Silencing cell wall remodeling enzymes in tomato and analysis of the effects on fruit softening

Fruits are the seed dispersal units of flowering plants and fleshy fruits form an important part of the human diet. Ripening is a complex developmental process and involves many events such as textural and constitutional changes. The texture of fleshy fruits is one of the major criteria for consumer choice. However, the molecular determinants of ripening- associated changes in texture or “softening” are relatively poorly understood and seem to involve a large number of cell wall remodelling factors. The recent completion of the tomato genome sequence has revealed more than 50 cell wall structure-related genes that are expressed during fruit development and ripening and may impact texture changes in this fruit (Tomato Genome Consortium, 2012). The aim of the project was to compare, on a genome-wide scale, ripening-related gene expression in a range of fleshy fruits and especially those linked with cell wall remodelling. Then by identifying orthologous genes in different fruit species, to make predictions about those genes important for the softening process in all fleshy fruits. We hypothesise that there are a core set of genes that is always associated with fruit softening. Once this core set was identified we planned to use transgenic approaches to test the role of these cell wall genes in texture changes using tomato (Solanum lycopersicum) as a model system. Comparative genomics analysis of tomato (S. lycopersicum), banana (Musa acuminata), melon (Cucumis melo) and grape (Vitis vinifera), was undertaken using Inparanoid software. This analysis showed that a total of 8,982 (25.86%) gene models could be identified in common between all four genomes based on comparison of amino acid sequences. However, comparison of the expression patterns of cell wall remodelling genes across the various species revealed that most were expressed in tissues other than ripening fruits and of the fruit expressed genes only a small number were common between different species. We selected a CELLULOSE SYNTHASE (CESA) gene that was up-regulated in all fruit species for further analysis. The PHYTOENE SYNTHASE 1 (PSY1) was selected as a control, and PECTATE LYASE (PL) and POLYGALACTURONASE (PG) as genes that were highly expressed in many fruits including tomato. We then used the recently developed CRISPR/ Cas9 DNA editing technology to generate mutations in the target PSY1, CESA, PL and PG genes. A range of either single base insertions or deletions of more than ten bases were identified at Cas9 cleavage site in T0 plants in all cases except for the CESA lines where no mutant plants were recovered; suggesting that knocking out this gene gives a lethal phenotype. The efficacy of CRISPR/Cas9 constructs in tomato fruits was readily apparent when observing the phenotype in PSY1 line where yellow or orange fruits occurred in T0 individuals. In the PL lines, substantially firmer tomato fruits were produced at the red ripe stage. There were some much smaller impacts on texture in the PG CRISPR lines. The CRISPR approaches described in this thesis provide methods to enhance texture and extend shelf life in tomato without compromising fruit quality

Identification of flowering genes of Stevia rebaudiana by using transcriptomic data

Stevia rebaudiana is a medicinal plant suggested as sucrose substitute ingredient in drinks as it lowers down the glucose level. It is recognized as zero calories sugar with 300 times sweeter than sugar cane. Stevia plant currently used as high non-nutritive high potency sweeteners. Research shows that stevioside level are the highest at the time of flower bud formation and lower at time preceding and following flower bud formation. Hence, the aim of this study is to identify gene involved in flowering of local S. rebaudiana accession MS 007 from transcriptomic data analysis. The samples were collected in Nursery and Glasshouse Complex (GNC), one sample of flowering (40 days after cultivation) and another is was collected before flowering bud formation. The clustering of the index-coded samples was performed on a cBot Cluster Generation System using PE Cluster Kit cBot-HS (Illumina) according to the manufacturer’s instructions. This process involved de novo transcriptome assembly of Illumina using Trinity. Data Quality Control and data filtering was done prior to annotation and analysis. The analysis of transcriptomic data manages to annotate total of 103524 of expressed genes with 9832 genes was found in flowering sample and 8871 genes non-flowering sample. Among flowering genes identified; PHYTOCHROME A (PHYA) genes, GIBBERELLIN (GA3) genes and LATE ELONGATED HYPOCOTYL (LHY) genes which involved in plant flowering pathway such as gibberellin signalling and circadian clock signalling pathway. The outcome of this study will help further study in manipulation of flowering process especially in increasing the yield of steviol glycosides during harvest process

In Silico development of CRISPR/Cas9 Construct for Oryza sativa subsp. indica

Oryza sativa, which is commonly known as rice, is one of the staple foods consumed by Asian. There are three subspecies of O. sativa that are different in their geographical adaptations which are Japonica, Javanica and Indica. Climate change causes increasing greenhouse gas emissions fluctuating rainfall, and drought which is the limiting factors of rice production in Malaysia. In rice, SUMO E2-Conjugating Enzyme (OsSCE1) gene plays a role as a negative regulator in the drought stress response. OsSCE1 gene is a type of small ubiquitin-like modifier (SUMO)-conjugating enzyme involved in a few regulatory processes such as SUMOylation. The crop improvement can be made possible through a genome editing approach which enables the manipulation of targeted genetic traits and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system is one of the genomes editing technologies that are accessible. In this study, the CRISPR construct of drought tolerance of Oryza sativa subsp. indica was developed. The CRISPR construct development involved several stages; OsSCE1 gene prediction, sgRNA design, and vector construction. FGENESH and GeneMarkS were used for OsSCE1 gene prediction. The predicted OsSCE1 gene has been validated using Polymerisation Chain Reaction (PCR) and sequencing. The BLASTN result shows high similarity with Oryza sativa in chromosome 10 with 99% identities. This step followed by sgRNA design which was carried out manually with the help of gRNA prediction tools such as WU-CRISPR, CCTop, Benchling, and CRISPR-P. Lastly, vector construction was executed virtually using Benchling. Overall, the OsSCE1 gene was successfully characterized and validated, and the in silico CRISPR construct for Oryza sativa subsp. indica was developed. To ensure a good outcome, in silico CRISPR construct should be verified through in vivo studies in further

In silico analysis of OSRR22 isolated from MR 219 rice and the strategy for developing CRISPR/CAS9 construct for genome editing

The rice response regulator 22 (RR22) has been reported to be negatively regulating salt tolerance in Oryza sativa and involved in the cytokinin signaling pathway; however, it has not been documented in any Malaysian rice and is deemed as an appealing subject for CRIPSR/Cas9 editing. This study analysed the OsRR22 gene from the Malaysian rice cultivar ‘MR 219’ to elucidate its function and determine the gRNA target site, in which the finding served as the most vital part for our current CRISPR/Cas9 genome editing experiment. In brief, the methods employed include total genome isolation, polymerase chain reaction (PCR) amplification, DNA sequencing, genome search, and computational analyses involving an array of in silico tools. The transcript of OsRR22 was 2,019 bp long, composed of six exons and it encoded a highly conserved 696 amino acid residues. Motif analysis revealed the gene product, RR22, contained response regulator (RR) receiver domain (position 27-142), disordered domain (position 154-214), and Myb-like DNA-binding domain (position 214-273). Analysis on the protein-protein interaction (PPI) using STRING revealed RR22 interacted with various proteins including RR24, RR B8A7T0, and a set of HPt domain-containing proteins (B8B4B1, B8AYV8, B8BEM5, B8A9E0 and B8B9H1). Analysis of gene expression profiles via the Rice Expression Database (RED) revealed the OsRR22 (Os06g0183100) gene was highly expressed (FPKM>10) in the leaf and root. In addition, there were 15 genes to be co-expressed (Pearson’s r value > 0.85) with the OsRR22 gene of which high-affinity potassium transporter 9 (HAK9, Os07g0679000) was one of them. Based on the first exon of OsRR22 that encoded a part of the RR receiver domain, a CRISPR-gRNA 20-bp spacer was generated through CCTop. The gRNA spacer was synthesised, annealed, and ligated into the CRSPR/Cas9 pRGEB32 vector. The CRISPR/Cas9 construct—targeting MR 219’s OsRR22 and intended for Agrobacterium-based delivery—was successfully developed. Our study here documents the upstream workflow involved in rice genome editing with an emphasis on the gene characterisation through multiple bioinformatics tools

In silico identification and characterization of Kaurene Synthase protein in Stevia rebaudiana MS007

Stevia rebaudiana (Sr), belonging to Asteraceae family is a plant native to Paraguay. It is currently being used as a healthier alternative for sugar. Sr produces steviol glycosides (SGs), a group of secondary metabolite compounds that is responsible for its sweetening taste. SGs act as sweetener due to the presence of two major compounds, Stevioside and Rebaudioside A. Biosynthesis of these compounds involve enzymes such as geranylgeranyl pyrophosphate (GPPS), copalyl diphosphate synthase (CPPS), kaurene synthase (KS) and kaurene oxidase (KO) in the pathway. In this study, the identification and characterization of Stevia rebaudiana MS007 kaurene synthase (SrKS) were done by in silico analysis of the transcriptomic dataset. Homology search from BLASTx resulting in SrKSfrom query Cluster-31069.42907 (Sr MS007) of transcriptomic dataset shows the highest similarity percentage identity (99.62%). ExPasy tools were used to translate the nucleotide sequence into protein sequence. The protein domain is predicted by protein domain search analysis using Interpro and shows IPR005630 (terpene synthase metal-binding domain) available at positions 454 to 719 and IPR001906 (terpene-synthase-N-terminal-domain) at position 222 to 411 as the domains. In constructing the phylogenetic analysis tree, multiple sequence alignment was initially done using MUSCLE and MEGA-X was used as phylogenetic tree analysis tools. Cluster-31069.42907 shows the relationship between the ancestors, based on the bootstrap value. Bootstrap value of Helianthus annuus and Stevia rebaudiana is 100% as both the sequences are from the Asteraceae family. This study contributes to a deeper understanding of S. rebaudiana MS007 Kaurene synthase through in silico analysis

Healing garden: Modul aktiviti dan pelaksanaan

Modul dibina bagi rujukan sekolah bekeperluan khas (pelajar autistik) yang boleh merujuk senarai aktiviti yang boleh dilakukan dalam Healing Garden

De novo transcriptome dataset of Stevia rebaudiana accession MS007

Stevia rebaudiana (S. rebaudiana) is a herbaceous and perennial plant belonging to Asteraceae family. The genus stevia is well known as a natural producer of sweetener comprising non-caloric and non-carcinogenic steviol glycosides. In recent years, the capability in producing natural sweetner has increased the demand for S. rebaudiana as substitute of processed sugars. Flowering phase of S. rebaudiana has shown to affect the content of steviol glycosides in the leaves. Steviol glycosides level is the highest at the time of flower bud formation and lowest at time preceding and following flower bud formation. Therefore, sequencing and analysing the genes that are involved in flowering phase will provide platform for gene manipulation in increasing steviol glycosides content. The Stevia transcriptome data that include two stages of growth (before flowering and after flowering), were obtained using Illumina RNA-seq technology and can be accessed at NCBI Sequence Read Archive under Accession No. SRX6362785 and SRX6362784

In silico development of CRISPR/Cas9 construct for oryza sativa subsp. indica

Oryza sativa, which is commonly known as rice, is one of the staple foods consumed by Asian. There are three subspecies of O. sativa that are different in their geographical adaptations which are Japonica, Javanica and Indica. Climate change causes increasing greenhouse gas emissions fluctuating rainfall, and drought which is the limiting factors of rice production in Malaysia. In rice, SUMO E2-Conjugating Enzyme (OsSCE1) gene plays a role as a negative regulator in the drought stress response. OsSCE1 gene is a type of small ubiquitin-like modifier (SUMO)-conjugating enzyme involved in a few regulatory processes such as SUMOylation. The crop improvement can be made possible through a genome editing approach which enables the manipulation of targeted genetic traits and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system is one of the genomes editing technologies that are accessible. In this study, the CRISPR construct of drought tolerance of Oryza sativa subsp. indica was developed. The CRISPR construct development involved several stages; OsSCE1 gene prediction, sgRNA design, and vector construction. FGENESH and GeneMarkS were used for OsSCE1 gene prediction. The predicted OsSCE1 gene has been validated using Polymerisation Chain Reaction (PCR) and sequencing. The BLASTN result shows high similarity with Oryza sativa in chromosome 10 with 99% identities. This step followed by sgRNA design which was carried out manually with the help of gRNA prediction tools such as WU-CRISPR, CCTop, Benchling, and CRISPR-P. Lastly, vector construction was executed virtually using Benchling. Overall, the OsSCE1 gene was successfully characterized and validated, and the in silico CRISPR construct for Oryza sativa subsp. indica was developed. To ensure a good outcome, in silico CRISPR construct should be verified through in vivo studies in further

Host range and control strategies of Phytophthora palmivora in Southeast Asia perennial crops

Phytophthora palmivora is a destructive plant pathogenic oomycete that has caused lethal diseases in a wide range of hosts. It is a pan-tropical distributed pathogen that can infect plants at all growth stages. Extensive studies have linked P. palmivora to severe diseases in several crops, such as black pepper, rubber, cocoa, and durian, causing global economic losses. This review covers the following topics in depth: (i) P. palmivora as phytopathogen; (ii) identification and infection mechanism in rubber, cocoa, and durian; and (iii) management and control applied for P. palmivora diseases. Effective management strategies were studied and practiced to prevent the spread of P. palmivora disease. Genetic resistance and biocontrol are the best methods to control the disease. A better understanding of P. palmivora infection mechanisms in our main crops and early disease detection can reduce the risk of catastrophic pandemics