Expression of Phytase gene in transgenic maize with seed-specific promoter 27-kDa γ Zein and constitutive promoter CaMV 35S

Phytase enzymes are applied to animal feed to help animals absorb more nutrients. The use of feed raw materials containing phytase enzymes is expected to reduce the cost of animal feed production. Efforts to increase the phytase content in maize were carried out by improving genetics, in the way of assembling transgenic plants containing high phytase content. The 27-kDa γ Zein promoter is a specific promoter that expresses genes in caryopsis, and promoter CaMV 35S is a constitutive promoter that controls gene expression in all tissues and generally does not depend on the growth phase. Transgenic maize was transformed using Agrobacterium tumefacien infection method on maize B104. The reverse transcriptase polymerase chain reaction (RT-PCR) approach was used to examine the expression of phytase genes in leaves, roots, and caryopsis was done 10, 20, and 30 days after pollination (DAP). The phytase enzyme activity test was also carried out by using the colorimetric phosphomolybdate analysis method to see the phytase enzyme activity in unit µg-1. The results showed that the phytase gene in transgenic plants with the 27-kDa γ Zein promoter was highly expressed in maize caryopsis, but in line Z6.10 was also expressed in leaves, while in the CaMV 35S promoter the phytase gene was only expressed on the leaves. Phytase enzyme activity showed that transgenic maize was higher than non-transgenic maize.

Functional Analysis of a Viral Promoter From the Strawberry Vein Banding Virus (SVBV) Chinese Isolate

BackgroundPromoter is an important factor during gene expression in cells. In this study, we cloned a full-length promoter from the strawberry vein banding virus (SVBV) Chinese isolate and produced several its deletion mutants.MethodsThe full-length promoter of SVBV (SP1) and its three deletion mutants (SP2, SP3, and SP4) were amplified using polymerase chain reaction (PCR). The expression activities controlled by the SVBV SP1, SP2, SP3, and SP4 were evaluated using β-glucuronidase (GUS) and green fluorescent protein (GFP) reporter genes.ResultsOur transient expression assays showed that the SVBV SP1 promoter as well as its three deletion mutants all expressed the reporter genes, but to very different levels. Interestingly, the expression activity driven by the SP1 promoter was much higher than that shown by the CaMV 35S promoter. After stable transformation of a GUS gene into Nicotiana tabacum plants, the transgene expression level driven by the SVBV SP1 promoter was about 2.6-fold greater than that driven by the CaMV 35S promoter. In addition, the GUS gene expression levels could be enhanced by co-infiltrating the plants with the SP1 promoter-driven vector carrying the GUS gene and the vector expressing the SVBV ORF V or ORF VI.ConclusionsThe SVBV Chinese isolate promoter SP1 is a stronger promoter than the CaMV 35S and FLt-US promoter, may be more useful for production of stable transgenic plants.

Targeted Transgene Expression in Rice Using a Callus Strong Promoter for Selectable Marker Gene Control

Precise expression of a transgene in the desired manner is important for plant genetic engineering and gene function deciphering, but it is a challenge to obtain specific transgene expression free from the interference of the constitutive promoters used to express the selectable marker gene, such as the Cauliflower mosaic virus (CaMV) 35S promoter. So, the solutions to avoid these inappropriate regulations are largely demanded. In this study, we report the characterization of a callus strong promoter (CSP1) in rice and its application for accurate transgene expression. Our results indicate that the high expression of the CSP1 promoter in the callus enables efficient selection of hygromycin equivalent to that provided by the CaMV 35S promoter, whereas its expression in other tissues is low. To evaluate possible leaky effects, the expression of a β-glucuronidase reporter driven by six specific promoters involving hormone signaling, pathogen response, cell fate determination, and proliferation was observed in transgenic rice plants generated by CSP1-mediated selection. Distinct β-glucuronidase expression was found consistently in most of the transgenic lines obtained for each promoter. In addition, we applied these specific marker lines to investigate the root cellular responses to exogenous cytokinin and auxin treatment. The results reveal that the root growth inhibition by cytokinin was differently regulated at high and low concentrations. In summary, we have established the feasibility of using callus-specific promoter-dependent selection to mitigate the transgene misexpression in rice. By enabling efficient transformation, rice plants with reliable transgene expression will be easily acquired for broad applications.

Evaluation of Plant-Derived Promoters for Constitutive and Tissue-Specific Gene Expression in Potato

Various plant-derived promoters can be used to regulate ectopic gene expression in potato. In the present study, four promoters derived from the potato genome have been characterized by the expression of identical cassettes carrying the fusion with the reporter β-glucuronidase (gusA) gene. The strengths of StUbi, StGBSS, StPat, and StLhca3 promoters were compared with the conventional constitutive CaMV 35S promoter in various organs (leaves, stems, roots, and tubers) of greenhouse-grown plants. The final amount of gene product was determined at the post-transcriptional level using histochemical analysis, fluorometric measurements, and Western blot analysis. The promoter strength comparison demonstrated that the StUbi promoter generally provided a higher level of constitutive β-glucuronidase accumulation than the viral CaMV 35S promoter. Although the StLhca3 promoter was predominantly expressed in a green tissue-specific manner (leaves and stems) while StGBSS and StPat mainly provided tuber-specific activity, a “promoter leakage” was also found. However, the degree of unspecific activity depended on the particular transgenic line and tissue. According to fluorometric data, the functional activity of promoters in leaves could be arranged as follows: StLhca3 > StUbi > CaMV 35S > StPat > StGBSS (from highest to lowest). In tubers, the higher expression was detected in transgenic plants expressing StPat-gusA fusion construct, and the strength order was as follows: StPat > StGBSS > StUbi > CaMV 35S > StLhca3. The observed differences between expression patterns are discussed considering the benefits and limitations for the usage of each promoter to regulate the expression of genes in a particular potato tissue.

Functional characterization of an endosperm specific promoter p1062 from common buckwheat (Fagopyrum esculentum Moench) for driving tissue specific gene expression / Funkcijske lastnosti endospermskega promotorja p1062 navadne ajde ...

Seed storage proteins of grain crops meet the major dietary protein requirement of over half of the world population. PCR based genome walking the 5’UTR of the gene coding for a lysine rich legumin type protein amplified a 1.1kb DNA fragment representing the promoter region of the gene. Clustal alignment of this sequence with other sequences in the Genbank database clearly showed 100 percent complementary base match of 282 bases at the 3’ end of the sequence, corresponding to nucleotide position 780-1062 with correspondingly similar number of bases on the 5’ end of the 1.7kb Bwleg gene.We detected one prolamin box and three RY-repeat motifs in the sequence. Seven deletion fragments of the putative promoter were generated by 5’ nested PCR and cloned in pCAMBIA1304 upstream of GUS gene after excising the CaMV 35S promoter from the vector. Arabidopsis plants plants harbouring the deletion construct pBwlDF1 to pBwlDF6 clearly showed seed specific expression of the reporter gene. Seeds harbouring the constructs pBwlDF3, pBwlDF4 and pBwlDF5 showed a nearly threefold decrease in GUS activity than those harbouring the construct with full length promoter. Key words: buckwheat, DNA, promoter, constructs Izvleček Založne beljakovine semen zrnastih poljščin ustrezajo glavnim potrebam po beljakovinah za več kot polovico svetovnega prebivalstva. S PCR in 5’UTR so za kodiranje kakovostnih beljakovin leguminskega tipa pomnožili odlomek 1,1 kb DNK, ki je promotorsko gensko območje. Vzporejanje te sekvence z drugimi sekvencami podatkovne baze genske banke jasno pokaže popolno komplementarnost 282 baz na 3’ koncu sekvence, kar ustreza pozicijam 780-1062 z ustreznim številom baz na 5’ koncu gena 1,7 kb Bwleg. V sekvenci smo odkrili eno prolaminsko škatljo in tri RY-ponovljene motive. Sedem delecijskih fragmentov putativnega promotorja smo generirali z 5’ PCR kloniranjem pCAMBIA1304 navzgor od GUS gena po izločitvi promotorja CaMV 35S iz vektorja. Semena s konstrukti pBwlDF3, pBwlDF4 in pBwlDF5 so izražali skoraj trikratno zmanjšanje GUS aktivnosti v primerjavi s konstrukti, ki so vsebovali polne dolžine promotorjev. Ključne besede: ajda, DNK, promotor, konstrukti

A simple and accurate PCR method for detection of genetically modified rice

Background Legislation regulating for labeling and use of genetically modified (GM) crops are increased considerably worldwide in order to health and safety assurance of consumers. For this purpose, a polymerase chain reaction (PCR) method has been developed for detection of GM rice in people’s food diet. Methods In this study, eighty-one non-labeled rice samples were collected randomly from different market sites of Tehran, Iran. In order to analysis, rice genomic DNA was extracted using MBST DNA extraction kit and subsequently, sucrose phosphate synthase (SPS) gene was used to confirm the quality of extracted DNA. Then, cauliflower mosaic virus (CaMV) 35S promoter and Agrobacterium nopaline synthase (NOS) terminator were selected as screening targets for detection of GM rice sequences by PCR. Results According to our results, 2 out of 81 (2.4%) samples tested were positive for CaMV 35S promoter while no positive result was detected for NOS terminator. Conclusion The obtained data indicated that this method is capable to identify the GM rice varieties. Furthermore, it can demonstrate the possibility of the presence of GM rice in Tehran’s market, thus putting emphasis on the requirement for developing a precise approach to evaluate this product.

CHUYỂN GEN CẢI BIẾN GDHA CÓ NGUỒN GỐC E.COLI VÀO CÂY THUỐC LÁ N. TABACUM

Gen gdhA mã hoá cho NAPH- GDH từ vi khuẩn E.coli khi biểu hiện trong cây thuốc lá đã làm tăng cường khả năng chống chịu thuốc bảo vệ thực vật, tăng sinh khối thực vật và tăng khả năng chịu hạn, ngoài ra khi biểu hiện ở cây ngô đã cho thấy gen gdhA giúp cây ngô tăng năng suất và sự tăng năng suất có được là nhờ cải thiện khả năng chịu đựng hạn của cây. Với mục đích có nguồn gen giá trị này, chúng tôi tiến hành phân lập gen gdhA từ chủng vi khuẩn E. coli JM109, tạo dòng và xác định trình tự gen gdhA để tìm sự khác biệt về gen trong chủng hiện có. Đồng thời, chúng tôi thực hiện cải biến trình tự gen gdhA bằng phần mềm OptimumGeneTM cho phù hợp với thực vật. Gen gdhA sau khi cải biến được ghép nối với promoter CaMV 35S và 35S terminator trong vector trung gian pRTRA 7/3 để tạo cấu trúc biểu hiện 35S::gdhA::35S. Cấu trúc này đã được chuyển vào vector pCAMBIA1300 tạo nên vector biểu hiện thực vật mang gen gdhA và biến nạp vector vào tế bào vi khuẩn A.tumefaciens chủng EHA105. Để kiểm tra sự biểu hiện của gen gdhA chúng tôi tiến hành chuyển cấu trúc biểu hiện gen gdhA vào cây mô hình thuốc lá N. tabacum K326 thông qua A. tumefaciens. Phân tích sự có mặt của gen trong 20 dòng cây thuốc lá chuyển gen bằng PCR và RT-PCR cho thấy 2/20 dòng thuốc lá chuyển gen có mang gen gdhA hoạt động ở mức độ phiên mã tạo mRNA. Sự biểu hiện của cấu trúc này ở cây thuốc lá cho thấy có thể sử dụng cấu trúc mang gen để chuyển gen gdhA vào cây trồng đích như cây ngô.

A short history of the CaMV 35S promoter

In an organism, be it plant, animal or human, almost every gene has its own promoter sequence, which is typified as a DNA stretch that controls how a gene is expressed in a cell. Hence, the activity of a promoter controls in which cell type, during which developmental stage or during what environmental condition a certain gene is expressed. However, the most widely used promoter in plant biotechnology is actually not derived from a plant, but a pathogenic virus. How and why did that happen? Here's a short history of the CaMV 35S promoter.