Browsing by Author "Rehna Augustine"

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CRISPR/Cas9 mediated editing of microRNA osa-miR1432 for enhanced grain filling in rice
(Department of Plant Biotechnology, Centre for Plant Biotechnology and molecular Biology, College of Agriculture , Vellanikkara, 2022-09-26) Varsha, K; Rehna Augustine
Rice (Oryza sativa L.) is the predominant food crop for almost half of the world population. In light of the rising world population, it has become imperative to increase rice grain yield per unit area to meet the global food demand. The CRISPR/Cas9 technology has recently emerged as a powerful tool for genetically modifying crops due to its simplicity, precision and genome editing capabilities like knock-in, knock-out, knock-down and expression activation. Recent research revealed that knock-down of rice microRNA, osa-miR1432 can simultaneously enhance resistance towards blast disease and overall yield in rice. Hence the current study, ‘CRISPR/Cas9 mediated editing of microRNA osa-miR1432 for enhanced grain filling in rice’ was undertaken at the Department of Plant Biotechnology, College of Agriculture, Vellanikkara, Kerala Agricultural University, Thrissur during the period from 2019-2022 with an objective to improve overall yield in rice by enhancing grain filling rate through targeted editing of microRNA osa-miR1432 using CRISPR/Cas9 system. The genomic sequence information of the osa-miR1432 gene was retrieved from rice genome annotation project. The stem-loop sequence of osa-miR1432 available in the microRNA database, miRBase was used for guide RNA (gRNA) designing. The gRNA was designed using the online design tool CRISPR-P v2.0. The best gRNA (osamiR1432 G1) was selected based on its features like location in the genome, on-score value, GC content and no. of off-target sites. Genomic region of osa-miR1432 gene, flanking the gRNA target was amplified using gene specific primers and sequence of the target region was confirmed using BLASTn and Clustal Omega analysis. The CRISPR/Cas9 binary vector pRGEB32 was used to clone the gRNA. The development of CRISPR/Cas9 construct was accomplished by annealing of the gRNA strands followed by ligating them into the digested and purified pRGEB32 vector using high fidelity BsaI restriction enzyme. The pRGEB32 vector containing the gRNA construct was cloned into E. coli strain DH5α by transformation using the heat shock method. Plasmid DNA was isolated from the colonies obtained on the transformed plate and were Sanger sequenced using universal M13 reverse primer. The third colony (osamiR1432 G1 #3) was confirmed to be a positive clone after analysing the sequence data. The CRISPR/Cas9 construct was then mobilized into Agrobacterium tumefaciens strain EHA105 using freeze-thaw method. The positive clones were confirmed by plasmid PCR using hygromycin gene specific primers as well as guide sequence specific primers. The positive colony in EHA105 (pRGEB32: osa-miR1432 G1#1) was then used for rice genetic transformation experiments. The rice cultivar Nipponbare (Oryza sativa ssp. japonica cv. Nipponbare) was chosen for the study. Nipponbare seeds were inoculated on to N6 medium supplemented with 2, 4- D (3.0 mg/L) for callus induction. Five days old calli were co-cultivated with Agrobacterium cultures harboring the desired gRNA construct. After 1.5 to 2 days of co-cultivation, the calli were washed thoroughly using bacteriostatic agents augmentin or carbenicillin to remove the excess Agrobacterium load. The calli were then placed on selection medium containing hygromycin and augmentin or carbenicillin. The hygromycin resistant calli showed proliferation after 14 days of incubation. These proliferating microcalli were then transferred to regeneration medium containing NAA (0.02 mg/L) and Kinetin (2.0 mg/L). The regenerated shoots were placed in rooting medium which developed proper roots after one week. The plantlets developed were properly hardened in containment polyhouse. Rice genetic transformation protocol was optimized to suit our laboratory and culture conditions. A total of 25 putative transformant T0 plants were examined for the presence of hygromycin resistance gene and 17 plants were found to be hygromycin positive. PCR amplification of the partial osa-miR1432 gene, flanking the gRNA target region was performed in the hygromycin positive plants and the PCR products were Sanger sequenced to detect mutation. Sequence data analysis using the software Sequencher 5.4.6 detected mutation in seven plants. The detected mutations were single base insertions of A (85.7%) or T (14.3%) exactly at 3 bp upstream to the PAM sequence. The mutation efficiency was found to be 41.18% and the mutations detected were classified as homozygous (71.43%) or heterozygous (28.57%). In the current study, using the CRISPR/Cas9 technology rice plants with mutation in the osa-miR1432 gene were successfully developed which can lead to enhanced grain filling in rice
Differential expression of genes involved in anthocyanin pigmentation in red banana and green-red clones
(Department of Plant Biotechnology, College of Agriculture, Vellayani, 2006) Rehna Augustine; Rajmohan, K
The study entitled “Differential expression of genes involved in anthocyanin pigmentation in Red banana and Green-red clones” was conducted at the Department of Plant Biotechnology, Vellayani, Thiruvananthapuram during the period from 2004 to 2006 with an objective of contrasting the expression of genes (chalcone synthase, dihydroflavonol 4-reductase and UDP: glucose 3-oxy-glucosyl transferase) involved in anthocyanin pigmentation in Red banana and Green-red clones. Experiments were also conducted to differentiate the clones based on total sugar concentration and protein profile. Reverse transcription-polymerase chain reaction was carried out to study the expression of genes. Heterologous primers designed based on the gene sequences of Oryza sativa and Vitis vinifera were used for RT-PCR. Total RNA from tissue culture shoot, leaves and fruit from three different stages of Red banana and Green-red clones were isolated using hot phenol method which gave an yield of 80 - 200 µg g -1 of the tissue and a A260/A280 ratio ranging between 1.6 –2.0. Messenger RNA was purified from the total RNA using the mRNA purification kit from GENEI (Bangalore). The RT-PCR amplified products of the two clones, representing chalcone synthase (CHS), dihydroflavonol 4- reductase (DFR), and UDP: glucose flavonoid 3-oxy-glucosyl transferase (UFGT) were eluted and purified. The cDNA fragments were cloned to pCRII vector (TA Cloning Kit, Invitrogen. Inc., USA) and sequenced. The nucleotide to nucleotide BLAST of cDNA clone of 183 bp from Red banana showed similarity with the chalcone synthase mRNA sequence of rice (Acc. No. X89859). cDNA from Green-red clone showed no significant similarity to any other chalcone synthase gene during homology search. The translated query vs. protein database (blastx) search exhibited similarity with shikimate 3- dehydrogenase, an enzyme in the biochemical pathway for aromatic amino acids. The cDNAs synthesized from Red banana and Green-red clone with gene specific primers for dihydroflavonol 4- reductase were having 354 bp and 325 bp length respectively. The homology search (blastn) revealed no similarities with any of the nucleotide sequences specific for dihydroflavonol 4- reductase. The cDNAs amplified from Red banana and Green-red clone with UDP: glucose 3-oxy-glucosyltransferase specific primers were of 361 bp and 345 bp, respectively. The homology search using blastn showed similarity with mRNA sequence for UDP: glucose 3-oxy-glucosyltransferase in grapes. In Red banana the blastx search revealed similarity with a glucosyltransferase from Synechocystis sp. There was no similarity for UDP: glucose 3-oxy-glucosyltransferase cDNA of Green-red clone in the NCBI database. SDS-PAGE analysis showed no difference in the protein profile of Red banana and Green-red clone. Total sugar content in the peel, pulp, peel together with the pulp of red banana and Green-red clone did not showed any significant difference. The expression analysis of the key genes of the general pathway showed no significant difference in both the clones. All the three genes selected for the study were present in both Red and Green-red clones. The genes isolated were not totally identical in the two banana clones. Further studies are needed to get a better insight into the cause of colour change.
Editing of rice transcription factor OsMADS26 for drought tolerance through CRISPR/Cas9 system
(Department of Plant Biotechnology, College of Horticulture, Vellanikkara, 2021) Anjala, K; Rehna Augustine
Rice (Oryza sativa L.) is the most widely consumed staple food of world’s human population belonging to Asia and Africa. Being a semi-aquatic annual plant, rice is highly prone to losses due to various environmental stresses. Many studies regarding this had revealed the need for developing varieties tolerant to abiotic and biotic stresses. Various methods like Marker Assisted Breeding, mutation breeding, RNAi, Antisense technology, ZFNs and TALENs were in use to develop elite traits for abiotic stress tolerance in crops like rice. But very recently, CRISPR/Cas9 system had come into the limelight as an efficient tool for the genetic manipulations of crops. Studies have identified OsMADS26 transcription factor as a negative regulator of drought tolerance in rice. Hence the current study, ‘Editing of rice transcription factor OsMADS26 for drought tolerance through CRISPR/Cas9 system’ was undertaken during the period from 2019 to 2021 at the Centre for Plant Biotechnology and Molecular Biology, CoA, Vellanikkara, Thrissur with an objective to develop drought tolerance in rice. The rice cultivar Nipponbare was selected for the study due to its competence in genetic transformation and regeneration. For CRISPR/Cas9 mediated targeted editing of OsMADS26 gene, guide RNAs (gRNAs) were designed using online software CRISPR-P v2. Genome sequence information of OsMADS26 gene available from rice genome annotation project was used for the study. Genomic region of OsMADS26 gene, flanking the gRNA target (~ 450 bp) was amplified using gene specific primers and sequence of the target region was confirmed using BLASTn and ClustalW analysis. The CRISPR/Cas9 binary vector pRGEB32 was used to clone the guide RNAs using BsaI restriction sites. Three gRNAs were selected for cloning based on features like on score value (higher the value better the editing efficiency), GC content, (40-60%), no. of off-target sites (Minimum number of off-target sites preferred), presence of secondary structure, location on the genome (towards 5' end of gene in exonic region is preferred) etc. The CRISPR/Cas9 construct for cloning was developed by annealing and ligating the gRNAs to the pRGEB32 vector followed by cloning in E. coli strain DH5α. The putative positive clones were identified by colony PCR and further confirmed by Sanger sequencing. The plasmids isolated from PCR positive colonies were sequenced using universal M13 Reverse primer which is present on pRGEB32 vector. The sequences of the clones were confirmed using multiple sequence alignment tool ClustalW. One colony of gRNA 1 construct (OsMADS26 #G1-1) and two colonies of gRNA 3 (OsMADS26 #G3-3 and OsMADS26 #G3-4) were found positive. The CRISPR/Cas9 constructs of OsMADS26 were then mobilized into Agrobacterium tumefaciens strain EHA105 following the Freeze-thaw method. The positive clones were identified using plasmid PCR using hygromycin gene specific primers. Positive colonies of OsMADS26 #G1-1 and OsMADS26 #G3-3 constructs in EHA105 were then used for rice genetic transformation. The seeds of Oryza sativa sub species japonica cultivar Nipponbare were inoculated into N6 medium supplemented with 3.0 mgL-1 2,4-D for callus induction. After five days, the calli were infected with Agrobacterium cultures harboring desired gRNA constructs for 1.5-2 min. Along with the gRNA constructs, an empty vector was also transformed to rice as vector control and a set of untransformed culture were also maintained. After around two days of co-cultivation, the excess Agrobacterium growth was washed-off thoroughly from the calli using the bacteriostatic agent Augmentin. The calli were then placed on selection medium containing Augmentin and Hygromycin. The hygromycin resistant calli showed proliferation after 14 days of incubation. The proliferating microcalli were then transferred to regeneration medium after 21 days. Proliferation of microcalli was observed in vector control, wild type as well as OsMADS26 #G1-1 and OsMADS26 #G3-3 co-transformed plates. The vector control and untransformed calli showed greening and shoot primordia initiation in regeneration medium. The regenerated shoots will be analyzed for mutation in future. Hence, in the current study, gRNA constructs for targeted editing of OsMADS26 gene was successfully developed and transformed in to rice cultivar Nipponbare. Rice genetic transformation suitable to our lab conditions were also optimized. Rice plants with mutations in the OsMADS26 gene is expected in future which can confer drought tolerance.
Glucosinolate profiling and molecular analysis of MYB28 gene for metabolome editing in Moringa oleifera Lam
(Department of Plant Biotechnology, College of Agriculture ,Vellanikkara, 2024-02-29) Muhammed Ameer; Rehna Augustine
Moringa (Moringa oleifera Lam.), an indigenous herb native to South Asia, recognized for its health benefits, is often referred to as "the miracle tree." Thriving in the foothills of the Himalayas, moringa belongs to the Moringaceae genus, comprising 14 known species. Each part of the plant possesses medicinal properties, contributing to antimicrobial, anti-inflammatory, detoxifying, and anticancer activities. Glucosinolates (GSLs) are stable secondary metabolites derived from sulfur and nitrogen-rich amino acids. Myrosinase, the sole known β-thioglucosidase, is responsible for driving their breakdown. The resulting product, isothiocyanates (ITCs), possesses fungicidal, bacteriocidal, nematocidal, and herbivore-deterrent qualities. The R2R3-MYB class of transcription factors significantly influences glucosinolate (GSL) biosynthesis. Studies manipulating the MYB28 gene, a key transcription factor, have been reported to regulate GSL levels in plants. GSLs are suggested to be produced in green tissues and then transported to developing reproductive tissues through the phloem. While extensive research explores the control of the GSL pathway in Brassica and Arabidopsis, in members of Brassicales like moringa, the regulatory mechanism of GSLs is yet unclear. It is reported that the GSLs or ITCs in M. oleifera are probably responsible for many of the therapeutic benefits that have long been associated with them in traditional medicine. M. oleifera is home to several unusual GSLs with unique properties. Glucomoringin, also known as 4-(α-L-rhamnopyranosiloxy) benzyl glucosinolate (4RBGS) is the most prevalent GSL in all of M. oleifera's components, especially the pulp seed, followed by 3-hydroxy- 4-(α-L-rhamnopyranosyloxy) benzyl glucosinolate (4-OHBGS). Due to the presence of a second saccharide residue in the aglycon side chain, this chemical could show biological effects that are very different when compared to those of other GSLs because of its unusual structure. Thus, one of the main goals of M. oleifera breeding is to improve nutritional and medicinal characteristics by creating high GSL lines. The objective of the current research is glucosinolate profiling, isolation and expression analysis of MYB28 gene from M. oleifera variety PKM-1 and development of CRISPR/Cas construct for functional analysis of MYB28 gene. Desulphoglucosinolate profiling using High Performance Liquid Chromatography showed a major peak of 4RBGS and a minor peak of 4-OHBGS in aerial tissues, suggesting them as the predominant GSLs. However, in roots, in addition to the benzyl GSLs, indole GSL (indol-3-ylmethyl GSL, I3M) was also detected. The highest concentration of total GSLs was found in moringa seeds (399.48 µmoles/g dry weight). This was followed by stem (63.95 µmoles/g dry weight) and flowers (30.61 µmoles/g dry weight) had the least amount of GSL accumulation. Isolation and sequence analysis of MYB28 gene in moringa proved that the sequence is diverse from that of its related species coming under the order Brassicales. Homology search using NCBI BLASTn showed 86% identity with the predicted mRNA sequence of Abelmoschus esculentus transcription factor MYB28. Bioinformatic analysis of the genomic and CDS sequences were performed using softwares like NCBI Splign, Molbiotools, ORFfinder and InterPro to characterize the MYB28 gene isolated from moringa. Expression profiling of the MYB28 gene was performed in different tissues of moringa using Real-time qRT-PCR. Highest level of MYB28 expression was found in the stem followed by immature pod tissue, flower and leaf tissue. Lowest level of gene expression was found in seeds, where no GSL synthesis occur, which act as only sink for GSLs. In order to elucidate the role of the putative MYB28 in GSL biosynthesis, a gene knock-out cassette based on CRISPR/Cas9 system was generated in the study. The genomic sequence of MYB28 obtained in the study was used to design gRNA using Cas-designer of ‘CRISPR RGEN Tools’ software. The MYB28 CRISPR/Cas9 construct was ligated to CRISPR/Cas9 binary vector pKSE401 and cloned in to E. coli strain DH5α. The positive clones were confirmed by Sanger sequencing of the plasmid DNA. The construct was further mobilized to A. tumefaciens strain GV3101. Positive clones were identified by colony PCR using vector and gRNA specific primers. The construct will be used for moringa genetic transformation. Callus and cell suspension cultures of moringa was established for moringa genetic transformation in future.
Targeted editing of rice micro RNA osa-miR396b through CRISPR/Cas9 system
(Department of Plant Biotechnology, Centre for Plant Biotechnology and Molecular Biology, College of Agriculture , Vellanikkara, 2022-10-15) Sanjay Sathian; Rehna Augustine
Rice (Oryza sativa L.) is one of the most produced and consumed food crops in the world. There is an urgent need to increase rice production to feed the increasing population. Rice yield is determined by several components like grain size, grain weight, number and architecture of panicles, number of spikelets per panicle and grain filling. The microRNA family osa-miR396 is known to suppress the expression of rice growth regulating factors (OsGRFs) resulting in reduced growth and yield. The miRNA osa-miR396b is reported to be a negative regulator of spikelet number and inflorescence development. CRISPR/Cas9 mediated knockout of osa-miR396b gene can thus possibly result in an enhanced yield in rice. Hence, the current study ‘Targeted editing of rice microRNA, osa-miR396b through CRISPR/Cas9 system’ was conducted during the period from 2019 to 2022 at the Department of Plant Biotechnology, College of Agriculture, Kerala Agricultural University, Vellanikkara, Thrissur. Oryza sativa ssp. japonica cultivar Nipponbare was selected for the study due to well established transformation protocols and higher transformation efficiency. Initially, the sequence information of the rice microRNA gene osa-miR396b was retrieved from ‘miRbase’. The stem loop sequence obtained was used to design guide RNAs (gRNAs) using the software CRISPR-P v2.0 and CRISPR-PLANT v2. The gRNAs were selected for further studies mainly based on GC content and number of off-target sites. The target osa-miR396b gene sequence was confirmed by amplifying the genomic region flanking the target using gene specific primers followed by sequencing and the sequence analysis using Clustal Omega and BLASTn showed 100% similarity with reported sequences. The osa-miR396b G1 CRISPR/Cas9 construct was generated by restriction digestion of CRISPR/Cas9 binary vector pRGEB32 using BsaI restriction enzyme followed by ligation with annealed and phosphorylated gRNA. The osa-miR396b G1 construct was cloned to E. coli strain DH5α. The positive clones were confirmed by Sanger sequencing of the plasmid DNA isolated from the colonies and sequence analysis using Clustal Omega. Three (osa-miR396b G1 #2, osa-miR396b G1 #3 and osa-miR396b G1 #4) out of four plasmids sequenced were having gRNA insertion. The osa-miR396b G1 #4 CRISPR/Cas9 construct was mobilized to A. tumefaciens strain EHA105. Positive clones were confirmed by PCR amplification of hygromycin resistance gene (hptII) using specific primers. Colony #1 of A. tumefaciens with osamiR396b G1 CRISPR/Cas9 construct out of the two positive colonies was used for rice transformation. Genetic transformation of rice was achieved through Agrobacterium-mediated transformation. The first step was induction of calli from dehusked and sterilized Nipponbare seeds. Five-day-old calli were infected with Agrobacterium harbouring osa-miR396b G1 CRISPR/Cas9 construct for 1.5-2 min. and co-cultivated for 48 hours. An empty vector was also transformed as control. After washing off excess Agrobacterium growth with sterile distilled water containing Augmentin (300 mgL-1 ) or carbenicillin (250 mgL-1 ), the calli were kept for selection in selection medium supplemented with hygromycin (50 mgL-1 ) and Augmentin (300 mgL-1 ) or carbenicillin (400 mgL-1 ). The calli showing proliferation of microcalli were transferred to regeneration medium supplemented with NAA (0.02 mgL-1 ) and kinetin (2.0 mgL-1 ) for inducing somatic embryogenesis. The somatic embryos were allowed to develop into small plantlets which were transferred to rooting medium for root development. The rooted plantlets were initially maintained in sterile distilled water and then hardened in sterile soil-cocopeat mixture in paper cups and transferred to pots with soilsand-cow dung mixture. A total of 94 putative transformed plants for osa-miR396b G1 CRISPR/Cas9 construct and four vector control plants were obtained. For confirming successful transformation, PCR amplification of hptII gene using hygromycin gene specific primers was done. DNA extracted from 35 plants and two vector control were used as PCR template. A total of 16 out of 35 transformed and two vector control plants were hygromycin positive, indicating successful transformation. The osa-mir396b partial gene sequence was amplified using gene specific primers and sequenced by Sanger sequencing for detecting mutation. Detection of mutation was carried out using ‘Inference of CRISPR Edits (ICE)’ software by Synthego. Analysis using ‘ICE’ detected indel mutations in seven plants. Five plants (71.42%) had deletions and two (28.57%) had insertions around the cut site. Four plants (57.14%) had heterozygous mutations (mutation in one allele) and three (42.86%) had chimeric (more than two) mutations. The mutation efficiency was calculated to be 43.75%. The mutations obtained could lead to a non-functional osa-miR396b gene in these plants. The study successfully demonstrated application of CRISPR/Cas9 system to mutate rice microRNA gene. The knockout of osa-miR396b gene will likely promote the expression of rice GRF genes improving the grain yield. Further studies should be conducted to study inheritance pattern of mutations in the subsequent generations. Genotypic and phenotypic analyses is to be done to study effect of mutated osamiR396b gene on its target genes and on yield and other agronomically important traits.