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Assam Agricultural University, Jorhat

Assam Agricultural University is the first institution of its kind in the whole of North-Eastern Region of India. The main goal of this institution is to produce globally competitive human resources in farm sectorand to carry out research in both conventional and frontier areas for production optimization as well as to disseminate the generated technologies as public good for benefitting the food growers/produces and traders involved in the sector while emphasizing on sustainability, equity and overall food security at household level. Genesis of AAU - The embryo of the agricultural research in the state of Assam was formed as early as 1897 with the establishment of the Upper Shillong Experimental Farm (now in Meghalaya) just after about a decade of creation of the agricultural department in 1882. However, the seeds of agricultural research in today’s Assam were sown in the dawn of the twentieth century with the establishment of two Rice Experimental Stations, one at Karimganj in Barak valley in 1913 and the other at Titabor in Brahmaputra valley in 1923. Subsequent to these research stations, a number of research stations were established to conduct research on important crops, more specifically, jute, pulses, oilseeds etc. The Assam Agricultural University was established on April 1, 1969 under The Assam Agricultural University Act, 1968’ with the mandate of imparting farm education, conduct research in agriculture and allied sciences and to effectively disseminate technologies so generated. Before establishment of the University, there were altogether 17 research schemes/projects in the state under the Department of Agriculture. By July 1973, all the research projects and 10 experimental farms were transferred by the Government of Assam to the AAU which already inherited the College of Agriculture and its farm at Barbheta, Jorhat and College of Veterinary Sciences at Khanapara, Guwahati. Subsequently, College of Community Science at Jorhat (1969), College of Fisheries at Raha (1988), Biswanath College of Agriculture at Biswanath Chariali (1988) and Lakhimpur College of Veterinary Science at Joyhing, North Lakhimpur (1988) were established. Presently, the University has three more colleges under its jurisdiction, viz., Sarat Chandra Singha College of Agriculture, Chapar, College of Horticulture, Nalbari & College of Sericulture, Titabar. Similarly, few more regional research stations at Shillongani, Diphu, Gossaigaon, Lakhimpur; and commodity research stations at Kahikuchi, Buralikson, Tinsukia, Kharua, Burnihat and Mandira were added to generate location and crop specific agricultural production packages.

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  • ThesisItemOpen Access
    Characterization of the Sinapis alba-Alternaria brassicicola interaction and, identification of associated defense response genes
    (2021) Ahmed, Reshma; Bhorali, Priyadarshini
    Alternaria blight caused by Alternaria brassicicola is one of the most devastating and widespread fungal diseases of the oilseed mustards. It causes yield losses up to 47% and has been reported from various parts of the country including the state of Assam. The non-host Sinapis alba, wild relatives of Brassicaceae, has been reported to have resistance against Alternaria blight disease. In order to understand this non-host mechanism, isolation and identification of pathogen, morphopathological, screening for resistance both in vitro and in vivo, histopathological study by using Scanning Electron Microscopy (SEM) and global gene expression using next generation sequencing method(NGS), RNA-seq, has been done to develop resistance variety of rapeseed mustard against Alternaria blight. For this, infected leaves, siliques and stems of the Toria variety TS-38, showing the initial conspicuous characteristic symptoms of Alternaria blight, were collected for isolation of the pathogen. The infected plant parts were surface sterilized and inoculated in petriplates containing Potato Dextrose Agar (PDA) medium under optimal conditions for fungal growth and sporulation. The mycelial growth of the fungus was observed after 3days of inoculation and the hyphal growth covered the petriplates within 15days of inoculation. On the basis of the conidial morphology as observed through microscopic studies, the pathogen was identified as Alternaria brassicicola. Purification of the fungal pathogen was done by single spore isolation method. Further, molecular detection of the fungus was successfully carried out by amplification of the fungal genomic DNA using reported ITS primers (ITS1/ITS2 & ITS2/ITS4). Sequencing of the fungal ITS region also confirmed the pathogen at the genus level. Further confirmation upto species level has been done with A. brassicicola specific primers (ABS28). The primary screening test both in vitro detached leaf assay showed the development of infection by showing cholorotic region after 72hpi with no chlorosis in S. alba. The light microscopy study of the infected portion showed the appearance of increase number fungal filaments in B. rapa with a few filaments in S. alba. The trypan blue staining showed the increase level of necrosis in B. rapa in comparison with S. alba. The SEM analysis also showed a similar type of result with light microscopy with a less hyphal penetration with a few spores in comparison with B. rapa by the presence of mass of hyphal filament with increased number of spores. Further Pathogenecity test was performed in vivo that showed successful development of disease in B. rapa after 72hpi whereas the infection developed after 7dpi in S. alba. The disease progression study by morphological study showed a slow and restricted with little infection in S. alba after 10dpi whereas in B. rapa with severe infection with complete death of the inoculated leaves within that period. The disease scoring has been studied from 0hrs to 10 days. Leaf disease incidence percentage (LDI%) was calculated that analyzed the number of incidence of spot after infection. LDI% for B. rapa was found to be 82.50% while in S. alba it only showed 15% after 10dpi which is very less and showed its resistance against A. brassicicola. Global transcritome profile has been done at early infection period viz. 48 and 72 hours post inoculation to identify the genes responsible for its resistance by deferential expression study of genes in both the cultivars. A mapping percentage of 79.41% and 78.46% was found in S. alba after 48 and 72hpi and of 96.63% and 92.87% was found in B. rapa after 48 and 72hpi. DEG analysis showed a large number of genes were up-regulated at 48hpi than 72hpi. Validation of transcriptome data was performed by selecting 12 defense related genes that showed a high fold change in S. alba in comparison with B. rapa which showed a similar trend that confirms the validation of the targeted data set.
  • ThesisItemOpen Access
    Mapping of drought tolerant QTLS in upland Rice variety Banglami
    (2021) Roy, Nabarun; Modi, Mahendra Kumar
    The study entitled ‘Mapping of drought tolerant QTLs in upland rice variety Banglami’ was taken up to identify the drought tolerant QTLs in Banglami, a local medium duration upland low yielding landrace of Assam which is drought tolerant. For this a mapping population was developed by crossing Banglami with Ranjit, an elite long duration high yielding photosensitive and drought susceptible variety of Assam. QTL mapping was done in this population with the use of SSR markers by Verma et al. (2017a) and Sharma et al. (2017) and several QTLs were mapped. But with the demand of latest technology which needs QTLs within narrow confidence intervals so that they can be used precisely in any breeding programme without much problem of linkage drag, the present work done became demanding as Genotyping by sequencing (GBS) technology was used to find out SNP markers which will now flank the mapped QTLs. For this, the mapping population was evaluated for 2 consecutive seasons for several yield and agronomic traits under both reproductive stage drought stress and control condition. The phenotypic data collected was associated with genotypic data to form a high density/saturated linkage map. 4646 polymorphic informative SNPs were assigned to 12 linkage groups (LGs) covering a total of 1306.424 cM of the rice genome at an average marker distance of 0.32 cM. A total of 65 QTLs were mapped which explained a PVE of 1.95- 13.80% with LOD scores ranging from 2.5- 31.6, out of which 30 QTLs were mapped from stress data, whereas 35 QTLs were mapped from non stress data. Out of these only 5 QTLs were major QTLs and 2 QTLs were found to be stable across environments. 3 QTLs were mapped for the trait grain yield per plant (GYP). 10 QTL clusters were identified among which cluster no. 10 on chromosome no. 12 had a congregation of 8 QTLs together within a region of 29 cM and can be considered as a QTL hotspot. Six genes within the QTL regions were found to be differentially expressed under stress condition. The genes were Calcium-transporting ATPase 9, Phosphoinositide binding protein, Histone demethylase JARID1C, Nuclear-pore anchor and OsWAK3 - OsWAK receptor-like cytoplasmic kinase and Cytochrome P450. Overall results from the present study sets a good achievement within the science of molecular breeding and can give a strong scientific background for the future researchers, the outcomes of which can help them to develop drought tolerant rice varieties.
  • ThesisItemOpen Access
    Mapping of quantitative trait locus (qtl) for drought tolerance in Rice
    (2021) CHANDRAKANT, KALDATE RAHUL; Modi, Mahendra Kumar
    Drought is a major abiotic constraint for rice production worldwide. The reproductive stage drought stress (RSDS) leads to a huge loss in grain yield. The prospecting of new donor cultivars with identification and introgression of large effect drought tolerance related QTLs is essential to develop drought-tolerant rice varieties. The present study was conducted with the aim of mapping quantitative trait locus (QTLs) associated with drought tolerance in rice. With a total of 3417 GBS (Genotyping by sequencing) based polymorphic SNP (Single nucleotide polymorphism) markers distributed over the rice chromosomes a saturated linkage map spanning 1924.136 cM was constructed with an average marker density of 0.56 cM using an F3 mapping population developed by crossing traditional Ahu rice cultivar Koniahu (drought tolerant) with Disang (drought susceptible). Using inclusive composite interval mapping (ICIM) approach 172 genomic regions associated with grain yield and related traits were detected in 198 F3 and F4 segregating lines evaluated for two consecutive seasons under both RSDS and irrigated control conditions. Of which, 102 QTLs were identified under RSDS with LOD (Logarithm of odds) score value ranging between 2.53 to 7.51 and phenotypic variance explained (PVE) value of 1.97 to 12.42 per cent. While 70 QTLs with LOD score value ranging between 2.50 to 10.07 and PVE value of 2.35 to 11.46 per cent were detected under control condition. In total, nine QTLs were major QTLs having a PVE value 10. Among the QTLs identified under RSDS, 72 (70.59%) QTLs were novel whereas 30 (29.41%) QTLs were observed to be co-localized or overlapped with genomic regions previously mapped for the same trait/ QTLs. Five putative QTLs namely, qGY2.00, qGY5.05, qGY6.16, qGY9.19, qGY10.20 were found to be associated with grain yield under drought. Further, fourteen CAPS (Cleaved Amplified Polymorphic Sequences) were developed from selected eight novel QTLs linked SNP regions and validated in parental cultivars along with ten F5 generation RILs. Putative gene identification within eight QTL regions detected a total of 3341 genes in which 1516 (45.63%) genes were annotated to at least one gene ontology (GO) term. The putative QTLs and candidate genes identified in the present study need to be further validated, which will be helpful for the improvement of drought tolerance in rice.
  • ThesisItemOpen Access
    Optimization of in vitro transformation protocol and RNAi based gene silencing for viral (Cucumber Mosaic Virus) resistance in Bhut jolokia (Capsicum chinense Jacq.)
    (2021) Deuri, Bharati; Bhorali, Priyadarshini
    Bhut jolokia (Capsicum chinense Jacq.), one of the most popular and hottest chillies in the world, is widely cultivated in Assam and other North Eastern regions in India. A member of the Solanaceae family, Bhut jolokia is characterized by very high pungency due to the presence of high amount of phenolic alkaloid ‘Capsaicinoids’. It is an ideal chilli variety for extraction of oleoresin and capsaicin, which have high market demand due to their industrial uses and medicinal properties. Bhut jolokia production is challenged by several biotic constraints, particularly viral diseases, which affect its quality and yield. Among the viruses, Cucumber Mosaic Virus (CMV) causes severe crop damage, leading to low productivity. Current control measures for CMV are mainly preventive through vector management strategies, which are not adequate in controlling the disease. An effective way to control the disease is the use of biotechnological tools such as RNA interference (RNAi) technology to engineer resistance against the virus. Plants expressing a copy of a viral gene in sense and/or antisense orientation have shown resistance upon infection with the virus via post-transcriptional gene silencing. In the present investigation, an in vitro regenerationtransformation strategy has been optimized for Bhut jolokia and, a hairpin RNA (hpRNA) based gene silencing construct has been developed using the Replicase gene from CMV. The study was initiated by callus induction from Bhut jolokia leaf segments in MS basal medium. Very good quality callus were induced in MS medium supplemented with 0.5 mg/l or 1 mg/l 2,4-D. Multiple shoot induction and regeneration from callus were obtained in MS medium supplemented with 8.5 mg/l KIN and 0.5 mg/l TDZ along with 5 mg/l AgNO3 with maximum shoot initiation frequency of 95% and regeneration frequency of 90%. Root regeneration was found to be optimum in half strength MS medium supplemented with 1.5 mg/l NAA within 4 weeks of culture with maximum rooting frequency of 70%. For standardization of an Agrobacterium-mediated genetic transformation system, the strain LBA4404 carrying pCAMBIA1301 binary vector construct with gusA as the reporter gene and hptII and nptII as selection marker genes was used. Transformation was carried out using 45 days old callus and also with intact Bhut jolokia seeds as explants. Hygromycin concentration of 9 mg/l was found to be optimum for efficient selection of putative transformants. From a total of 30 nos. of callus infected by Agrobacterium, 9 numbers of putative transformed shoots were regenerated in presence of selection agent. Finally, only 2 (6.66%) fully rooted plants survived out of which, only 1 plant finally survived during hardening in the green house. Moreover, out of 30 nos. of infected seeds, a total of 7 numbers of putative transformed seedlings were developed. Finally, only 1 (3.33%) seedling survived, which was transferred to the green house for hardening. Thus, both callus and seeds could be used as explants for transformation in Bhut jolokia, although the frequency of putative transformants obtained using callus explants was higher than that in seed transformation. The putative transformants were confirmed by GUS histochemical assay and PCR analysis. For developing the RNAi construct, a 323 bp Replicase gene sequence was cloned into pHANNIBAL vector both in sense and anti-sense orientations. The construct was then transferred to pBI121 binary vector, which was electroporated into Agrobacterium strain LBA4404 for plant transformation. Functional validation of the CMV Replicase hp-RNA construct was done through bioassay in model plant Nicotiana benthamiana by Agro-infiltration. Transgene expression in N. benthamiana was confirmed by RT-PCR analysis. The bioassay results indicated suppression of CMV infection in Agro-infiltrated N. benthamiana plants when mechanically inoculated with CMV sap. Further, DAS-ELISA established the functional efficiency of the hpRNA construct in providing considerable level of resistance against CMV infection. The in vitro regeneration-transformation strategy and the hpRNA based gene silencing construct, developed through this study would serve as a foundation towards future studies on engineering resistance against CMV in Bhut jolokia.
  • ThesisItemOpen Access
    Morphogenetic, Metabolic and Molecular Dynamics during Mycelial Interactions among Fungal species
    (AAU, Jorhat, 2021) Dullah, Samim; Boro, Robin Chandra
    In nature, microorganisms interact/compete with one other for food and space and the type of interactions are unique to each interacting species. Fungal-fungal interactions are complex, and different types of secondary metabolites are secreted during interaction. In this study, 14 fungal isolates were facilitated in 105 possible combinations to interact in potato dextrose agar (PDA). Ten interactions between different fungal isolates showed mutual replacement with each fungus; capturing territory from the other. Thirty-five interactions showed complete replacement as growth of one of the fungal partners was inhibited. In forty-six interactions, formation of barrage was observed leading to deadlock type of interaction wherein both fungi have restricted growth. The barrage formation during interaction was further studied with two fungal interactions viz., (i) T. coccinea vs. L. lactinea and (ii) T. coccinea vs. T. versicolor. Microscopic changes were observed in the hyphal growth during interaction like hyphal coiling, dense mycelial network, pore formation. Fungal-fungal interaction often leads to the change in metabolite profile of both the interacting fungus which may have potential implication in industry or agriculture. The metabolites produced during interaction of Trametes coccinea (F3) with Leiotrametes lactinea (F9) and Trametes coccinea (F3) with Trametes versicolor (F1) was analysed through Liquid Chromatography coupled with Mass Spectroscopy (LC-MS). Most of the metabolites secreted during interaction are associated with defensive response. The bipartite fungal interaction resulted in the production of a dark brown colour pigment – melanin as confirmed by the LC-MS, FTIR and NMR analysis. Moreover, the fungal-fungal interaction also led to increase in the production of laccase, a group of multicopper oxidases involved in detoxification of toxic compounds. Further increased activity of superoxide dismutase, an enzyme that catalyzes the dismutation of the superoxide anion to hydrogen peroxide was also recorded during fungal–fungal interaction. There was significant increase in the activities of hydrolytic enzymes including cellulase, xylanase and chitinase during in vitro fungal-fungal interaction, suggesting the importance of such interactions for commercial enzyme production. Quantitative real-time PCR revealed upregulation of lcc1 (encoding a laccase enzyme) and few other stress related genes of T. versicolor during its hyphal interaction with T. coccinea, suggesting a direct correlation between laccase production and melanin production. The study helped to gain a better understanding on the morpho-physiological, biochemical and gene expression profiles during in vitro fungal-fungal interaction. Such interactions induce the production and secretion of an array of metabolites and enzymes which can be prospected towards biotechnological applications.
  • ThesisItemOpen Access
    MAPPING OF QUANTITATIVE TRAIT LOCUS (QTL) FOR DROUGHT TOLERANCE IN RICE
    (AAU, Jorhat, 2021) CHANDRAKANT, KALDATE RAHUL; Modi, Mahendra Kumar
    Drought is a major abiotic constraint for rice production worldwide. The reproductive stage drought stress (RSDS) leads to a huge loss in grain yield. The prospecting of new donor cultivars with identification and introgression of large effect drought tolerance related QTLs is essential to develop drought-tolerant rice varieties. The present study was conducted with the aim of mapping quantitative trait locus (QTLs) associated with drought tolerance in rice. With a total of 3417 GBS (Genotyping by sequencing) based polymorphic SNP (Single nucleotide polymorphism) markers distributed over the rice chromosomes a saturated linkage map spanning 1924.136 cM was constructed with an average marker density of 0.56 cM using an F3 mapping population developed by crossing traditional Ahu rice cultivar Koniahu (drought tolerant) with Disang (drought susceptible). Using inclusive composite interval mapping (ICIM) approach 172 genomic regions associated with grain yield and related traits were detected in 198 F3 and F4 segregating lines evaluated for two consecutive seasons under both RSDS and irrigated control conditions. Of which, 102 QTLs were identified under RSDS with LOD (Logarithm of odds) score value ranging between 2.53 to 7.51 and phenotypic variance explained (PVE) value of 1.97 to 12.42 per cent. While 70 QTLs with LOD score value ranging between 2.50 to 10.07 and PVE value of 2.35 to 11.46 per cent were detected under control condition. In total, nine QTLs were major QTLs having a PVE value 10. Among the QTLs identified under RSDS, 72 (70.59%) QTLs were novel whereas 30 (29.41%) QTLs were observed to be co-localized or overlapped with genomic regions previously mapped for the same trait/ QTLs. Five putative QTLs namely, qGY2.00, qGY5.05, qGY6.16, qGY9.19, qGY10.20 were found to be associated with grain yield under drought. Further, fourteen CAPS (Cleaved Amplified Polymorphic Sequences) were developed from selected eight novel QTLs linked SNP regions and validated in parental cultivars along with ten F5 generation RILs. Putative gene identification within eight QTL regions detected a total of 3341 genes in which 1516 (45.63%) genes were annotated to at least one gene ontology (GO) term. The putative QTLs and candidate genes identified in the present study need to be further validated, which will be helpful for the improvement of drought tolerance in rice.
  • ThesisItemOpen Access
    Agrobacterium mediated genetic transformation of Citrus reticulata cv. Khasi mandarin
    (2020-02) Bhandari, Sangeeta; Singh, Salvinder
    Citrus is number one fruit of the world on accounts of its high nutritional value. India is the fourth largest producer of Citrus in the world. The north-eastern region of India is a rich treasure of various Citrus species. Khasi mandarin is the most economically important one and plays a vital role in the socio-economic development of the people in this region. Khasi mandarins are declining at a very high rate due to its vulnerability to different pathogen and insect/ pest. Conventional breeding for overcoming these problems are limited in Citrus and are directly associated with the reproductive biology of Citrus. Recent advances in genetic engineering have made it possible to incorporate desirable genes from elite genotype mainly through Agrobacterium-mediated genetic transformation. Citrus species showed varied response to in vitro regeneration and genetic transformation. Cultivar specific optimization of in vitro regeneration and transformation protocol is very important. In the present investigation, in vitro regeneration and Agrobacterium mediated genetic transformation protocol for Khasi Mandarin was optimized using different explants like epicotyl, hypocotyl, nodal and inter nodal segment obtained from six-week-old in vitro grown zygotic seedling. Explants were transformed wih Agrobacterium strain LBA4404, harbouring plasmid pBI121-AtSUC-GUS containing nptII as a selectable marker and gus as a reporter gene. Hypocotyl was found to be the best explants for khasi mandarin transformation and regeneration. MS medium supplemented with BAP (2mg/L), NAA (0.5 mg/L), 2, 4-D (1mg/L), MES (0.5g/L), sucrose (30g/L) and acetosyringone (100μM) was found to be best medium for co-cultivation. Modified MS medium containing BAP (4mg/L), MES (0.5g/L), sucrose (30g/L), phytagel (4g/L), kanamycin (50mg/L) and timentin (150mg/L) showed highest regeneration efficiency (18%). Modified MS medium containing BAP (4mg/L), GA3 (0.5mg/L), MES (0.5g/L), sucrose (30g/L), phytagel (4g/L), kanamycin (50mg/L) and timentin (150mg/L) showed highest multiple shoot induction (6%). In vitro regenerated shoots that survived up to 3rd selection cycle were subjected to GUS assay for confirmation of GUS expression in the phloem tissues. Present investigation is a preliminary study for optimization of an in vitro regeneration and genetic transformation protocol in Khasi Mandarin.
  • ThesisItemOpen Access
    GENETIC STUDIES FOR IMPROVING YIELD UNDER DROUGHT STRESS ENVIRONMENTS IN RICE OF ASSAM
    (2019-07) PARRAY, ROUF AHMAD; Baruah, Akhil Ranjan
    Drought is a major limiting factor for rice under rainfed ecosystem in Assam. In this context, thirteen rice cultivars with varied level of drought tolerance were chosen from a set of 272 different rice genotypes based on a field experiment conducted during 2014-15 season under drought. The thirty days old seedlings of 13 cultivars were tested for extensive morpho-physiological, biochemical parameters, relative transcript accumulation and global gene expression using next generation sequencing (NGS) method, and data were recorded at fifth, tenth and fifteenth day of withholding water (DWW) in order to obtain detail trait based gene architecture and to improve high yielding variety of Assam using transcript dynamics. Among the physiological traits studied, stomatal conductance decreased as the dehydration stress increased but the effect was minimum in Apo, Dumai and Tepi Dumai compared to others. Photosynthetic rate decreased with increasing water deficit, but the effect was less pronounced in Apo, Dumai and Tepi Dumai. The rate of transpiration decreased upto 5DWW but gradual increase was observed in later stage. Moreover, the fall in transpiration rate was less in Apo. Water use efficiency (WUE) of rice plants was enhanced significantly under moisture stress at all the three periods of stress (5DWW, 10DWW, 15DWW) in Apo, Tepi Dumai and Dumai. Reduction in RWC was experienced across all genotypes but the decrease was less prominent in Apo, Dumai and Tepi Dumai. Drought stress condition led to increased proline content across genotypes as compared to irrigated condition. Apo, Tepi Dumai, Dumai and Kali Murali showed rapid increase compared to others. Increase in root length was observed across all cultivars with Apo being the longest followed by Dumai and Ranjit. Then, five drought responsive pathway genes (OsDREB2, OsNAC1, bZIP16, OsbZIP 23, OsbZIP72) were chosen to check the differential expression patern in the cultivars at the same data point as mentioned above. Expression profiling of OsDREB2 showed significant increase in gene expression with increase in drought stress in the case of Apo and Dumai. Significant expression of the OsNAC1 was found in Apo, Dumai at different time points of dehydration stress whereas expression of ARC 10372 was prominent in 15DWW. Apo showed significant difference in expression of bZIP16 under all the three stages of water stress whereas Dumai and Ranjit showed enhanced expression compared to other cultivars. Expression profile of OsbZIP23 showed significant accumulation of transcripts in Apo in all stages followed by Dumai. Significant expression of OsbZIP72 was observed in Apo at 10DWW and 15DWW followed by Ranjit and Dumai. Based on the results of morpho-physiological, biochemical and expression analysis, three cultivars, viz., Ranjit, Apo and Dumai were chosen to study the detailed transcriptome at only 10DWW. Transcriptome profile revealed highest mapped genes in Dumai followed by Ranjit and Apo, however, only 14.5% genes were in common. Ranjit was found to be more responsive to abiotic stimulus including water stress. Gene ontology (GO) suggested no significant change of pathway genes upto 10 DWW among the three cultivars. The transcriptome data were validated using five differentially expressed genes in these three cultivars along with a F4 mapping population. It revealed similar trend, suggesting the present transcriptome data set was in good fit. However, detail transcriptome study in vital plant parts at different stages under drought stress will throw more light about the interaction of pathway genes to adress the problem better.
  • ThesisItemOpen Access
    Molecular cloning and in silico characterization of linalool synthase gene from Cymbopogon winterianus
    (AAU, Jorhat, 2018-07) Saha, Oliva; Sen, Priyabrata
    Citronella (Cymbopogon winterianus), an aromatic and medicinal grass from Poaceae family, is grown for the commercial and industrial purpose. It has the large repository of isoprenoid compounds known for its anti-tumoral, anti-bacterial, anti-fungal, anti-viral, anticancerous, detoxifying and natural insect repellent properties. Out of the different components of citronella oil, linalool (3,7-Dimethylocta-1,6-dien-3-ol) is one of the significant aromatic ingredient. Formation of linalool is catalysed by linalool synthase (LIS) which is encoded by linalool synthase gene. This gene has been widely studied in many medicinal plants along with sorghum, rice, maize, setaria, etc.However, till date, no studies on molecular cloning, characterization and tissue-specific expression profiling of LIS gene from C. winterianus (CwLIS) has been reported.Therefore, the present study aims at cloning and characterization of full-length cDNA of LIS gene from C. winterianus. The full-length sequence of CwLIS was obtained by primer walking using several pairs of degenerate primers and the sequence fragments were aligned to acquire full length sequence. Cloning of the desired gene (1758bp) was performed using TOPO TA vector (3.9kb), and the transformation was done in E. coli DH5α competent cells. The sequence analysis revealed 1758 bps cDNA, which has a Coding Sequence (CDS) of 1422 bps that encodes a protein of 473 amino acids. The domain analysis revealed that CwLIS is a single domain protein under terpene synthase superfamily. The multiple sequence alignment (MSA) based on PSI-BLAST search against RefSeq with 62% sequence identity revealed the presence of two aspartate-rich regions, which are supposed to coordinate 3 Mg2+ ions. The phylogenetic analysis revealed that the CwLIS is closely related to Sorghum bicolor linalool synthase. To understand the molecular mechanism behind Mg2+ and linalool binding, first, the 3D model of CwLIS was generated and validated with their stereo chemical parameters. Further, to find out the expression profile of CwLIS in different tissues, Reverse Transcriptase-PCR was performed and the results revealed a higher expression in leaf sheath followed by leaf, root and flower and further conformed by qRTPCR analysis. The result from the present study provide basic information for further research about linalool synthase and comprehensive sequence resource for study, such as gene expression, genomics and functional genomics in Cymbopogon winterianus.