants to 400 mM NaCl and to drought.

Controlled Over-Expression of AtDREB1A Enhances Tolerance against Drought and Salinity in Rice

Engineering transcription factors (TF) hold promise in enhancing abiotic stress tolerance in plants. In this study, one of the popular rice varieties of South India, namely ADT 43, was engineered with a TF AtDREB1A driven by a stress-inducible rd29A promoter. PCR and Southern hybridization were employed to confirm the integration and copy number of the transgene. Transgenic lines (T1) of ADT 43 showed enhanced tolerance to drought and salinity compared to the non-transgenic ADT 43. Transgenic lines were found to maintain higher RWC %, lower leaf temperature, and partially closed stomata, enabling better survival under stress conditions. qRT-PCR analysis revealed the strong induction of AtDREB1A transcripts during drought. Transgenic lines of ADT 43 exhibited increased germination and retention of chlorophyll in their leaves under salinity. Evaluation of transgenic lines under transgenic screen house conditions revealed that line # A16 exhibited on par agronomic performance against its non-transgenic counterpart under normal conditions. Under drought, non-transgenic ADT 43 showed >20% reduction in the total number of spikelets per panicle, whereas transgenic line # A16 registered only a 2% reduction. Non-transgenic ADT 43 recorded 80% yield reduction under drought, whereas line # A16 recorded only 54% yield loss. The above results demonstrated the effectiveness of controlled expression of DREB1A in regulating dehydration responses in rice.

Expression of Oryza sativa galactinol synthase gene in maize (Zea may L.)

Galactinol synthase (GolS) is a key biological catalyst for the synthesis of the raffinose oligosaccharides (RFOs) which play important roles in abiotic stress adaptation of plants, especially drought tolerance. GolS gene has been isolated on a variety of plants in order to create material resources for generating transgenic plants resistant to adverse environmental factors. In our previous research, we have isolated a GolS gene from drought stress cDNA library of Oryza sativa L. Moctuyen (named OsGolS). In this study, the expression vector pCAM-Rd/OsGolS carrying the isolated OsGolS gene under the control of stress-inducible Rd29A promoter was constructed and introduced into Agrobacterium tumefaciens LBA4404, which was used for maize transformation. PCR and Real-time PCR assay indicated that transgene was integrated in the genome of the regenerated Zea mays plants. Reverse transcription-PCR showed that the OsGolS was transcribed into mRNA in Zea mays and was highly expressed. These results provide a basis for the study of the function of OsGolS in drought responses and for the development of drought stress tolerant crops.

Construction an expression vector and Agrobacterium tumefaciens strain carrying ZmbZIP72 gene isolated from maize

In plants, members of bZIP transcription factors are involved in various biological processes such as organ and tissue differentiation or vascular development. Moreover, the basic leucine zipper (bZIP) is one of the largest transcription factor families which play an important role in environmental stress responses. However, few bZIP genes are related to abiotic stress responses. Some studies have found that transgenic plants overexpressing the bZIP genes enhanced tolerance to abiotic stresses. Several of stress-related genes in maize have been identified and characterized. A ZmbZIP72 gene belonged to A group could be strongly induced by ABA and abiotic stresses. Some previous studies have shown that overexpression of the ZmbZIP72 gene resulted in improved drought and salt tolerance of a transgenic Arabidopsis. In this work, the ZmbZIP72 gene from drought-treated maize was isolated and sequenced with 894 bp in full-length of the coding region. Nucleotide comparison of the ZmbZIP72 gene and a GenBank sequence (accession number HQ328839) revealed that two changes were found at positions 486 (A to C) and 493 (C to T), respectively. Changes in predicted amino acid sequence were at positions 102 (Lys>Asp) and 105 (Pro>Ser). The newly isolated ZmbZIP72 gene was ligated with RD29A promoter and 35S terminator to create a RD29::ZmbZIP72::35S cassette in pRTL2 vector. Afterwards, this cassette was constructed into pCAMBIA1301 and the new recombinant pCAMBIA1301 vector carrying ZmbZIP72 construct has transformed into A. tumefaciens strain EHA105 which is material for plant transformation.

Preparation of Graphene Oxide and Its Mechanism in Promoting Tomato Roots Growth

Graphene oxide is a new kind of nanomaterial. The graphene oxide was prepared and its quality detected by atomic force microscopy (AFM) and transmission electron microscopy (TEM), for better understanding of effects of the nanomaterial on plants. Wild type (WT) tomato (Solanum lycopersicum) germplasm ‘New Yorker’ and corresponding transgenic plants (Prd29A::LeNCED1) were treated with prepared graphene oxide. 9-cis-epoxycarotenoid dioxygenase (NCED) is a key gene for ABA biosynthesis and overexpression of the NCED resulted in ABA accumulation and higher drought tolerance. Seminal root length in the WT tomato was longer than that in the control samples when the seedlings were treated with 20 mg/L graphene oxide for 15 days. In contrast, the same treatment resulted in shorter seminal root length in the transgenic plants compared with control samples. The graphene oxide treatments led to lower Superoxide Dismutase (SOD), Peroxidase (POD), Catalase (CAT) activity and Malondialdehyde (MDA) content in the WT and transgenic plants. 20 mg/L graphene oxide treatment also affected the transcript levels of IAA7, IAA4 and IAA10 but the effect on the wild type and corresponding transgenic plants was different. IAA4 transcription level decreased both in the WT and Prd29A::LeNCED1 transgenic plants while the IAA7 transcription level decreased in the transgenic plants and increased in the WT tomato. The IAA10 transcription level decreased in the WT tomato and increased in the Prd29A::LeNCED1 transgenic plants. Graphene oxide treatments resulted in higher transcription level of ABCG25 and ABCG40 in the WT plants but had no significant effect on transgenic plants. The transcription level of NCED in the WT and Prd29A::LeNCED1 transgenic plants treated with graphene oxide increased significantly, however, it was higher in the transgenic plants than in the WT tomato after 15 d treatment, indicating that the graphene oxide activated the rd29A promoter as does drought and salt. The HD-ZIP transcription level only decreased significantly in the treated Prd29A::LeNCED1 transgenic plants. All these results suggested that there was a crosstalk between ABA and graphene oxide and the graphene oxide affected plant growth through the ABA and IAA pathway.

Expression of the DREB1A gene in lentil (Lens culinaris Medik. subsp. culinaris) transformed with the Agrobacterium system

Until now three publications have reported the development of transgenic lentil plants through protocol optimisation using the gusA gene, but there are no reports of the introduction of a gene with agronomic importance. In the present study we report the introduction of the DREB1A gene into lentil to enhance drought and salinity tolerance. Decapitated embryos were immersed in Agrobacterium suspension and then co-cultivated for 4 days. Direct organogenesis was induced from the apical meristems and cotyledonary buds. Subsequently, the explants were subjected to selection in medium containing 10 mg/L phosphinothricin for nine rounds with 2-week intervals. The putative transgenic explants were micro-grafted onto non-transformed rootstocks to establish transgenic plants. The PCR results confirmed the insertion and stable inheritance of the gene of interest and bar marker gene in the plant genome. The Southern blot analysis revealed the integration of a single copy of the transgenes. T0 plants and progeny up to T2 generations showed complete resistance to the herbicide Basta. The DREB1A gene driven by the rd29A promoter was induced in transgenic plants by salt stress from sodium chloride solution. The total RNA was extracted and cDNA synthesised. The results showed that DREB1A mRNA was accumulated and thus the DREB1A transgene was expressed in the transgenic plants, whereas no expression was detected in the non-transformed parents.