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A Comparison of transgenic and wild type soybean seeds: analysis of transcriptome profiles using RNA-Seq.

Lambirth KC, Whaley AM, Blakley IC, Schlueter JA, Bost KL, Loraine AE, Piller KJ - BMC Biotechnol. (2015)

Bottom Line: However, the effects of expressing recombinant protein at high levels on bean physiology are not well understood.ST77 (hTG) and ST111 (hMBP) had significantly less differences with 52 and 307 differentially expressed genes respectively.Gene ontology enrichment analysis found that the upregulated genes in the 764 line were annotated with functions related to endopeptidase inhibitors and protein synthesis, but suppressed expression of genes annotated to the nuclear pore and to protein transport.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, University of North Carolina at Charlotte, Charlotte, NC, 28223, USA. kclambirth@uncc.edu.

ABSTRACT

Background: Soybean (Glycine max) has been bred for thousands of years to produce seeds rich in protein for human and animal consumption, making them an appealing bioreactor for producing valuable recombinant proteins at high levels. However, the effects of expressing recombinant protein at high levels on bean physiology are not well understood. To address this, we investigated whether gene expression within transgenic soybean seed tissue is altered when large amounts of recombinant proteins are being produced and stored exclusively in the seeds. We used RNA-Seq to survey gene expression in three transgenic soybean lines expressing recombinant protein at levels representing up to 1.61 % of total protein in seed tissues. The three lines included: ST77, expressing human thyroglobulin protein (hTG), ST111, expressing human myelin basic protein (hMBP), and 764, expressing a mutant, nontoxic form of a staphylococcal subunit vaccine protein (mSEB). All lines selected for analysis were homozygous and contained a single copy of the transgene.

Methods: Each transgenic soybean seed was screened for transgene presence and recombinant protein expression via PCR and western blotting.  Whole seed mRNA was extracted and cDNA libraries constructed for Illumina sequencing.  Following alignment to the soybean reference genome, differential gene expression analysis was conducted using edgeR and cufflinks.  Functional analysis of differentially expressed genes was carried out using the gene ontology analysis tool AgriGO.

Results: The transcriptomes of nine seeds from each transgenic line were sequenced and compared with wild type seeds. Native soybean gene expression was significantly altered in line 764 (mSEB) with more than 3000 genes being upregulated or downregulated. ST77 (hTG) and ST111 (hMBP) had significantly less differences with 52 and 307 differentially expressed genes respectively. Gene ontology enrichment analysis found that the upregulated genes in the 764 line were annotated with functions related to endopeptidase inhibitors and protein synthesis, but suppressed expression of genes annotated to the nuclear pore and to protein transport. No significant gene ontology terms were detected in ST77, and only a few genes involved in photosynthesis and thylakoid functions were downregulated in ST111. Despite these differences, transgenic plants and seeds appeared phenotypically similar to non-transgenic controls. There was no correlation between recombinant protein expression level and the quantity of differentially expressed genes detected.

Conclusions: Measurable unscripted gene expression changes were detected in the seed transcriptomes of all three transgenic soybean lines analyzed, with line 764 being substantially altered. Differences detected at the transcript level may be due to T-DNA insert locations, random mutations following transformation or direct effects of the recombinant protein itself, or a combination of these. The physiological consequences of such changes remain unknown.

No MeSH data available.


Related in: MedlinePlus

T-DNA transcript levels and coverage in transgenic seeds. (a) The number of reads aligned to the T-DNA sequence by Tophat from the reference genome comprising an extra scaffold containing the respective gene of interest cassette. Numbers on the y-axis represent RPKM normalized expression values for the gene of interest in each sample. (b-d) Coverage graphs over the annotated region of each added scaffold are shown for lines ST111 (b), 764 (C), and ST77 (d). The annotated components correspond to those shown in Fig. 1. The highest expressing seed is shown for each transgenic plant. The y-axis of each coverage graph ranges from 0 to 100
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Fig2: T-DNA transcript levels and coverage in transgenic seeds. (a) The number of reads aligned to the T-DNA sequence by Tophat from the reference genome comprising an extra scaffold containing the respective gene of interest cassette. Numbers on the y-axis represent RPKM normalized expression values for the gene of interest in each sample. (b-d) Coverage graphs over the annotated region of each added scaffold are shown for lines ST111 (b), 764 (C), and ST77 (d). The annotated components correspond to those shown in Fig. 1. The highest expressing seed is shown for each transgenic plant. The y-axis of each coverage graph ranges from 0 to 100

Mentions: Illumina sequencing was performed on libraries prepared from seed cDNA from each set of nine transgenic seeds as well as nine wild type seeds of the same genotype. Single-end, 100 base sequencing generated between 7 and 12 million reads per library. Reads were aligned to the reference genome and transcriptome and mRNA expression levels for transgenes and native soybean genes were assessed. Normalized expression values per sample for the transgenes are shown in Fig. 2a. Coverage maps from the highest expressing seed from each transgenic plant are depicted in Fig. 2b–d. The ST111 line which accumulated the least amount of recombinant protein showed the fewest aligned T-DNA reads, while the ST77 and 764 lines expressed greater levels of recombinant protein and showed a higher number of aligned reads. Note that the transcript levels of ST77 and 764 are similar despite ST77 expressing twice as much recombinant protein by mass as 764. This observation is likely due to the large size of the hTG transgene coupled with fewer aligned reads in the upstream portion of the gene, and can be visualized in the coverage maps (Fig. 2c–d). Analysis of the coverage data revealed accurate transcript initiation and termination of each transgene. Similarly, accurate initiation and termination of the selectable marker gene transcripts (BAR) was also observed in all cases.Fig. 2


A Comparison of transgenic and wild type soybean seeds: analysis of transcriptome profiles using RNA-Seq.

Lambirth KC, Whaley AM, Blakley IC, Schlueter JA, Bost KL, Loraine AE, Piller KJ - BMC Biotechnol. (2015)

T-DNA transcript levels and coverage in transgenic seeds. (a) The number of reads aligned to the T-DNA sequence by Tophat from the reference genome comprising an extra scaffold containing the respective gene of interest cassette. Numbers on the y-axis represent RPKM normalized expression values for the gene of interest in each sample. (b-d) Coverage graphs over the annotated region of each added scaffold are shown for lines ST111 (b), 764 (C), and ST77 (d). The annotated components correspond to those shown in Fig. 1. The highest expressing seed is shown for each transgenic plant. The y-axis of each coverage graph ranges from 0 to 100
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
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getmorefigures.php?uid=PMC4591623&req=5

Fig2: T-DNA transcript levels and coverage in transgenic seeds. (a) The number of reads aligned to the T-DNA sequence by Tophat from the reference genome comprising an extra scaffold containing the respective gene of interest cassette. Numbers on the y-axis represent RPKM normalized expression values for the gene of interest in each sample. (b-d) Coverage graphs over the annotated region of each added scaffold are shown for lines ST111 (b), 764 (C), and ST77 (d). The annotated components correspond to those shown in Fig. 1. The highest expressing seed is shown for each transgenic plant. The y-axis of each coverage graph ranges from 0 to 100
Mentions: Illumina sequencing was performed on libraries prepared from seed cDNA from each set of nine transgenic seeds as well as nine wild type seeds of the same genotype. Single-end, 100 base sequencing generated between 7 and 12 million reads per library. Reads were aligned to the reference genome and transcriptome and mRNA expression levels for transgenes and native soybean genes were assessed. Normalized expression values per sample for the transgenes are shown in Fig. 2a. Coverage maps from the highest expressing seed from each transgenic plant are depicted in Fig. 2b–d. The ST111 line which accumulated the least amount of recombinant protein showed the fewest aligned T-DNA reads, while the ST77 and 764 lines expressed greater levels of recombinant protein and showed a higher number of aligned reads. Note that the transcript levels of ST77 and 764 are similar despite ST77 expressing twice as much recombinant protein by mass as 764. This observation is likely due to the large size of the hTG transgene coupled with fewer aligned reads in the upstream portion of the gene, and can be visualized in the coverage maps (Fig. 2c–d). Analysis of the coverage data revealed accurate transcript initiation and termination of each transgene. Similarly, accurate initiation and termination of the selectable marker gene transcripts (BAR) was also observed in all cases.Fig. 2

Bottom Line: However, the effects of expressing recombinant protein at high levels on bean physiology are not well understood.ST77 (hTG) and ST111 (hMBP) had significantly less differences with 52 and 307 differentially expressed genes respectively.Gene ontology enrichment analysis found that the upregulated genes in the 764 line were annotated with functions related to endopeptidase inhibitors and protein synthesis, but suppressed expression of genes annotated to the nuclear pore and to protein transport.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, University of North Carolina at Charlotte, Charlotte, NC, 28223, USA. kclambirth@uncc.edu.

ABSTRACT

Background: Soybean (Glycine max) has been bred for thousands of years to produce seeds rich in protein for human and animal consumption, making them an appealing bioreactor for producing valuable recombinant proteins at high levels. However, the effects of expressing recombinant protein at high levels on bean physiology are not well understood. To address this, we investigated whether gene expression within transgenic soybean seed tissue is altered when large amounts of recombinant proteins are being produced and stored exclusively in the seeds. We used RNA-Seq to survey gene expression in three transgenic soybean lines expressing recombinant protein at levels representing up to 1.61 % of total protein in seed tissues. The three lines included: ST77, expressing human thyroglobulin protein (hTG), ST111, expressing human myelin basic protein (hMBP), and 764, expressing a mutant, nontoxic form of a staphylococcal subunit vaccine protein (mSEB). All lines selected for analysis were homozygous and contained a single copy of the transgene.

Methods: Each transgenic soybean seed was screened for transgene presence and recombinant protein expression via PCR and western blotting.  Whole seed mRNA was extracted and cDNA libraries constructed for Illumina sequencing.  Following alignment to the soybean reference genome, differential gene expression analysis was conducted using edgeR and cufflinks.  Functional analysis of differentially expressed genes was carried out using the gene ontology analysis tool AgriGO.

Results: The transcriptomes of nine seeds from each transgenic line were sequenced and compared with wild type seeds. Native soybean gene expression was significantly altered in line 764 (mSEB) with more than 3000 genes being upregulated or downregulated. ST77 (hTG) and ST111 (hMBP) had significantly less differences with 52 and 307 differentially expressed genes respectively. Gene ontology enrichment analysis found that the upregulated genes in the 764 line were annotated with functions related to endopeptidase inhibitors and protein synthesis, but suppressed expression of genes annotated to the nuclear pore and to protein transport. No significant gene ontology terms were detected in ST77, and only a few genes involved in photosynthesis and thylakoid functions were downregulated in ST111. Despite these differences, transgenic plants and seeds appeared phenotypically similar to non-transgenic controls. There was no correlation between recombinant protein expression level and the quantity of differentially expressed genes detected.

Conclusions: Measurable unscripted gene expression changes were detected in the seed transcriptomes of all three transgenic soybean lines analyzed, with line 764 being substantially altered. Differences detected at the transcript level may be due to T-DNA insert locations, random mutations following transformation or direct effects of the recombinant protein itself, or a combination of these. The physiological consequences of such changes remain unknown.

No MeSH data available.


Related in: MedlinePlus