GENETIC DIVERSITY ANALYSIS OF CHICK PEA
(Cicer arietinum L.) USING SEED PROTEIN PROFILING
(Short Title: PROTEIN PROFILING OF CHICK PEA)
Dipinte Guptaa, Bhardwaj Chellapilab and Rajiv Ranjana* aPlant Molecular Biology Laboratory, Department of Botany, Faculty of Science, Dayalbagh Educational Institute (Deemed University), Dayalbagh, Agra-282005, India, Tel: 0562-2801545, Fax: 0562-2801226 b Division of Genetics, Indian Agricultural Research Institute, Pusa, New Delhi 110 012
*Corresponding Author: rajivranjanbt@gmail.com
Abstract
From ancient time onward chickpea (Cicer arietinum) is most important source of nutrients in the human diet. Along with the nutritional importance it possesses many medicinal values. It is very much essential to improve the quality of chickpea, an economic important crop, hereby in this study a biochemical approach had been employed to determine taxonomic and evolutionary information of chickpea. Forty Five accession of Cicer arietinum were taken for the study of genetic variation within intragenic varieties based on their seed protein using SDS PAGE. A total of 20 and 16 distinct bands were detected for albumin and globulin protein respectively, from the obtained band pattern a binary matrix was created and subjected to combined cluster analysis of albumin and globulin protein using UPGMA method. Based on analysis of cluster, FLIP-90-160 showed distinct relation from other cultivars. Present study concluded both globulin and albumin protein
Rubisco is the most abundant protein on earth that is essential for carbon fixation in plants. For the protein to function at its optimal level, it needs to be isolated from the mixture of proteins and in its purest form. The three isolation techniques carried out in this lab are salting out, ion exchange chromatography, and SDS-PAGE. Rubisco will be purer as each technique is conducted and will be in its purest form after the last isolation technique is carried out.
fischeri genomic, pUC 18, and lux operon, from previous experiments, were purified from agarose gel. To begin, 3 volumes of Buffer QG was added into 1 volume of agarose gel in microcentrifuge tube. To dissolve the gel, the mixtures were incubated in 50 ºC water bath for 10 minutes or until the gel dissolved completely. The color of the mixtures was yellow indicating the pH is equal or less than 7.5. Next, 1 gel volume of isopropanol was added to each tube and the final mixture was transferred to the QIA columns.
In order to test whether the curry leaf possesses one of the 6 alleles and whether it has a Ruby gene promoter, PCR and gel electrophoresis was used. Primers, short DNA sequences designed specifically for each allele of the Ruby gene, were used to isolate the gene and amplify it for PCR. Multiple PCRs were performed, each using a different primer set. Primers attach to specific regions surrounding the gene and amplify only the region that is flanked. The forward primer is bound to a specific sequence before the Ruby promoter, found to precede the actual gene sequence, and the reverse primer is bound to the DNA after the gene. In order for visible bands for gel electrophoresis to be produced, a forward primer must bind to one strand of DNA and a reverse primer must bind to the strand complementary to the one the forward primer binds to. If both primers bind to the same strand, the PCR product formed will not produce a result after gel electrophoresis. The size of the gel band of the DNA amplified by the primer will determine the identity of the allele. Band sizes will
Ph.D. Korea Advanced Institute of Science and Technology (KAIST), Biological Science and Engineering (Dewey D. Ryu) (1983)
Genomic DNA was extracted from the fungal mat of Sclerotium rolfsii. Thirty mg of freeze-dried mycelium was ground to a fine powder in an Eppendorf tube in liquid nitrogen. The ground mycelium was resuspended and lysed in 500 µl of lysis buffer (40 mM Tris-acetate, 20 mM sodium acetate,1 mM EDTA, 1% w/v SDS pH 8) (Lerner and Model1981). RNase A (2 µl of 10 mg/ml; Sigma USA) was added and the mixture was incubated for 5 min at 37 °C. To facilitate the precipitation of most polysaccharides, protein and cell debris, 165 ml of 5 mol/l NaCl solution was added and the components mixed by inverting the tube several times. The suspension was centrifuged at 6700 x g for 20 min at 4 °C, the supernatant was immediately transferred to a fresh tube and
which serve as the bases for many related studies. In this experiment , the specimen of E. chlorotica were collected from salt marsh located at Martha Vineyartd., being starved for more than 2 months and kept at 10 °C with fluorescent light under 14:10 light-dark illumination cycle [40]. The RNA were extracted from E. chlorotica and transported to BGI in Hong Kong for analysis. Researchers have also sequenced the genome of V. litorea. E. chlorotica transcriptom were aligned with V. litorea as a reference source and detect for region of similarity between sequences by BLAST. Once matching sequencies in V. litorea were detected , BlastX algorithm were applied
leucophylla DNA was added to the master mix in rbcL and ITS2 tubes. The PCR was set to start with (1) 2 minutes at 94 ℃, (2) 30 seconds at 94 ℃ (3) 30 seconds at 51 ℃, (4) 90 seconds at 71℃ (5) 10 minutes at 71 ℃ and held at 4℃ until gel electrophoresis can be conducted. Steps 2-4 will be repeated a total of 35 times.
1Department of Veterinary Physiology and Biochemistry; Karnataka Veterinary, Animal & Fisheries Sciences University; Veterinary College, Vinobanagar, Shimoga- 577 204, India, 2Division of Biochemistry, 3 & 4Division of Veterinary Biotechnology, Indian Veterinary Research Institute, Izatnagar-243 122, Bareilly, Uttar Pradesh, India
In this Agarose Gel Electrophoresis (AGE) Analysis, we run our entire DNA from the previous experiment 2A, 2B, and 2C; bacterial, plant and also animal’s DNA sample. Based on the Figure 1(focus on the red circle), the separation of DNA fragment for 7a, 7b, and 7c from the AGE diagram
From cotton genome database, 150 protein amino acid sequences of GhTRX were aligned by Clustalx1.81 program and a phylogenetic tree was constructed using the MEGA6 program with the neighbor joining (NJ) method and internal branch support was estimated with 1000 bootstrap replicates(Tamura, Stecher, Peterson, Filipski, & Kumar, 2013). Motifs were generated using MEME (http://meme.sdsc.edu/meme4_6_1/cgi-bin/meme.cgi/) and visualized with Logo (http://weblogo.berkeley.edu/logo.cgi/). MEME was run from the web server with the minimum and
The fragments were amplified using Phusion High-Fidelity DNA polymerase with standard reaction conditions and either genomic or plasmid templates. All fragments shown on the gel are of the expected size indicating the correct fragment has been amplified. The expected sizes are indicated on the gel with mCherry 737 bp, TtrpC 589 bp, noxA 1976 bp, mssD 3097 bp and PgpdA 2338 bp. After gel extraction and purification mCherry was shown to be of very low concentration 13.5 ng/L by the nanodrop. This was not sufficient for successful Gibson assembly of the plasmid. The other fragments had sufficient amplification with TtrpC containing 82.3 ng/L, noxA containing 51.2 ng/L and PgpdA containing 160.1 ng/L. From the initial PCR protocol mssD was not
Results of the first PCR reaction and gel electrophoresis with all primer sets can be seen in image 2. The first well contained the molecular weight marker, well 2; primer set 1, well 3; primer set 2, well 4; primer set 3, well 5; primer set 4 and well 6; primer set 5. The primer bands can be seen to have run to the end of the gel in wells 2 through 6, however no cDNA band were visible, only the weight maker bands were seen in the first well. The process was redone with a lower annealing temperature in the PCR stage with the aim to increase binding of the primers to the cDNA. Only primer set 2 was used, and again only marker bands in well 1 and primer bands at the end of well 2 were visible, which does not give any conclusive results, that can be seen in Image 2. Still determined, a third PCR reaction was performed with marker in well 1 and primer set 2 in wells 2 through 6, but with a PCR cycling conditions and temperature gradient. But, alas Image 3 revealed no useful data. As the GFP gene product was absent in al performed tests. Validation of extracted Zoanthid cDNA cannot proceed.
To get high-quality clean reads, the assessment of sequencing quality and read processing were performed by fastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) and FASTX-Toolkit (http://hannonlab.cshl.edu/fastx_toolkit/). The low-quality reads were removed using the same filtering criteria as described previously []. The clean reads were aligned on the F. vesca reference genome [1] using HISAT2 using default parameters [2]. Stringtie with higher accuracy and precision was used to generate transcript assembly [2]. Meanwhile, all unmapped reads derived from the above libraries were de novo assembled using the Trinity under default parameters to obtain comprehensive
Background: Rice (Oryza sativa, L.) is one of the strategic crops in Egypt, and improvement of its productivity is an essential requirement to ensure food security. Rice blast is one of the most important diseases of rice causing an average yield loss of 30-50% annually in rice growing areas of the world. Many resistant rice cultivars are short-lived after deployment because of the highly variation in Magnaporthe oryzae population, due to a high level of genomic instability of the pathogen. So that, the rice breeders trying to overcome this problem by developing resistant varieties by genetic approaches to maintain sustainability of rice production.
Cassava production in Africa is constrained with various pests and viral diseases especially cassava brown streak and cassava mosaic diseases. Research efforts to genetically improve resistance against viral diseases, drought, postharvest deterioration and nutrients fortification of this crop are underway (Zang et al., 2005; Vanderschuren et al., 2009; Nyaboga et al., 2013). All these efforts are through genetic transformation method which relies on the ability of cassava to regenerate from somatic embryos to full plant. Due to heterozygozity nature of cassava, it is difficult to use its zygotic embryos as starting materials for genetic transformation protocols (Raemakers et al., 1997; Sarria et al., 2000; Taylor et al., 2001; Siritunga and Sayre 2003). Instead, organised structures from somatic embryo cotyledons and friable embryogenic calli (FECs) have been developed (Schöpke et al., 1996; Taylor et al., 2004). FECs