Functional Characterization of a Bidirectional Plant  Promoter from Cotton Leaf Curl Burewala Virus Using an Agrobacterium-Mediated Transient Assay

Ashraf, Muhammad Aleem; Shahid, Ahmad Ali; Rao, Abdul Qayyum; Bajwa, Kamran Shehzad; Husnain, Tayyab

doi:10.3390/v6010223

Cited by 23 publications

(19 citation statements)

References 79 publications

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“…An agro-infiltration assay was used to check the efficacy of the cloned genes transient expression [ 11 ], resulting in a bluish-green color in the infiltrated region. Similar results were obtained by Ashraf, Bakhsh, and Pathi [ 26 - 28 ]. Transformation of the Cry genes with transit peptides not only makes it possible to localize transgene proteins in green parts of the plant, but it is also helpful in overcoming health and biosafety issues.…”

Section: Discussionsupporting

confidence: 90%

Chloroplast localization of Cry1Ac and Cry2A protein- an alternative way of insect control in cotton

et al. 2015

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BackgroundInsects have developed resistance against Bt-transgenic plants. A multi-barrier defense system to weaken their resistance development is now necessary. One such approach is to use fusion protein genes to increase resistance in plants by introducing more Bt genes in combination. The locating the target protein at the point of insect attack will be more effective. It will not mean that the non-green parts of the plants are free of toxic proteins, but it will inflict more damage on the insects because they are at maximum activity in the green parts of plants.ResultsSuccessful cloning was achieved by the amplification of Cry2A, Cry1Ac, and a transit peptide. The appropriate polymerase chain reaction amplification and digested products confirmed that Cry1Ac and Cry2A were successfully cloned in the correct orientation. The appearance of a blue color in sections of infiltrated leaves after 72 hours confirmed the successful expression of the construct in the plant expression system. The overall transformation efficiency was calculated to be 0.7%. The amplification of Cry1Ac-Cry2A and Tp2 showed the successful integration of target genes into the genome of cotton plants. A maximum of 0.673 μg/g tissue of Cry1Ac and 0.568 μg/g tissue of Cry2A was observed in transgenic plants. We obtained 100% mortality in the target insect after 72 hours of feeding the 2nd instar larvae with transgenic plants. The appearance of a yellow color in transgenic cross sections, while absent in the control, through phase contrast microscopy indicated chloroplast localization of the target protein.ConclusionLocating the target protein at the point of insect attack increases insect mortality when compared with that of other transgenic plants. The results of this study will also be of great value from a biosafety point of view.

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Section: Discussionsupporting

confidence: 90%

Chloroplast localization of Cry1Ac and Cry2A protein- an alternative way of insect control in cotton

et al. 2015

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“…Though several groups have carried out similar studies in tobacco and cotton plants, they did not quantify the expression level driven by these promoters at transcriptional level [ 15 , 16 , 19 ]. Ashraf et al .…”

Section: Discussionmentioning

confidence: 99%

“…Ashraf et al . [ 15 ] quantified the transiently expressed GUS activity driven by CLCuBuV C1 gene promoter by fluorometric assay. Evidence was provided exhibiting 2–3 fold higher GUS activity of CLCuBuV promoter than that of 35S.…”

Section: Discussionmentioning

confidence: 99%

“…The genome of monopartite begomovirus is organized into six ORFs viz. AC1 (Rep), AC2 (TrAp), AC3 (REn), AC4 , AV1 (CP) and AV2 which are transcribed bidirectionally from the LIR [ 15 ]. Rep is involved in viral replication and acts as transcriptional repressor of its own expression by binding to the iterated elements located in the LIR [ 12 , 27 – 29 ].…”

Section: Introductionmentioning

confidence: 99%

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Functional Characterization of a Strong Bi-directional Constitutive Plant Promoter Isolated from Cotton Leaf Curl Burewala Virus

2015

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Cotton leaf curl Burewala virus (CLCuBuV), belonging to the genus Begomovirus, possesses single-stranded monopartite DNA genome. The bidirectional promoters representing Rep and coat protein (CP) genes of CLCuBuV were characterized and their efficacy was assayed. Rep and CP promoters of CLCuBuV and 35S promoter of Cauliflower mosaic virus (CaMV) were fused with β-glucuronidase (GUS) and green fluorescent protein (GFP) reporter genes. GUS activity in individual plant cells driven by Rep, CP and 35S promoters was estimated using real-time PCR and fluorometric GUS assay. Histochemical staining of GUS in transformed tobacco (Nicotiana tabacum cv. Xanthi) leaves showed highest expression driven by Rep promoter followed by 35S promoter and CP promoter. The expression level of GUS driven by Rep promoter in transformed tobacco plants was shown to be two to four-fold higher than that of 35S promoter, while the expression by CP promoter was slightly lower. Further, the expression of GFP was monitored in agroinfiltrated leaves of N. benthamiana, N. tabacum and cotton (Gossypium hirsutum) plants using confocal laser scanning microscopy. Rep promoter showed strong consistent transient expression in tobacco and cotton leaves as compared to 35S promoter. The strong constitutive CLCuBuV Rep promoter developed in this study could be very useful for high level expression of transgenes in a wide variety of plant cells.

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“…Gene expression process in geminivirus happens by an early expression of complimentary sense gene, whose products are then participating in viral replication such as AC1 (Rep) or they may act as transcription activator for the virion sense gene expression, such as AC2 (TrAp). In contrast, the expression of virion sense genes usually appears later and requires complimentary sense gene product/products for activation (Ashraf et al 2014 ). We have observed that the MMPTY-pro, which had the highest expression levels, was TATA-less like the CP which means that the TATA box had no great effect on the promoter activity in our case.…”

Section: Discussionmentioning

confidence: 99%

Impact of cis-acting elements’ frequency in transcription activity in dicot and monocot plants

et al. 2015

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The production of new cultivars via recombinant DNA technology is important in applied agriculture. Promoters play fundamental roles in successful transformation and gene expression. Fragments of the upstream regulatory region of the movement protein gene of the Tomato yellow leaf curl virus (TYLCV; two fragments) and Watermelon chlorotic stunt virus (WmCSV, two fragments) and one fragment of the coat protein putative promoter of TYLCV (CPTY-pro) were isolated to assess their abilities to drive expression in monocot and dicot plants. We used bioinformatic analyses to identify tentative motifs in the fragments. The five promoter fragments were isolated, fused with the GUS reporter gene, and transformed into tomato, watermelon, and rice plantlets via Agrobacterium infiltration. GUS expression driven by each putative promoter was analysed using histochemical and fluorometric analyses. In both dicots and the monocots, the highest level of GUS expression was obtained using a truncated regulatory region from TYLCV (MMPTY-pro) followed by a truncated regulatory region from WmCSV (MMPWm-pro). However, the corresponding full-length fragments from TYLCV and WmCSV showed essentially equivalent expression levels in the fluorometric GUS assay compared with the enhanced Cauliflower mosaic virus e35S-pro. In addition, CPTY-pro showed no expression in either the dicots or the monocot. This study demonstrated that MMPTY-pro and MMPWm-pro may be useful as plant promoters.

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Functional Characterization of a Bidirectional Plant Promoter from Cotton Leaf Curl Burewala Virus Using an Agrobacterium-Mediated Transient Assay

Cited by 23 publications

References 79 publications

Chloroplast localization of Cry1Ac and Cry2A protein- an alternative way of insect control in cotton

Chloroplast localization of Cry1Ac and Cry2A protein- an alternative way of insect control in cotton

Functional Characterization of a Strong Bi-directional Constitutive Plant Promoter Isolated from Cotton Leaf Curl Burewala Virus

Impact of cis-acting elements’ frequency in transcription activity in dicot and monocot plants

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