Gene expression analysis of Thaumatotibia leucotreta in response to the Cryptophlebia leucotreta granulovirus
- Authors: Ridgeway, Jaryd Antony
- Date: 2015
- Subjects: Gene expression , Insects -- Viruses , Tortricidae -- Viruses
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:5931 , http://hdl.handle.net/10962/d1017809
- Description: Gene expression studies provide baseline information on the interactions of insects with their environment. Despite the importance of this information, limited gene expression data are available for most insect pests, including the family Tortricidae (Lepidoptera), which includes Thaumatotibia leucotreta (Meyr), an important agricultural pest in Africa. Because T. leucotreta can be controlled successfully by a granulovirus, this system is a good model for exploring insect-virus susceptibility. The main aim of this study was to investigate gene expression of T. leucotreta in responce to virus infection. However, before pursuing this aim, two objectives required completion. First, the most suitable RNA extraction method for insects needed to be determined and second, the most suitable reference genes for qPCR for Tortricidae pests needed to be identified. Once these objectives were accomplished, the response of T. leucotreta to its granulovirus was evaluated at different temperatures and points after infection.Four RNA extraction methods, the RNeasy® Mini Kit, SV Total RNA isolation system, TRIzol® reagent, and a CTAB-based method, were compared using two beetle and two moth species, including T. leucotreta. The quality of extracted RNA was similar for all four species for all extraction methods. Based on several criteria, the best RNA extraction method was the SV Total RNA isolation system. Six candidate reference genes were evaluated for qPCR using different tissue types of T. leucotreta and two other Tortricidae pests. Additionally, reference genes were evaluated for T. leucotreta with and without its granulovirus at different temperatures. Reference gene stability was found to be dependent on species and tissue type. Overall the most suitable combination of reference genes for T. leucotreta were α-actin, arginine kinase and elongation factor 1-α.Gene expression of T. leucotreta in response to granulovirus infection at different temperatures and intervals after infection was evaluated by qPCR using 13 target genes associated with the infection process. Most genes were down-regulated after 24 and 48 h.p.i. However, after 72 h.p.i most genes were up-regulated. The same trend was observed at different temperatures, where most genes were down-regulated at 15°C and 25°C but up-regulated at 35°C. These results show that there is a dynamic gene expression response in T. leucotreta due to granulovirus infection under different conditions. Not only do these findings provide insight into the control of this tortricid pest, they also contribute further to our knowledge of insect-virus interactions.
- Full Text:
- Date Issued: 2015
- Authors: Ridgeway, Jaryd Antony
- Date: 2015
- Subjects: Gene expression , Insects -- Viruses , Tortricidae -- Viruses
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:5931 , http://hdl.handle.net/10962/d1017809
- Description: Gene expression studies provide baseline information on the interactions of insects with their environment. Despite the importance of this information, limited gene expression data are available for most insect pests, including the family Tortricidae (Lepidoptera), which includes Thaumatotibia leucotreta (Meyr), an important agricultural pest in Africa. Because T. leucotreta can be controlled successfully by a granulovirus, this system is a good model for exploring insect-virus susceptibility. The main aim of this study was to investigate gene expression of T. leucotreta in responce to virus infection. However, before pursuing this aim, two objectives required completion. First, the most suitable RNA extraction method for insects needed to be determined and second, the most suitable reference genes for qPCR for Tortricidae pests needed to be identified. Once these objectives were accomplished, the response of T. leucotreta to its granulovirus was evaluated at different temperatures and points after infection.Four RNA extraction methods, the RNeasy® Mini Kit, SV Total RNA isolation system, TRIzol® reagent, and a CTAB-based method, were compared using two beetle and two moth species, including T. leucotreta. The quality of extracted RNA was similar for all four species for all extraction methods. Based on several criteria, the best RNA extraction method was the SV Total RNA isolation system. Six candidate reference genes were evaluated for qPCR using different tissue types of T. leucotreta and two other Tortricidae pests. Additionally, reference genes were evaluated for T. leucotreta with and without its granulovirus at different temperatures. Reference gene stability was found to be dependent on species and tissue type. Overall the most suitable combination of reference genes for T. leucotreta were α-actin, arginine kinase and elongation factor 1-α.Gene expression of T. leucotreta in response to granulovirus infection at different temperatures and intervals after infection was evaluated by qPCR using 13 target genes associated with the infection process. Most genes were down-regulated after 24 and 48 h.p.i. However, after 72 h.p.i most genes were up-regulated. The same trend was observed at different temperatures, where most genes were down-regulated at 15°C and 25°C but up-regulated at 35°C. These results show that there is a dynamic gene expression response in T. leucotreta due to granulovirus infection under different conditions. Not only do these findings provide insight into the control of this tortricid pest, they also contribute further to our knowledge of insect-virus interactions.
- Full Text:
- Date Issued: 2015
Differential expression and regulation of sucrose transporters in rice (Orzya sativa L, cv Nipponbare) during environmental stress conditions
- Authors: Ibraheem, Omodele
- Date: 2011
- Subjects: Crops -- Effect of stress on , Plant molecular genetics , Gene expression , Sucrose , Rice
- Language: English
- Type: Thesis , Doctoral , PhD (Biochemistry)
- Identifier: vital:11249 , http://hdl.handle.net/10353/330 , Crops -- Effect of stress on , Plant molecular genetics , Gene expression , Sucrose , Rice
- Description: Plant productivity is greatly affected by environmental stresses such as drought, salinity and insect herbivory. Plants respond and adapt to these stresses by exhibiting physiological as well as biochemical changes at the cellular and molecular levels in order to survive. Expression of a variety of genes which encode numerous membrane transporters have been demonstrated to be induced by these stresses in a variety of plants. The nutritional status of plants is controlled by these transporters, which are regulated by the transcription of the corresponding genes. In spite of these adverse stress effects on agricultural yield, only a few studies have focused on gene transcriptional and translational regulation of membrane transporters during environmental stress situations. Rice, like other plants, contains a number of sucrose transporters encoded by a family of genes. However, detailed knowledge of their roles, localization and regulation during environmental stress conditions is lacking. Bioinformatic tools were used to identify putative cis-acting regulatory elements that may be involved in the regulation of rice and Arabidopsis thaliana sucrose transporters. The possible cis-acting regulatory elements were predicted by scanning genomic sequences 1.5 kbp upstream of the sucrose transporter genes translational start sites, using Plant CARE, PLACE and Genomatix Matinspector professional data bases. Several cis-acting regulatory elements that are associated with plant development, plant hormonal regulation and stress response were identified, and were present in varying frequencies within the 1.5 kbp of 5′ regulatory region. The putative cis-acting regulatory elements that possibly are involved in the expression and regulation of sucrose transporter gene families in rice and Arabidopsis thaliana during cellular development or environmental stress conditions were identified as: A-box, RY, CAT, Pyrimidine-box, Sucrose-box, ABRE, ARF, ERE, GARE, Me-JA, ARE, DRE, GA-motif, GATA, GT-1, MYC, MYB, W-box, and I-box. Expression analysis was used to elucidate the role of rice (Oryza sativa L. cv Nipponbare) sucrose transporter (OsSUT) genes during drought and salinity treatments of three week old rice plants ( at four leaf stage) over a 10 days. Among the five rice OsSUT genes identified, only OsSUT2 was observed to be progressively up-regulated during drought and salinity treatments, while OsSUT1, OsSUT4 and OsSUT5 were expressed at low levels, and OsSUT3 showed no detectable transcript expression. Sucrose transport will be essential to meet the cellular energy demands and also for osmoprotectant activities during drought and salinity stresses. It therefore indicates that OsSUT2 which facilitates transport of sucrose from photosynthetic cells will be III essential for rice plants to cope with drought and salinity stresses, and cultivars with a higher OsSUT2 expression should be able to tolerate these environmental stresses better. The role of OsSUT in assimilate transport during rusty plum aphids (Hysteroneura setariae; Thomas) infestation on the leaves of three week old rice (Orzya sativa L. cv Nipponbare) cultivar plants, over a time-course of 1 to 10 days of treatments, was also examined by combination of gene expression and β-glucuronidase (GUS) reporter gene analysis. Real Time PCR analysis of the five OsSUT genes revealed that the expression of OsSUT1 was progressively up-regulated during the course of aphid infestation. OsSUT2 and OsSUT4 expression were comparatively low in both the control and treated plants. OsSUT5 showed no clear difference in transcript expression in both control and treated plants, while no detectable transcript expression of OsSUT3 could be found. The up-regulation of OsSUT1 gene was verified at protein level by western blot analysis in both the control and treated plants. OsSUT1 protein expression was found to increase with time during aphid infestation. A similar trend was noticeable in the control plants, however at a lower expression level. These demonstrate that the cellular expression of OsSUT1is regulated by both developmental and environmental factors. OsSUT1-promoter:::GUS reporter gene expression was observed within the vascular parenchyma and/or companion cells associated with phloem sieve elements of the large and small bundles in the phloem tissues of the flag leaf blade regions where feeding aphids were confined, which progressively increased with time of infestation. It is suggested that OsSUT1 may primarily play an essential role in phloem transport of assimilate to wounded tissues from adjacent health tissues or may be involved in the retrieval of assimilate back into the phloem to minimize loss caused by the infestation. Some OsSUT1-promoter:::GUS expression was also found in the metaxylem at 10 days after infestation, which could signify a recovery system in which sucrose lost into the xylem as a result of aphids feeding are retrieved back into the phloem through the vascular parenchyma. This was supported by the exposure of cut ends of matured OsSUT1-promoter:::GUS rice plant leaf to 2% sucrose solution. OsSUT1-promoter:::GUS expression was observed within the protoxylem, xylem and phloem parenchyma tissues. This indicates that sucrose translocating within the xylem tissues are retrieved into the phloem via the OsSUT1 localized within the parenchyma tissues. In conclusion, the differential expression and regulation of rice (Orzya sativa L. cv Nipponbare) sucrose transporters as reported here suggest that OsSUT2 and OsSUT1 were constitutively expressed compared to other rice sucrose transporters during drought and salinity, and rusty plum aphids (Hysteroneura setariae; Thomas) infestation stresses respectively. Thus, the expression and regulation of the sucrose transporters could be related to the physiological and nutritional requirements of the cells during plant developmental or environmental stress state that allows their differential expression.
- Full Text:
- Date Issued: 2011
- Authors: Ibraheem, Omodele
- Date: 2011
- Subjects: Crops -- Effect of stress on , Plant molecular genetics , Gene expression , Sucrose , Rice
- Language: English
- Type: Thesis , Doctoral , PhD (Biochemistry)
- Identifier: vital:11249 , http://hdl.handle.net/10353/330 , Crops -- Effect of stress on , Plant molecular genetics , Gene expression , Sucrose , Rice
- Description: Plant productivity is greatly affected by environmental stresses such as drought, salinity and insect herbivory. Plants respond and adapt to these stresses by exhibiting physiological as well as biochemical changes at the cellular and molecular levels in order to survive. Expression of a variety of genes which encode numerous membrane transporters have been demonstrated to be induced by these stresses in a variety of plants. The nutritional status of plants is controlled by these transporters, which are regulated by the transcription of the corresponding genes. In spite of these adverse stress effects on agricultural yield, only a few studies have focused on gene transcriptional and translational regulation of membrane transporters during environmental stress situations. Rice, like other plants, contains a number of sucrose transporters encoded by a family of genes. However, detailed knowledge of their roles, localization and regulation during environmental stress conditions is lacking. Bioinformatic tools were used to identify putative cis-acting regulatory elements that may be involved in the regulation of rice and Arabidopsis thaliana sucrose transporters. The possible cis-acting regulatory elements were predicted by scanning genomic sequences 1.5 kbp upstream of the sucrose transporter genes translational start sites, using Plant CARE, PLACE and Genomatix Matinspector professional data bases. Several cis-acting regulatory elements that are associated with plant development, plant hormonal regulation and stress response were identified, and were present in varying frequencies within the 1.5 kbp of 5′ regulatory region. The putative cis-acting regulatory elements that possibly are involved in the expression and regulation of sucrose transporter gene families in rice and Arabidopsis thaliana during cellular development or environmental stress conditions were identified as: A-box, RY, CAT, Pyrimidine-box, Sucrose-box, ABRE, ARF, ERE, GARE, Me-JA, ARE, DRE, GA-motif, GATA, GT-1, MYC, MYB, W-box, and I-box. Expression analysis was used to elucidate the role of rice (Oryza sativa L. cv Nipponbare) sucrose transporter (OsSUT) genes during drought and salinity treatments of three week old rice plants ( at four leaf stage) over a 10 days. Among the five rice OsSUT genes identified, only OsSUT2 was observed to be progressively up-regulated during drought and salinity treatments, while OsSUT1, OsSUT4 and OsSUT5 were expressed at low levels, and OsSUT3 showed no detectable transcript expression. Sucrose transport will be essential to meet the cellular energy demands and also for osmoprotectant activities during drought and salinity stresses. It therefore indicates that OsSUT2 which facilitates transport of sucrose from photosynthetic cells will be III essential for rice plants to cope with drought and salinity stresses, and cultivars with a higher OsSUT2 expression should be able to tolerate these environmental stresses better. The role of OsSUT in assimilate transport during rusty plum aphids (Hysteroneura setariae; Thomas) infestation on the leaves of three week old rice (Orzya sativa L. cv Nipponbare) cultivar plants, over a time-course of 1 to 10 days of treatments, was also examined by combination of gene expression and β-glucuronidase (GUS) reporter gene analysis. Real Time PCR analysis of the five OsSUT genes revealed that the expression of OsSUT1 was progressively up-regulated during the course of aphid infestation. OsSUT2 and OsSUT4 expression were comparatively low in both the control and treated plants. OsSUT5 showed no clear difference in transcript expression in both control and treated plants, while no detectable transcript expression of OsSUT3 could be found. The up-regulation of OsSUT1 gene was verified at protein level by western blot analysis in both the control and treated plants. OsSUT1 protein expression was found to increase with time during aphid infestation. A similar trend was noticeable in the control plants, however at a lower expression level. These demonstrate that the cellular expression of OsSUT1is regulated by both developmental and environmental factors. OsSUT1-promoter:::GUS reporter gene expression was observed within the vascular parenchyma and/or companion cells associated with phloem sieve elements of the large and small bundles in the phloem tissues of the flag leaf blade regions where feeding aphids were confined, which progressively increased with time of infestation. It is suggested that OsSUT1 may primarily play an essential role in phloem transport of assimilate to wounded tissues from adjacent health tissues or may be involved in the retrieval of assimilate back into the phloem to minimize loss caused by the infestation. Some OsSUT1-promoter:::GUS expression was also found in the metaxylem at 10 days after infestation, which could signify a recovery system in which sucrose lost into the xylem as a result of aphids feeding are retrieved back into the phloem through the vascular parenchyma. This was supported by the exposure of cut ends of matured OsSUT1-promoter:::GUS rice plant leaf to 2% sucrose solution. OsSUT1-promoter:::GUS expression was observed within the protoxylem, xylem and phloem parenchyma tissues. This indicates that sucrose translocating within the xylem tissues are retrieved into the phloem via the OsSUT1 localized within the parenchyma tissues. In conclusion, the differential expression and regulation of rice (Orzya sativa L. cv Nipponbare) sucrose transporters as reported here suggest that OsSUT2 and OsSUT1 were constitutively expressed compared to other rice sucrose transporters during drought and salinity, and rusty plum aphids (Hysteroneura setariae; Thomas) infestation stresses respectively. Thus, the expression and regulation of the sucrose transporters could be related to the physiological and nutritional requirements of the cells during plant developmental or environmental stress state that allows their differential expression.
- Full Text:
- Date Issued: 2011
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