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20195
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Subject: Agricultural Biotechnology × End date: 2019 × Start date: 2019 × Thesis Type: Ph.D ×
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    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.
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    ThesisItemOpen Access
    Molecular characterization and nutritional equivalence evaluation of transgenic chickpea expressing either a cry1Ac or cry2Aa gene
    (AAU, Jorhat, 2019-10) Gupta, Rubi; Sarmah, Bidyut Kumar
    Biosafety assessment of transgenic chickpeas having B.thuringiensis genes for resistance to pod borers is a regulatory requirement and mandatory to document before releasing in the field. Therefore, Bt chickpea lines harbouring either a cry1Ac or cry2Aa gene were characterized for the presence and expression of the transgene in their advanced generations, biosafety assessments and transcript profile were studied. The homozygous lines were selected for comparative nutritional equivalence assay. Biochemical estimations of major nutritional components such as proximates, vitamins, minerals, fatty acids and anti nutrients confirmed that the Bt chickpeas lines are nutritionally equivalent to their non-transgenic counterparts and the seed composition is similar or within the range reported, previously. Seed protein quality was investigated by separating the proteins in PAGE and eluted proteins after mass spectrometry (MS) showed expected fractions of 11S legumin, 7S vicilin, and 2S albumin of chickpea storage proteins in the transgenic lines. The protein digestibility was assayed using the multi-enzyme system and transient pepsin hydrolysis to mimic simulated gastric fluid followed by trypsin hydrolysis to mimic simulated intestinal fluid. Total seed proteins of both the transgenic and nontransgenic lines were digested at a similar rate and enzyme-resistant peptides were not observed in transgenic Bt chickpea lines. The unintended changes in the whole transcriptome profile of Bt chickpeas were surveyed using a homozygous transgenic line expressing a cry1Ac gene. The differentially expressed genes (DEGs) profiling confirmed a low (0.69%, log2fold change≥2) frequency of differentially expressed in the transgenic chickpea line. Only a small (34 upregulated) proportion of genes showed > 2 fold (P-value of 0.05, FDR of 0.05) change in their expression, while only 23 genes down-regulated by >2 fold. Furthermore, no transcripts for potential allergenic proteins were represented in the DEGs. Most of these genes appeared to be developmentally regulated or stress-related which was expected because absolute synchronous growth and development even under a controlled environment are challenging. A few upregulated (AT-hook motif nuclear-localized protein 17-like, probable inactive 2-oxoglutaratedependent dioxygenase AOP2, protein EXORDIUM-like) and down-regulated (histone H2B, gonadal, embryonic abundant protein VF30.1-like, fasciclin-like arabinogalactan protein 12) genes were subjected to qPCR. The qPCR data validated the fold change of the up-regulated (>2) and down-regulated (>-2) genes. Thus, the above data revealed no potential alterations in the nutritional equivalence or transcript profile of transgenic Bt chickpeas.
  • Thumbnail Image
    ThesisItemOpen Access
    Molecular characterization and nutritional equivalence evaluation of transgenic chickpea expressing either a cry1Ac or cry2Aa gen
    (AAU, Jorhat, 2019-10) Gupta, Rubi; Sarmah, Bidyut Kumar
    iosafety assessment of transgenic chickpeas having B.thuringiensis genes for resistance to pod borers is a regulatory requirement and mandatory to document before releasing in the field. Therefore, Bt chickpea lines harbouring either a cry1Ac or cry2Aa gene were characterized for the presence and expression of the transgene in their advanced generations, biosafety assessments and transcript profile were studied. The homozygous lines were selected for comparative nutritional equivalence assay. Biochemical estimations of major nutritional components such as proximates, vitamins, minerals, fatty acids and anti nutrients confirmed that the Bt chickpeas lines are nutritionally equivalent to their non-transgenic counterparts and the seed composition is similar or within the range reported, previously. Seed protein quality was investigated by separating the proteins in PAGE and eluted proteins after mass spectrometry (MS) showed expected fractions of 11S legumin, 7S vicilin, and 2S albumin of chickpea storage proteins in the transgenic lines. The protein digestibility was assayed using the multi-enzyme system and transient pepsin hydrolysis to mimic simulated gastric fluid followed by trypsin hydrolysis to mimic simulated intestinal fluid. Total seed proteins of both the transgenic and nontransgenic lines were digested at a similar rate and enzyme-resistant peptides were not observed in transgenic Bt chickpea lines. The unintended changes in the whole transcriptome profile of Bt chickpeas were surveyed using a homozygous transgenic line expressing a cry1Ac gene. The differentially expressed genes (DEGs) profiling confirmed a low (0.69%, log2fold change≥2) frequency of differentially expressed in the transgenic chickpea line. Only a small (34 upregulated) proportion of genes showed > 2 fold (P-value of 0.05, FDR of 0.05) change in their expression, while only 23 genes down-regulated by >2 fold. Furthermore, no transcripts for potential allergenic proteins were represented in the DEGs. Most of these genes appeared to be developmentally regulated or stress-related which was expected because absolute synchronous growth and development even under a controlled environment are challenging. A few upregulated (AT-hook motif nuclear-localized protein 17-like, probable inactive 2-oxoglutaratedependent dioxygenase AOP2, protein EXORDIUM-like) and down-regulated (histone H2B, gonadal, embryonic abundant protein VF30.1-like, fasciclin-like arabinogalactan protein 12) genes were subjected to qPCR. The qPCR data validated the fold change of the up-regulated (>2) and down-regulated (>-2) genes. Thus, the above data revealed no potential alterations in the nutritional equivalence or transcript profile of transgenic Bt chickpeas.
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    ThesisItemOpen Access
    Cloning, Characterization and RNAi mediated silencing of gene encoding 1-deoxy-D-xylulose 5-phosphatereductoisome rase (DXR) in Centella asiatica
    (AAU, Jorhat, 2019-01) Sharma, Richa; Sen, Priyabrata
    Centella asiatica (L.) is one of the most valuable medicinal plants which belong to the family Apiaceae. The medicinal importance of this green leafy vegetable is known since prehistoric times. The pharmaceutical importance of this herb is due to the accumulation of large quantities of pentacyclic triterpenoid saponins, collectively known as centelloids synthesized by the isoprenoid biosynthesis pathway. Biosynthesis of triterpenoid in the plants proceeds via either of the two pathways, viz. Mevalonate (MVA) pathway (in the cytosol) or 2-C-methyl-Derythritol 4-phosphate (MEP) pathway (in plastid). In Centella, the pathway leading to the accumulation of triterpenoid is still not known. Thus, to know whether the MVA or MEP pathway or contribution of both has a role in the biosynthesis of triterpenoid, silencing the key regulatory enzyme gene using RNAi tool, of each of the pathway and then to analyze a metabolite is an efficient approach. In our lab, HMGR (a key enzyme of MVA pathway) RNAi construct has already been designed, confirmed by RT-PCR and validated by Agro-infiltration. 1-deoxy-D-xylulose-5- phosphate reductoisomerase (DXR) play a role in catalyzing the first committed step of the MEP pathway. The present study is the first step aimed to delineate the MEP pathway using RNAi silencing approach to knock down rate limiting 1-deoxy-Dxylulose- 5-phosphate reductoisomerase (DXR) enzyme. The full-length DXR gene sequence (JQ965955) of Centella has been characterized using in silico approach. CaDXR is a 1425bp ORF encoding a peptide of 474 amino acids and of molecular weight of 51.5 KDa. Multiple sequence comparison using MEGA tool showed the presence of two NADPH binding motif, two substrates binding motif, and one cleavage site motif. In this study, the 3-D structure of CaDXR was identified and validated along with this molecular dynamics simulation and finally docking with cofactor NADPH was done. The expression analysis suggests that CaDXR is differentially expressed in different tissues (with maximal expression in node and lowest in the roots). Our result suggests that nodes may be crucial to terpenoid biosynthesis in Centella asiatica. The RNAi-DXR construct was designed using the pHANNIBAL vector and subsequently cloned into a binary vector pART27. The binary vector pART27 containing RNAi-CaDXR construct was transformed into Agrobacterium strain AGL1. The transient analysis of the RNAi-CaDXR using semiquantitative RT-PCR confirmed the silencing of the endogenous DXR gene in Nicotiana and further confirmed in Centella asiatica. Thus, further incorporation of both the RNAi construct (HMGR and DXR) in transformed Centella shall shed light into the pathway that leads to the synthesis of principal secondary metabolites i.e centelloids.
  • Thumbnail Image
    ThesisItemOpen Access
    GENETIC STUDIES FOR IMPROVING YIELD UNDER DROUGHT STRESS ENVIRONMENTS IN RICE OF ASSAM
    (AAU, Jorhat, 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, Photosynthetic rate decreased with increasing water deficit, but the effect was less pronounced in Apo, Dumai and Tepi Dumai. The rate of transpiration decreased in all the cultivars under study. 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.