“…The male gametophyte-specific promoters from A. thaliana include those for the genes AtSTP6 (Starke et al 2003), BCP1 (Xu et al 1993), AtVEX, and AtGEX2 (Engel et al 2005). Similarly, some pollen-specific promoters reported from other plants include those driving the expression of genes Zm908 from Maize, LGC1 from lily, and Os10g22450 from Rice (Smirnova and Kochetov 2020). Most of these promoters were discovered based on the reports of their tissue-specific expression.…”
Section: Discussionmentioning
confidence: 99%
“…There are previous reports of functional characterization of promoters specific for the male gametophytic generation. These include the promoters for the genes LAT52 and LAT59 from tomato (Twell et al 1991), OSIPA (Swapna et al 2011) and OSIPK (Gupta et al 2007) from rice, AtVEX, and AtGEX2 (Engel et al 2005), HAP2 (von Besser et al 2006), DUO3 (Brownfield et al 2009), and WRKY34 (Zou et al 2010) from Arabidopsis and many others as reviewed in Smirnova and Kochetov (2020). The reports on female gametophyte-specific promoters are very few in comparison to their male counterparts.…”
Gametophyte-specific promoters drive the expression of genes in male and/or female gametophytes. These have applications in breeding experiments, gene function identification, developmental biology-related studies, and off-lately in genome editing also. ThePlant Intracellular Ras-group Leucine-Rich-Repeat6 (PIRL6) gene is known to be necessary for male and female gametogenesis. Using theGUS-based deletion analysis, we have identified thePIRL6promoter length that is essential for exclusive expression in the male gametophyte ofArabidopsis thaliana.We studied the strength of various lengths ofPIRL6promoters in different tissues, by GUS expression quantification. The male-gametophyte-specific promoter segment (PA1) exhibited stronger expression in mature anthers than the younger ones. We identified 50 other genes that co-expressed withPIRL6inArabidopsisusing the Expression Angler tool. Gene ontology (GO) analysis shows that these 51 co-expressing genes were predominantly involved in cell differentiation. By comparing the promoter sequences of these 51 genes, the presence of three over-represented known motifs, POLLEN1LELAT52, ACGTATERD1 and CIACADIANLELHC was identified. We could also predict the presence of three novelcis-regulatory elements in the co-expressing gene network using the MEME suite tool. Additionally, we confirmed the functionality of the ABRE and P-box elements present inPIRL6promoter using the tobacco leaf transient assay. Thus, we cloned and functionally confirmed the promoter region ofPIRL6required for male gametophyte-specific expression, compared this promoter with those of 50 other co-expressed genes and predicted their functions, and analyzed theircisregulatory regions and predicted three novel motifs also.Key messageA pollen-specific promoter fragment ofPIRL6was functionally characterized usingGUS-based deletion analysis. Fifty other co-expressed genes were compared and novelcis-regulatory elements were predicted. Two hormone-responsive elements in thePIRL6promoter were found to influence theGUSexpression. PA1 promoter can be used in experiments that require male gametophyte-specific expression of genes.
“…The male gametophyte-specific promoters from A. thaliana include those for the genes AtSTP6 (Starke et al 2003), BCP1 (Xu et al 1993), AtVEX, and AtGEX2 (Engel et al 2005). Similarly, some pollen-specific promoters reported from other plants include those driving the expression of genes Zm908 from Maize, LGC1 from lily, and Os10g22450 from Rice (Smirnova and Kochetov 2020). Most of these promoters were discovered based on the reports of their tissue-specific expression.…”
Section: Discussionmentioning
confidence: 99%
“…There are previous reports of functional characterization of promoters specific for the male gametophytic generation. These include the promoters for the genes LAT52 and LAT59 from tomato (Twell et al 1991), OSIPA (Swapna et al 2011) and OSIPK (Gupta et al 2007) from rice, AtVEX, and AtGEX2 (Engel et al 2005), HAP2 (von Besser et al 2006), DUO3 (Brownfield et al 2009), and WRKY34 (Zou et al 2010) from Arabidopsis and many others as reviewed in Smirnova and Kochetov (2020). The reports on female gametophyte-specific promoters are very few in comparison to their male counterparts.…”
Gametophyte-specific promoters drive the expression of genes in male and/or female gametophytes. These have applications in breeding experiments, gene function identification, developmental biology-related studies, and off-lately in genome editing also. ThePlant Intracellular Ras-group Leucine-Rich-Repeat6 (PIRL6) gene is known to be necessary for male and female gametogenesis. Using theGUS-based deletion analysis, we have identified thePIRL6promoter length that is essential for exclusive expression in the male gametophyte ofArabidopsis thaliana.We studied the strength of various lengths ofPIRL6promoters in different tissues, by GUS expression quantification. The male-gametophyte-specific promoter segment (PA1) exhibited stronger expression in mature anthers than the younger ones. We identified 50 other genes that co-expressed withPIRL6inArabidopsisusing the Expression Angler tool. Gene ontology (GO) analysis shows that these 51 co-expressing genes were predominantly involved in cell differentiation. By comparing the promoter sequences of these 51 genes, the presence of three over-represented known motifs, POLLEN1LELAT52, ACGTATERD1 and CIACADIANLELHC was identified. We could also predict the presence of three novelcis-regulatory elements in the co-expressing gene network using the MEME suite tool. Additionally, we confirmed the functionality of the ABRE and P-box elements present inPIRL6promoter using the tobacco leaf transient assay. Thus, we cloned and functionally confirmed the promoter region ofPIRL6required for male gametophyte-specific expression, compared this promoter with those of 50 other co-expressed genes and predicted their functions, and analyzed theircisregulatory regions and predicted three novel motifs also.Key messageA pollen-specific promoter fragment ofPIRL6was functionally characterized usingGUS-based deletion analysis. Fifty other co-expressed genes were compared and novelcis-regulatory elements were predicted. Two hormone-responsive elements in thePIRL6promoter were found to influence theGUSexpression. PA1 promoter can be used in experiments that require male gametophyte-specific expression of genes.
“…The type of promoter used in the chimeric gene construct for plant transformation is essential to achieve adequate temporal or spatial regulated expression of the desired trait. Although the number of promoter sequences is rather limited, the selection of an adequate promoter is not a trivial issue (reviewed in Smirnova and Kochetov, 2020 ). Different promoters derived from virus, bacteria, or plant species have been employed for citrus genetic transformation.…”
Section: Promoter Sequences Used For Citrus Transgene Expressionmentioning
Citrus are among the most prevailing fruit crops produced worldwide. The implementation of effective and reliable breeding programs is essential for coping with the increasing demands of satisfactory yield and quality of the fruit as well as to deal with the negative impact of fast-spreading diseases. Conventional methods are time-consuming and of difficult application because of inherent factors of citrus biology, such as their prolonged juvenile period and a complex reproductive stage, sometimes presenting infertility, self-incompatibility, parthenocarpy, or polyembryony. Moreover, certain desirable traits are absent from cultivated or wild citrus genotypes. All these features are challenging for the incorporation of the desirable traits. In this regard, genetic engineering technologies offer a series of alternative approaches that allow overcoming the difficulties of conventional breeding programs. This review gives a detailed overview of the currently used strategies for the development of genetically modified citrus. We describe different aspects regarding genotype varieties used, including elite cultivars or extensively used scions and rootstocks. Furthermore, we discuss technical aspects of citrus genetic transformation procedures via Agrobacterium, regular physical methods, and magnetofection. Finally, we describe the selection of explants considering young and mature tissues, protoplast isolation, etc. We also address current protocols and novel approaches for improving the in vitro regeneration process, which is an important bottleneck for citrus genetic transformation. This review also explores alternative emerging transformation strategies applied to citrus species such as transient and tissue localized transformation. New breeding technologies, including cisgenesis, intragenesis, and genome editing by clustered regularly interspaced short palindromic repeats (CRISPR), are also discussed. Other relevant aspects comprising new promoters and reporter genes, marker-free systems, and strategies for induction of early flowering, are also addressed. We provided a future perspective on the use of current and new technologies in citrus and its potential impact on regulatory processes.
“…В связи с этим поиск и последующее использование новых растительных промоторов при создании генетически модифицированных растений картофеля входит в число актуальных задач современной биотехнологии. В последние годы конститутивные, а также органо-и тканеспецифичные растительные промоторы находят все более широкое применение в различных прикладных и фундаментальных исследованиях в качестве эффективной альтернативы вирусным и бактериальным промоторам [9][10][11][12][13].…”
The effectiveness of plant genetic transformation is determined by the choice of genetic structures and their regulatory sequences that cause a high and stable expression level of heterologous genes. In this regard, the actual task of biotechnology is the use of highly effective plant promoters. The choice of promoter determines not only the level of the expression gene, but also the effectiveness of genetic transformation. The purpose of our study was to evaluate the influence of explant type and 5-deletion variants of the plant strong pro-SmAMP1 promoter, on the Agrobacterium -mediated transformation efficiency of potato ( Solanum tuberosum L.) cv. Udacha. To analyze the regenerative capacity of potato stem and leaf explants, AGL0 strain carrying constructs containing the 5-deletion variants of the promoter fragment of gene encoding antimicrobial peptide from Stellaria media L. ( pro-SmAMP1 ) was carried out. Four genetic constructs based on the plant expression vector pCAMBIA1381Z were used in this work, containing the selectable gene hptII and reporter gene uidA under different 5-deletion variants of the pro-SmAMP1 promoter (-442, -675, -732 and -1196 bp relative to the transcription initiation site); as well as two binary vectors based on the expression vector pCAMBIA1302 with 5-deletion pro-SmAMP1 promoter variants (-442 and -1196 bp), controlling the expression of gfp reporter gene. It was found that the effectiveness of Agrobacterium -mediated transformation depended on the type of genetic construction used, but not on the type of explant being cultivated. The insertion of the promoter region pro-SmAMP1 gene, hptII , as well as the absence of the bacterial Vir E gene was confirmed by PCR. Depending on the type of genetic construct, the transformation efficiency for the reporter gene varied from 2.0 to 7.2 %. The results are compared with previously conducted few studies, according to which the choice of promoter determines not only the expression level of marker genes, but also has a significant influence on the genetic transformation efficiency.
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