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CaMV 35S promoter-driven GFP expression profile in floral tissues in transgenic cotton plant expressing CaMV 35S:mGFP5-ER. The results presented here are from transformed line H3. Images of negative control are also provided for comparison. A and AA. Light and fluorescence images, respectively, of a portion of a longitudinal section through an unopened flower from negative control plant; b, bract; p, petal; s, sepal. B, C and D. Fluorescence images of a bract, sepal and petal, respectively, from a flower from the transgenic plant. E and EE. Light and fluorescence images, respectively, of a stamen. F. Fluorescence image of non-transgenic control (left) and transgenic (right) pollen. G and GG. Light and fluorescence images, respectively, of a staminal column. H and HH. Light and fluorescence images, respectively, of a transverse
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The CaMV 35S promoter is the most commonly used promoter for driving transgene expression in plants. Though it is presumed to be a constitutive promoter, some reports suggest that it is not expressed in all cell types. In addition, the information available on its expression profile in all possible cell and tissue types and during early stages of d...
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Strigolactones (SLs) are a class of phytohormones that regulate plant shoot branching and adventitious root development. However, little is known regarding the role of SLs in controlling the behavior of the smallest unit of the organism, the single cell. Here, taking advantage of a classic single-cell model offered by the cotton (Gossypium hirsutum...
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... Since the expressions of 6HTU and 6HU were driven by UBQ10 and 35S promoters, respectively, the dynamic difference in HTU-and HU-conjugated proteins further implied a transcriptional regulation of endogenous UBQ genes in silique development, as we discovered previously [36]. It was reported that the 35S promoter was not active in immature embryos [42]. The strong HU-conjugates detected in immature siliques indicated that most of these products originated from maternal or non-embryonic tissues. ...
... Since the expressions of 6HTU and 6HU were driven by UBQ10 and 35S promoters, respectively, the dynamic difference in HTU-and HU-conjugated proteins further implied a transcriptional regulation of endogenous UBQ genes in silique development, as we discovered previously [36]. It was reported that the 35S promoter was not active in immature embryos [42]. The strong HU-conjugates detected in immature siliques indicated that most of these products originated from maternal or nonembryonic tissues. ...
In plants, the ubiquitin (Ub)-26S proteasome system (UPS) regulates numerous biological functions by selectively targeting proteins for ubiquitylation and degradation. However, the regulation of Ub itself on plant growth and development remains unclear. To demonstrate a possible impact of Ub supply, as seen in animals and flies, we carefully analyzed the growth and developmental phenotypes of two different poly-Ub (UBQ) gene overexpression plants of Arabidopsis thaliana. One is transformed with hexa-6His-UBQ (designated 6HU), driven by the cauliflower mosaic virus 35S promoter, while the other expresses hexa-6His-TEV-UBQ (designated 6HTU), driven by the endogenous promoter of UBQ10. We discovered that 6HU and 6HTU had contrasting seed yields. Compared to wildtype (WT), the former exhibited a reduced seed yield, while the latter showed an increased seed production that was attributed to enhanced growth vigor and an elevated silique number per plant. However, reduced seed sizes were common in both 6HU and 6HTU. Differences in the activity and size of the 26S proteasome assemblies in the two transgenic plants were also notable in comparison with WT, suggestive of a contributory role of UBQ expression in proteasome assembly and function. Collectively, our findings demonstrated that exogenous expression of recombinant Ub may optimize plant growth and development by influencing the UPS activities via structural variance, expression patterns, and abundance of free Ub supply.
... It is widely used for genetic modification and gene editing. Scions and/or rootstocks consisted of Coker 312 (Coker), TAM66274 [an RNAi-mediated, ultra-low gossypol cottonseed event (ULGCS) (Rathore et al., 2020)]; LCT237 [a CRISPR-mediated knockout event devoid of glands (KO-gl) (Janga et al., 2019)]; and LCT29 [a GFP overexpression event (GFP-cot) (Sunilkumar et al., 2002)]. ...
Intervarietal grafting, and in some cases interspecific grafting, practiced mainly in horticultural crops has many practical applications. Grafting can also be a useful tool to perform basic physiological, biochemical, and molecular studies to better understand communication between shoot and root in a row crop, such as cotton. We have established a grafting protocol for upland cotton (Gossypium hirsutum L.) genotypes. Although less efficient, it can be used to create grafts between upland and Pima (G. barbadense) or Old-world cottons (G. arboreum or G. herbaceum). The procedure used 8-day-old seedlings grown in culture under sterile conditions to obtain the scion and rootstock. The scion consisted of the shoot apical meristem with a few leaf primordia, epicotyl, and a small portion of the hypocotyl with the cotyledons removed. The rootstock retained 2 to 3 cm of basal hypocotyl portion with the roots trimmed. Cleft grafts were assembled under aseptic conditions followed by their culture in vitro. Once established, as indicated by the healthy growth of three to four true leaves and roots, the grafts were moved to soil in small pots. After hardening the grafts on a laboratory bench for few weeks, they were transferred to larger pots and grown to maturity in a greenhouse to obtain seeds. The success rate using our protocol ranged from 90 to 100% for Coker 312 genotype and its derivative transgenic and gene edited lines. The method was also applicable to generate intervarietal and interspecific graft unions among various types of cotton plants.
... Both studies, however, used mature seeds as the explant for analysis. When a detailed developmental survey was conducted in cotton plants expressing a green fluorescent protein (GFP) driven by a CaMV 35S promoter, intense GFP fluorescence was observed in both the embryo and endosperm during mid through late seed development, but was undetectable during early seed development (Sunilkumar et al. 2002). Our results suggest that tobacco is likely similar to cotton in this respect, and that the 35S promoter is not expressed at a high enough level during early seed development for the 35S:nATP1 construct to compensate for the missing atp1 gene, leading to aborted seed development. ...
Allotopic expression is the term given for the deliberate relocation of gene function from an organellar genome to the nuclear genome. We hypothesized that the allotopic expression of an essential mitochondrial gene using a promoter that expressed efficiently in all cell types except those responsible for male reproduction would yield a cytoplasmic male sterility (CMS) phenotype once the endogenous mitochondrial gene was inactivated via genome editing. To test this, we repurposed the mitochondrially encoded atp1 gene of tobacco to function in the nucleus under the transcriptional control of a CaMV 35S promoter (construct 35S:nATP1), a promoter that has been shown to be minimally expressed in early stages of anther development. The endogenous atp1 gene was eliminated (Datp1) from 35S:nATP1 tobacco plants using custom-designed meganucleases directed to the mitochondria. Vegetative growth of most 35S:nATP1/Datp1 plants appeared normal, but upon flowering produced malformed anthers that failed to shed pollen. When 35S:nATP1/Datp1 plants were cross-pollinated, ovary/capsule development appeared normal, but the vast majority of the resultant seeds were small, largely hollow and failed to germinate, a phenotype akin to the seedless trait known as stenospermocarpy. Characterization of the mitochondrial genomes from three independent Datp1 events suggested that spontaneous recombination over regions of microhomology and substoichiometric shifting were the mechanisms responsible for atp1 elimination and genome rearrangement in response to exposure to the atp1-targeting meganucleases. Should the results reported here in tobacco prove to be translatable to other crop species, then multiple applications of allotopic expression of an essential mitochondrial gene followed by its elimination through genome editing can be envisaged. Depending on the promoter(s) used to drive the allotopic gene, this technology may have potential application in the areas of: (1) CMS trait development for use in hybrid seed production; (2) seedless fruit production; and (3) transgene containment.
... RT−qPCR analysis showed that the red coloration of RUBY was directly related to the level of expression. Although the CaMV 35S promoter has been widely used as a constitutive promoter to study gene function, there are differences in expression abundance in different tissues and abiotic stresses at each developmental stage of plants [54][55][56]. This was a good explanation for each of the regions that had different types of red coloration in the same transgenic hairy root of P. volubilis; white inflorescence appeared in fully colored transgenic Arabidopsis line #3, and small numbers of red seeds appeared in noncolored line #6, which might be caused by differential expression of RUBY driven by the CaMV 35S promoter. ...
Betalains can be conveniently observed and quantified and, accordingly, have the potential as naked-eye visual screening reporters during plant transformation. RUBY is a new reporter system that uses “2A” peptides to fuse three key genes, CYP76AD1, DODA, and glucosyl transferase, for betalain biosynthesis, and has been successfully used for transformation of rice, Arabidopsis, and cotton, but its potential applications in the genetic transformation of various other plant species remain to be verified. In this study, RUBY was transferred into the hairy roots of Plukenetia volubilis and Nicotiana benthamiana, and was transferred into Arabidopsis by the floral-dip method. The expression levels of CYP76AD1, DODA, and glucosyl transferase were detected by RT−PCR and RT−qPCR, the relationship between the expression level of RUBY and red coloration was analyzed, and the genetic stability of RUBY in transgenic Arabidopsis was studied. The results showed that the expression of RUBY could reconstruct the betalain biosynthesis pathway in the hairy roots of P. volubilis, N. benthamiana, and Arabidopsis plants, indicating that it has the potential for versatile use across species. As a reporter, betalain did not affect callus induction, plant regeneration, development, or fertility. However, when used in plant transformation for observation and visual screening, it needed to accumulate to a certain extent to show red coloration, and it was positively correlated with gene expression. In general, RUBY is a convenient reporter for plant transformation, and has no obvious side effects during plant growth and development. However, the potential application of RUBY for visual screening is highly determined by the expression level, and further improvement is needed.
... The expression of KTI2, KTI3, and KTI7 were the highest, whereas for KTI1, KTI5, and BBI5 were lower (Supplemental Figure S5). The varied expression profile driven by the 35S promoter in different tissue types and plant species was also shown in other studies (Benfey and Chua, 1989;Sunilkumar et al., 2002;Kiselev et al., 2021). ...
Trypsin inhibitors (TIs) are widely distributed in plants and are known to play a protective role against herbivores. TIs reduce the biological activity of trypsin, an enzyme involved in the breakdown of many different proteins, by inhibiting the activation and catalytic reactions of proteins. Soybean (Glycine max) contains two major TI classes: Kunitz trypsin inhibitor (KTI) and Bowman-Birk inhibitor (BBI). Both genes encoding TI inactivate trypsin and chymotrypsin enzymes, which are the main digestive enzymes in the gut fluids of Lepidopteran larvae feeding on soybean. In this study, the possible role of soybean TIs in plant defense against insects and nematodes was investigated. A total of six TIs were tested, including three known soybean trypsin inhibitors (KTI1, KTI2 and KTI3) and three genes encoding novel inhibitors identified in soybean (KTI5, KTI7, and BBI5). Their functional role was further examined by overexpression of the individual TI genes in soybean and Arabidopsis. The endogenous expression patterns of these TI genes varied among soybean tissues, including leaf, stem, seed, and root. In vitro enzyme inhibitory assays showed significant increase in trypsin and chymotrypsin inhibitory activities in both transgenic soybean and Arabidopsis. Detached leaf-punch feeding bioassays detected significant reduction in corn earworm (Helicoverpa zea) larval weight when larvae fed on transgenic soybean and Arabidopsis lines, with the greatest reduction observed in KTI7 and BBI5 overexpressing lines. Whole soybean plant greenhouse feeding bioassays with H. zea on KTI7 and BBI5 overexpressing lines resulted in significantly reduced leaf defoliation compared to non-transgenic plants. However, bioassays of KTI7 and BBI5 overexpressing lines with soybean cyst nematode (SCN, Heterodera glycines) showed no differences in SCN female index between transgenic and non-transgenic control plants. There were no significant differences in growth and productivity between transgenic and non-transgenic plants grown in the absence of herbivores to full maturity under greenhouse conditions. The present study provides further insight into the potential applications of TI genes for insect resistance improvement in plants.
... The Cauliflower Mosaic Virus 35S (CaMV35S) promoter has been extensively employed in plant molecular biology-based research ever since it was discovered [7][8][9][10][11][12][13][14][15][16]; still, it is regarded as a 'dark horse' in this field. With the gradual expansion of transgenic research, there has been an increased demand for a versatile promoter with a higher transcriptional activity. ...
Native/endogenous promoters have several fundamental limitations in terms of their size, Cis-elements distribution/patterning, and mode of induction, which is ultimately reflected in their insufficient transcriptional activity. Several customized synthetic promoters were designed and tested in plants during the past decade to circumvent such constraints. Such synthetic promoters have a built-in capacity to drive the expression of the foreign genes at their maximum amplitude in plant orthologous systems. The basic structure and function of the promoter has been discussed in this review, with emphasis on the role of the Cis-element in regulating gene expression. In addition to this, the necessity of synthetic promoters in the arena of plant biology has been highlighted. This review also provides explicit information on the two major approaches for developing plant-based synthetic promoters: the conventional approach (by utilizing the basic knowledge of promoter structure and Cis-trans interaction) and the advancement in gene editing technology. The success of plant genetic manipulation relies on the promoter efficiency and the expression level of the transgene. Therefore, advancements in the field of synthetic promoters has enormous potential in genetic engineering-mediated crop improvement.
... The Cauliflower Mosaic Virus 35S (CaMV35S) promoter has been extensively employed in plant molecular biology-based research ever since it was discovered [7][8][9][10][11][12][13][14][15][16]; still, it is regarded as a 'dark horse' in this field. With the gradual expansion of transgenic research, there has been an increased demand for a versatile promoter with a higher transcriptional activity. ...
Native/endogenous promoters have several fundamental limitations in terms of their size,Cis-elements distribution/patterning, and mode of induction, which is ultimately reflected in theirinsufficient transcriptional activity. Several customized synthetic promoters were designed andtested in plants during the past decade to circumvent such constraints. Such synthetic promotershave a built-in capacity to drive the expression of the foreign genes at their maximum amplitude inplant orthologous systems. The basic structure and function of the promoter has been discussed inthis review, with emphasis on the role of the Cis-element in regulating gene expression. In additionto this, the necessity of synthetic promoters in the arena of plant biology has been highlighted. Thisreview also provides explicit information on the two major approaches for developing plant-basedsynthetic promoters: the conventional approach (by utilizing the basic knowledge of promoterstructure and Cis-trans interaction) and the advancement in gene editing technology. The success ofplant genetic manipulation relies on the promoter efficiency and the expression level of thetransgene. Therefore, advancements in the field of synthetic promoters has enormous potential ingenetic engineering-mediated crop improvement
... YUC6 is expressed in the stamen, it was unknown if the 35S promoter would drive expression in the stamen of T. joelii at the outset of this experiment. This promoter is not expressed in the anthers or pollen of Arabidopsis thaliana [57], but it has been reported to be expressed in the anthers [58,59] and pollen [60,61] of a number of other plant species. The pollen mating type in heterostyly is genetically sporophytically encoded, i.e., it is determined by the diploid genotype of the mother plant. ...
In heterostylous, self-incompatible Turnera species, a member of the YUCCA gene family, YUC6, resides at the S-locus and has been hypothesized to determine the male mating type. YUCCA gene family members synthesize the auxin, indole-3-acetic acid, via a two-step process involving the TAA gene family. Consequently, it has been speculated that differences in auxin concentration in developing anthers are the biochemical basis underlying the male mating type. Here, we provide empirical evidence that supports this hypothesis. Using a transgenic knockdown approach, we show that YUC6 acts pleiotropically to control both the male physiological mating type and pollen size, but not the filament length dimorphism associated with heterostyly in Turnera. Using qPCR to assess YUC6 expression in different transgenic lines, we demonstrate that the level of YUC6 knockdown correlates with the degree of change observed in the male mating type. Further assessment of YUC6 expression through anther development, in the knockdown lines, suggests that the male mating type is irreversibly determined during a specific developmental window prior to microsporogenesis, which is consistent with the genetically sporophytic nature of this self-incompatibility system. These results represent the first gene controlling male mating type to be characterized in any species with heterostyly.
... The transformation protocol utilized was essentially that described by Chetty et al. (2015). A. tumefaciens (LBA4404) carrying the binary vector, pBINmGFP5-ER (Sunilkumar et al. 2002), was used to transform leaf and internodes explants obtained from in vitro cultured plantlets. The T-DNA within pBINmGFP5-ER harbors a kanamycin-resistance gene (nptII) cassette and also a modified green fluorescence protein (mgfp) expression cassette (Siemering et al. 1996;Haseloff et al. 1997). ...
CRISPR/Cas9 has emerged as a simple, yet efficient gene editing tool to generate targeted mutations in desired genes in crops plants. Agrobacterium tumefaciens, a reliable and inexpensive DNA-delivery mechanism into plant cells, has been used for the generation of CRISPR/Cas9-mediated mutations in crop plants, including potato. However, little information is available as to the progression of gene knockout during various stages of culture following the introduction of CRISPR components in this species. In the current study, the green fluorescent protein (gfp) transgene was first introduced in the genome of a potato variety, Yukon Gold. Two GFP-expressing lines, one with a single gfp copy integrated and another with four gfp copies integrated, were subjected to CRISPR/Cas9-mediated mutations in the transgene(s) using three different gRNAs. Disappearance of GFP fluorescence was monitored during the entire culture/regeneration process. Although all three gRNAs successfully knocked out the transgene(s), their efficiencies differed greatly and did not completely match the predicted scores by some guide RNA prediction tools. The nature of mutations in various knockout events was analyzed. Several lines containing four gfp-copies showed four different types of mutations. These findings suggest that it is possible to target all four alleles of a desired native gene in the tetraploid potato.
... The use of promoters and enhancers with specific regulatory effects is essential for the efficiency of exogenous gene expression in recipient plants. The constitutive expression promoters such as rice Actin1 [80,81], maize Ubi [82,83], and 35S [84][85][86] has been used in plant transgenic engineering. However, these promoters tended to be transcribed in all plant tissues with poor spatio-temporal specificity, which increased plant energy consumption and also tended to result in gene silencing [87,88]. ...
Maize is one of the most important food crops, and maize kernel is one of the important components of maize yield. Studies have shown that the rice grain-size affecting gene GS5 increases the thousand-kernel weight by positively regulating the rice grain width and grain grouting rate. In this study, based on the GS5 transgenic maize obtained through transgenic technology with specific expression in the endosperm, molecular assays were performed on the transformed plants. Southern blotting results showed that the GS5 gene was integrated into the maize genome in a low copy number, and RT-PCR analysis showed that the exogenous GS5 gene was normally and highly expressed in maize. The agronomic traits of two successive generations showed that certain lines were significantly improved in yield-related traits, and the most significant changes were observed in the OE-34 line, where the kernel width increased significantly by 8.99% and 10.96%, the 100-kernel weight increased by 14.10% and 10.82%, and the ear weight increased by 13.96% and 15.71%, respectively; however, no significant differences were observed in the plant height, ear height, kernel length, kernel row number, or kernel number. In addition, the overexpression of the GS5 gene increased the grain grouting rate and affected starch synthesis in the rice grains. The kernels’ starch content in OE-25, OE-34, and OE-57 increased by 10.30%, 7.39%, and 6.39%, respectively. Scanning electron microscopy was performed to observe changes in the starch granule size, and the starch granule diameter of the transgenic line(s) was significantly reduced. RT-PCR was performed to detect the expression levels of related genes in starch synthesis, and the expression of these genes was generally upregulated. It was speculated that the exogenous GS5 gene changed the size of the starch granules by regulating the expression of related genes in the starch synthesis pathway, thus increasing the starch content. The trans-GS5 gene was able to be stably expressed in the hybrids with the genetic backgrounds of the four materials, with significant increases in the kernel width, 100-kernel weight, and ear weight. In this study, the maize kernel size was significantly increased through the endosperm-specific expression of the rice GS5 gene, and good material for the functional analysis of the GS5 gene was created, which was of great importance in theory and application.
