Progress 10/01/03 to 09/30/09
Outputs
OUTPUTS: Rice (Oryza sativa cv. Nipponbarre) was transformed with a construct for human papillomavirus type 16 protein E7 (HPV16E7), under the control of the rice seed-specific glutelin-1 promoter. An additional construct contained the rice lectin carboxyl terminal propeptide (CTPP) sequence for vacuolar targeting. Inclusion of the CTPP increased HPV16E7 expression 3-4 fold, up to 2.3% total seed protein. However, the signal peptide, which became glycosylated, was not efficiently cleaved from the HPV16E7 protein. The HPV16E7 protein formed large aggregates that required the presence of beta-mercaptoethanol for solubilization. The presence of the CTPP targeting signal did not influence localization of the HPV16E7. With both constructs, protein was localized to protein storage vacuoles and prolamin storage bodies. The specialized nature of rice seed storage tissue appears to influence recombinant protein trafficking and localization. Two homologs of isoflavone synthase (VuIFS1 and VuIFS2) were cloned from cowpea (Vigna unguiculata), including their promoter regions and 5' and 3' UTRs. VuIFS1 contained three poly (A) signals in the 3' UTR, resulting in three transcripts of different lengths. RT-PCR suggested that both genes were expressed in stem and leaf tissues, but not in seeds. Analysis of the cowpea promoter regions revealed the absence of ABRE motifs, which are present in soybean IFS promoters. Transformation of cowpea callus with the soybean IFS1 gene (with its promoter), resulted in a several-fold increase in isoflavone production in the callus. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Rice seed was shown to be a suitable plant-based system for the production of pharmaceutical proteins. Inclusion of a carboxyl terminal propeptide was found to be undesirable due to non-cleavage. Similar localization in protein storage vacuoles, regardless of the presence or absence of this targeting signal, suggests recombinant proteins in seeds will be localized in a manner common to native seed storage proteins. Lack of ABRE motifs in cowpea IFS promoters, and hence negligible expression in seeds, may explain in part the lack of isoflavones in cowpea seeds. Increased isoflavone levels in cowpea callus transformed with soybean IFS suggests that seed levels may be appreciably enhanced once transformed plants are obtained.
Publications
- Kaur, N. Genetic engineering of cowpea to enhance isoflavone biosynthesis. Ph.D. Dissertation, Univ. of Arkansas. May 2009.
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Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: Cowpea, a highly nutritious legume, is an important dietary staple in many developing countries, and is widely consumed in the U.S. While related to soybean, cowpea does not accumulate large amounts of isoflavones in its seeds as does soybean. Isoflavones are a class of phytochemicals which, when consumed, have demonstrated significant health benefits, including reduced risk of certain cancers and cardiovascular disease. The objective of this project is to determine whether cowpea can be engineered to accumulate isoflavones by the introduction of soybean isoflavone synthase (IFS), a key enzyme in isoflavone biosynthesis. The entire gene sequence, including the promoter region, for IFS1 was cloned from soybean. Cowpea explants have been transformed with this construct via Agrobacterium co-cultivation, but plant regeneration from transgenic callus has not yet been achieved. Using degenerate primers, the cowpea genes for IFS1 and IFS2 have been cloned for the first time. Human papillomavirus (HPV) Type 16 E7 protein has been implicated in the initiation and maintenance of tumorous cells in cervical cancer. Development of a therapeutic vaccine requires the safe, economical production of 16E7, such as can be obtained in a plant-based system. The objective of this project was to genetically engineer rice to accumulate HPV 16E7 in rice seed protein storage bodies. An expression cassette was designed, comprised of the sequences for the rice glutelin 1 promoter, a 6x-HIS tag and FXa protease site for purification, the coding sequence for 16E7, and the NOS terminator. An additional cassette was designed that included the vacuolar targeting sequence for the rice lectin CTPP at the 3' terminus of 16E7. Callus of the rice cultivar Nipponbare was transformed by co-cultivation with Agrobacterium, and transgenic plants were regenerated on selection media. Successful transformation and expression were verified by PCR and Western blotting. PARTICIPANTS: J. Brad Murphy, PI Teddy Morelock, UAF, collaborator on cowpea project Tim O'Brien, UAMS, collaborator on HPV project TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Comparison of the promoter regulatory elements in cowpea IFS with those in soybean revealed that the cowpea IFS promoter lacks any ABRE domains, which do occur in the soybean IFS promoter. This may contribute in part to the lack of isoflavone accumulation in cowpea seeds. Following transformation with the soybean IFS gene, cowpea callus exhibited a three-fold increase in isoflavone accumulation. Due to the seed-specific regulatory elements in the soybean IFS promoter, even higher levels of isoflavone accumulation might be expected in cowpea seeds when transgenic plants are obtained. Transgenic rice seeds accumulated HPV 16E7, which could be purified by affinity chromatography of the HIS tag. Attempts to further purify the protein revealed that it spontaneously formed high molecular-weight aggregates. It was determined that high levels of β-mercaptoethanol were absolutely necessary to extract and maintain any monomeric protein. While addition of the CTPP targeting sequence appeared to increase the level of accumulation, failure to cleave this sequence from the mature protein suggests that CTTPs may not be suitable for targeting of pharmaceutical proteins.
Publications
- Mehra, A. 2008. The production of cervical cancer-related HPV16E7 oncoprotein in rice seeds. Ph.D. Dissertation, Univ. of Arkansas. Dec. 2008.
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Progress 01/01/07 to 12/31/07
Outputs
OUTPUTS: Cowpea, a highly nutritious legume, is an important dietary staple in many developing countries, and is widely consumed in the U.S. While related to soybean, cowpea does not accumulate large amounts of isoflavones in its seeds as does soybean. Isoflavones are a class of phytochemicals which, when consumed, have demonstrated significant health benefits, including reduced risk of certain cancers and cardiovascular disease. The objective of this project is to determine whether cowpea can be engineered to accumulate isoflavones by the introduction of soybean isoflavone synthase (IFS), a key enzyme in isoflavone biosynthesis. The entire gene sequence, including the promoter region, for IFS1 was cloned from soybean. Cowpea explants have been transformed with this construct via Agrobacterium co-cultivation, but plant regeneration from transgenic callus has not yet been achieved. Using degenerate primers, the cowpea genes for IFS1 and IFS2 have been cloned for the first time. Human
papillomavirus (HPV) Type 16 E7 protein has been implicated in the initiation and maintenance of tumorous cells in cervical cancer. Development of a therapeutic vaccine requires the safe, economical production of 16E7, such as can be obtained in a plant-based system. The objective of this project was to genetically engineer rice to accumulate HPV 16E7 in rice seed protein storage bodies. An expression cassette was designed, comprised of the sequences for the rice glutelin 1 promoter, a 6x-HIS tag and FXa protease site for purification, the coding sequence for 16E7, and the NOS terminator. An additional cassette was designed that included the vacuolar targeting sequence for the rice lectin CTPP at the 3 feet terminus of 16E7. Callus of the rice cultivar Nipponbare was transformed by co-cultivation with Agrobacterium, and transgenic plants were regenerated on selection media. Successful transformation and expression were verified by PCR and Western blotting.
PARTICIPANTS: J. Brad Murphy, University of Arkansas-Fayetteville. Teddy Morelock, University of Arkansas-Fayetteville, collaborator on cowpea project. Tim O'Brien, University of Arkansas Medical Sciences, collaborator on HPV project.
Impacts
The cowpea coding sequences for IFS1 and IFS2 are highly homologous to soybean. Comparison of the promoter regulatory elements in cowpea IFS with those in soybean may shed light on why cowpea does not normally accumulate isoflavones. Successful regeneration of transgenic cowpea plants will allow us to determine if the expression of the soybean IFS does result in enhanced isofavone accumulation in the seeds. Transgenic rice seeds accumulated HPV 16E7, which could be purified by affinity chromatography of the HIS tag. The recombinant protein was the same size as the original HPV 16E7. Addition of the CTPP targeting sequence appeared to increase the level of accumulation. The recombinant protein can now be tested for its antigenicity to induce an immune response.
Publications
- No publications reported this period
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Progress 01/01/06 to 12/31/06
Outputs
Rice plantlets transformed with HPV proteins E6 and E7 have been obtained through tissue culture and transferred to a greenhouse. While surviving on selection media, the transgenic nature of these plants must still be verified. Cowpea shoots transformed with soybean isoflavone synthase have been obtained through tissue culture. These shoots will be screened by rooting on selection media. Transgenic cowpea callus has been obtained (verified by GUS staining), but plant regeneration from callus has not yet been accomplished.
Impacts
Successful transformation/regeneration of rice and cowpea will pave the way for further endeavors to produce pharmaceutical proteins and/or enhance the nutraceutical levels in these two crops.
Publications
- No publications reported this period
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Progress 01/01/05 to 12/31/05
Outputs
HPLC analysis of antioxidant carotenoids (lutein and Beta carotene) in spinach and other edible greens (kale, collards, turnip, and mustard) from nine separate studies has been completed. The intent of these studies encompassed plant-to-plant variation, leaf maturity effects, harvest date influence, and environmental (growing site, year) effects. Plant-to-plant variability was typically high within lines, which translated to considerable variation between lines with respect to the other parameters investigated. Relative ranking of lines changes depending on the individual study. However, the presence of such variability provides the opportunity to improve carotenoid levels through breeding. The fact that other edible greens such as kale had lutein levels around 50 percent higher than spinach suggests that there shouldn't be any physical limitation to significantly increasing lutein levels in spinach.
Impacts
These studies provide the foundation for further breeding efforts to increase the level of carotenoid antioxidants in spinach and edible greens, eventually enhancing the health benefits of these vegetables in the human diet.
Publications
- No publications reported this period
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Progress 01/01/04 to 12/30/04
Outputs
An HPLC analysis of lutein levels from whole spinach plants from 31 lines collected at two harvest dates, Dec 20, 2001 and Jan 30, 2002 at Crystal City, TX, and Dec 1,2001 and Feb 1, 2002 at Uvalde, TX, was conducted. At each harvest date, some lines had higher levels of lutein at Crystal City, others at Uvalde. At both sites, lutein levels on average decreased before the second harvest date, more severely at Crystal City (average -17.5 % over 31 lines) than at Uvalde (only -5.0%). The range of differences at Crystal City was -42.7% to +8.1%, while at Uvalde it was -44.3% to +25.3%, so not all lines showed a decline. ARK 97-159 had the highest lutein level at both sites, but showed a 33.5% loss at Crystal City and a 14.9% loss at Uvalde, knocking it out of the first place rank. ARK 91-415 was at the lower end of the lutein levels, but declined less than 10% at both sites. Over many studies with spinach and other edible greens, it was determined that there was a
consistently high correlation (r2 = 0.82) between lutein levels and chlorophyll a levels with a relatively constant slope (Y = .07X). Because this slope was so consistent, we explored whether some indirect method of estimating chlorophyll a could be used to select lines for high lutein. We tried using digital imaging and a chlorophyll meter to predict chlorophyll a content. Neither method was found to have the desired accuracy. Using a digital image analysis, a covariate analysis of red, green, and blue values gave an r2 = .6079. The chlorophyll meter readings gave an r2 = .1546. Leaves of highly variable chlorophyll a levels gave identical meter readings. In both cases, surface characteristics of the leaves must contribute to the lack of discernibility.
Impacts
Spinach shows a high genotype by environment interaction, making it difficult to identify breeding lines that will be consistently high in lutein at various growing sites.
Publications
- No publications reported this period
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Progress 01/01/03 to 12/31/03
Outputs
Lutein analysis in spinach has been completed for two additional experiments. These are the plant-to-plant variation and the leaf-age-comparison studies, both harvested 11-27-01 at Kibler, AR. The plant-to-plant variation study consisted of 32 selections with 10 plants per selection and three subsamples per plant. Percent variability among plants within spinach selections ranged from 4% in Coho to 12% in 97-139. Values were evenly distributed within the range observed, suggesting that the full range within a selection may still not be adequately defined even with ten plants. The leaf-age-comparison study consisted of 32 selections with five plants per selection, three leaf ages, one leaf per leaf age, and one subsample per leaf. Young leaves averaged 0.36 mg/g DW, medium-maturity leaves averaged 0.45 mg/g DW, and fully-mature leaves averaged 0.50 mg/g DW. However, there were a number of lines in which the young and medium-maturity leaves contained the highest levels.
Such varieties would be well suited for baby leaf production. In our study to produce pharmaceutical proteins in plants, Western blotting verified the accumulation of the target recombinant protein in seeds of Brassica carinata. The transgene has been stable through at least the third generation. Regeneration of rice and cowpea were accomplished using the thin cell layer method. We are currently developing transformation protocols for use in conjunction with this method.
Impacts
Consumption of lutein, a carotenoid antioxidant, has been associated with the prevention of a number of chronic illnesses, in particular, age-related macular degeneration. Analysis of the levels of lutein in various genotypes of spinach and other leafy greens will serve as the foundation for selections in a breeding program to enhance the level of this desirable antioxidant in the natural diet. Genetic engineering of recombinant proteins into plants offers a safe, economical means of producing high-value biopharmaceuticals.
Publications
- Secks, M.E., Murphy, J.B., & Morelock, T.E. 2003. Lutein and beta-carotene levels in selections of spinach and edible greens. Ark. Agric. Expt. Sta. Res. Ser. 506: 51-55.
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Progress 01/01/02 to 12/31/02
Outputs
Lutein analysis for spinach and greens includes three completed experiments and two experiments in progress. Whole plant samples of spinach were harvested 2-1-01 at Crystal City, TX. Lutein analysis of 20 selections, three subsamples per selection has been completed. Lutein levels of whole plants ranged from 0.34 mg/g dry weight (DW) to 0.48 mg/g DW among the 20 selections. Composite samples of spinach (five fully expanded leaves from five different plants comprised each composite sample) were harvested 11-27-01 at Kibler, AR, and all 32 selections, three subsamples per selection were analyzed. Lutein amounts among selections ranged from 0.36 mg/g DW to 0.58 mg/g DW. Also completed are the composite samples of assorted greens harvested 11-27-01 at Kibler, AR. There were 10 collard, 10 kale, six mustard, and six turnip selections, as well as three miscellaneous greens selections with three subsamples per selection. 'Green curled' kale, 'Tuscan' kale, 'Toscano' kale and
'Savannah' mustard contained the highest amounts of lutein at 0.76, 0.70, 0.63, and 0.57 mg/g DW, respectively. In progress are the plant-to-plant variation and the leaf-age-comparison studies, both harvested 11-27-01 at Kibler, AR. The plant-to-plant variation study consists of 32 selections with 10 plants per selection and three subsamples per plant, and has 18 selections completed to date. Percent variability among plants within spinach selections ranged from 4% in 'Coho' to 12% in 97-152. The leaf-age-comparison study consists of 32 selections with five plants per selection, three leaf ages, one leaf per leaf age, and one subsample per leaf, and has 17 selections completed. Young leaves averaged 0.35 mg/g DW, medium-maturity leaves averaged 0.45 mg/g DW, and fully-mature leaves averaged 0.49 mg/g DW. Regardless of the study, two spinach selections, 97-159 and 88-212 ranked consistently high (within the six highest lutein levels) among all four spinach experiments.
Impacts
Consumption of lutein, a carotenoid antioxidant, has been associated with the prevention of a number of chronic illnesses, in particular, age-related macular degeneration. Analysis of the levels of lutein in various genotypes of spinach and other leafy greens will serve as the foundation for selections in a breeding program to enhance the level of this desirable antioxidant in the natural diet.
Publications
- Secks, M.E., Murphy, J.B., & Morelock, T.E. 2002. What's hot? What's not? Evaluation of capsaicinoids in Capsicum spp. fruit using high performance liquid chromatography. Ark. Agric. Expt. Sta. Res. Ser. 494: 58-60.
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Progress 01/01/01 to 12/31/01
Outputs
Variability in pungency levels of chile pepper (Capsicum spp.) fruits was determined by the HPLC analysis of capsaicinoids. Comparing chile pepper types using a composite fruit sample from several plants, pungency varied over 10-fold from less than 1 mg/g DW total capsaicinoids (Anaheim) to almost 10 mg/g DW (Habanero). In terms of perceived heat (Scoville Heat Units, SCH), the values were around 12,000 for Anaheim and 145,000 for Habanero. Plant to plant variation was analyzed in detail on fruit from 129 plants of JM3, a breeding line developed from C. annuum L. var. okala. Total fruit capsaicinoids from these genetically similar plants varied 4-fold, ranging from 6.6 mg/g DW (98,000 SHU) to 25.6 mg/g DW (385,000 SHU). These values were lower than those from similar JM3 plants grown at a hotter, drier location. Thus, there are both significant genotypic and environmental effects on capsaicinoid levels.
Impacts
Products incorporating the pungency of chile peppers are continuing to gain in popularity. In order to provide a more consistent consumer product, the factors affecting capsaicinoid accumulation in chile pepper fruit need to be more fully understood.
Publications
- No publications reported this period
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Progress 01/01/00 to 12/31/00
Outputs
We have engineered plants to produce two human proteins that have been shown to be tumor-associated and may be involved in the regulation of cancer metastasis. Clones of a human tumor-associated protease and its natural inhibitor were linked to the promoter and gene for oleosin, a protein found in the membrane of seed oil bodies. The resulting gene construct was introduced into Brassica carinata, an oil-seed mustard. The gene construct included a recognition site that allows proteolytic cleavage of the introduced protein from the oleosin, allowing efficient purification of the protein of interest. Seed oil body proteins from transformed plants were analyzed by gel electrophoresis and probed with antibodies against the protease and its inhibitor, and the plants were found to be expressing the foreign proteins.
Impacts
The use of genetically modified plants to produce pharmaceutical proteins of direct benefit to human health represents the second generation of GMOs. Large-scale, low-cost production of these two cancer-related proteins in plants will allow rapid advances in their biochemical characterization and, more importantly, may facilitate their practical use in early cancer diagnosis or treatment.
Publications
- Murphy, J.B., & Morelock, T.E. 2000. Spinach breeding program yields lines containing high levels of carotenoid antioxidants. In: M.D. Richardson and J.R. Clark (eds.), Hort. Studies - 1999. Ark. Agri. Exp. Sta. Res. Ser. 475: 36-39.
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Progress 01/01/99 to 12/31/99
Outputs
Dark-green leafy vegetables, such as spinach and kale, are relatively high in carotenoids, especially lutein. Lutein is of particular interest because it has high antioxidant activity and has been directly implicated in cancer prevention and reducing macular degeneration. To determine the feasibility of increasing overall carotenoid antioxidants and lutein content in spinach, an HPLC analysis of carotenoid content and composition was conducted on 18 spinach lines including commercial cultivars and University of Arkansas breeding lines. Our study found that two lines coming out of the UA breeding program, Fall Green and F380, contain higher than average levels of both lutein and beta-carotene. This is particularly true for lutein, for which their contents were roughly 53% higher than the average. The variability within and between lines suggests that carotenoid antioxidant and, in paticular, lutein content of spinach can be further enhanced through a directed breeding
program.
Impacts
A comparison of commercial spinach cultivars and University of Arkansas breeding lines revealed that the UA program has produced several lines exhibiting higher than average contents of both lutein and beta-carotene, important dietary antioxidants implicated in the health benefits of fruits and vegetables. These findings suggest that carotenoid antioxidant levels in spinach could be further enhanced by a directed breeding program.
Publications
- Kirkpatrick, J.A., Murphy, J.B., Morelock, T.E., & Burden, B.D. 1999. Carotenoid antioxidant levels in spinach. HortSci. 34: 407. (Abstract)
- Murphy, J.B., & Morelock, T.E. 1999. Carotenoid antioxidant levels in spinach: preliminary screening. In: J.R. Clark and M.D. Richardson (eds.), Hort. Studies - 1998. Ark. Agri. Exp. Sta. Res. Ser. 466: 106-108.
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