To retrieve sequence information for cry genes and to compare native and cry genes. We are also going to relate various cry proteins using bioinformatics tools.
Recent advances in molecular biology and genetic engineering helps scientists to do better research in this fields. There are 3 different databases that are mainly used for DNA that is freely accessible to do their work very easily in very short time in plant growth and in plant developments. So, many scientists are developing many ideas in development of new plants that are resistant to many bacteria. BACILLUS THURINGENESIS is a naturally occurring bacterial disease, which is the active ingredient in some insecticides and, which is commonly used against some leaf and needle-feeding caterpillars. . Soil bacterium, a source of insecticidal toxins produced in transgenic plants is BACILLUS THURINGENESIS (BT) and the growth occurs where nutrients are available.
Cry genes are used for the production of transgenic plants since the level of expression of transgenic genes are very low in the plants. They are successfully transferred into potato and tobacco plants but insects were resistant for those genes. BACILLUS THURINGENESIS is used commercially to control several insects and pests. Its insecticidal activity is due to proteins that form a parasporal crystal during sporulation. Those parasporal crystal proteins are mainly encoded by cry genes usually located on plasmids of large molecular weight.
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The crystal proteins of Bacillus thuringiensis have pesticide properties for the production. These domain Cry Proteins have the mechanism which involves in photolytic activation step. Here it is ingested in the gut of the insect and this process now is followed by the following two domains. Domain 1 and Domain 2 have receptors on the surface of the cell. Here the Domain 1 is responsible for the formation of a channel through cell membrane. Novel Bt toxins, have no sequence similarity to three-domain Cry proteins expressed in transgenic plants. The specificity of Bt Cry toxins is a advantage in agriculture, as they is no target organism effected in the ecosystem. Bt formulations used in agriculture against coleopteran and lepidopteron pests are directed towards the surface of plants for control of dipterans pests. The characteristics of specific endotoxins determine insects by each product.
Since the first cloning of an insecticidal crystal protein gene from bacillus thuringiensis, many other such genes have been isolated. Each newly characterized genes or protein received an arbitrary designation from its discoveries.
In the following experiment, I have worked onto 5 protocols, the 1st is to retrieve a sequence from a database, the 2nd protocol is to align the sequence similarity of the two sequences, the 3rd is to translate a sequence of the base, the 4th is to find a similar sequences of the two accession numbers and, the 5th protocol is to align the multiple sequences and study about the relationship of different Cry toxins. Bioinformatics tools and a database is tested by analyzing Bacillus thuringiensis insecticidal proteins.
MATERIALS AND METHODS:
There are various bioinformatics tools to find the sequence relation between the various genes. The tools that were used in this experiment were:
National Centre for Biotechnology Information (NCBI):
It is a national centre for biotechnological information which gives the relative sequence between the various genes. Every gene has its own accession number so it is easy to find the nucleotide sequence and it was used in finding the coding sequence for the cry proteins. This was used for finding the sequence of cry gene. At the end the result was copied in the notepad and saved it.
It was used to find the significance of the alignment inbetween the two genes. It helped us to find the more significantly aligned genes and the E values between various genes.
Multiple Sequence Alignment- ClustalW:
Clustalw is the tool in the EBI software that helped us to do the cladogram and to find the similarity between the two genes. It showed the similarity and difference between various genes that given in the search bar.
This program achieves a high level of sensitivity for similarity searching at high speed for local alignments using a substitution matrix.
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Sequence similarity searching against protein databases using FASTA
Sequence similarity searching against nucleotide databases using FASTA
The URL http://www.ncbi.nlm.nih.gov/ was entered into the address bar of the web browser. The nucleotide database is selected from the drop down box and the accession(DQ241675.1) is entered into the search box and the search button is clicked.The accession is recorded for the next step and,scrolled down to 'CDS' link.The link is clicked for further step to appear the FASTA format.The FASTA format tab is clicked to get the sequence and is noted to the notepad and saved to the folder on the desktop.
The same process is repeated for the accession (AY376665.1) to get the whole process of the particular database.
The URL http://www.ebi.ac.uk/muscle is entered into the address bar of the web browser. The 'cds' for DQ241675.1 is taken from the saved protocol 1 and attached it to the sequence field,the enter button is pressed to note the other sequence i.e.,AY376665.1 from the saved protocol 1 and,the programme is run by clicking the run button.The two sequences are aligned that are entered into the dialogue box.The jalview is opened in the new window by clicking the jalview button, observed beside the jalview in the box ,containing the alignment of both the sequences.
Sequence translation using Transeq:
Transeq tells about one or more nucleotide sequences to the corresponding protein sequence translations to file.
The URL http://www.ebi.ac.uk/emboss/transeq was entered into the address bar of the web browser.The 'cds' for AY376665.1 was copied and attached to the sequence field.The run button was clicked and copied and attached the sequence into the notepad that has (*)character,represents the end codon of the sequence.The same process was repeated for the accession(DQ241675.1).
Homology searching using BLAST:
The 'homology' is defined as the boundaries of a region of sequence homology containing no insertions or deletions. In general it is said that it is similar sequeence.
The Basic Local Alignment Search Tool (BLAST) is the region that finds the local similarity between sequences.BLAST consists various other tools which are as follows:
It is used to search a nucleotide database using a nucleotide query.
It is used to search protein database using a protein query.
It is used to search protein database using a translated nucleotide query.
It is used to search translated nucleotide database using a protein query.
It is used to search translated nucleotide database using a translated nucleotide query.
The URL http://www.ncbi.nlm.nih.gov/ was entered into the address bar of the web browser. The BLAST option was clicked which was present at right of the screen. Upon all the tools present BLAST p was chosen for the protein sequence query. The saved sequence was taken from the protocol 1 and is attached to the sequence field for the accession (DQ241675.1).The BLAST button was clicked and wait for the results to appear on the screen, which tells the significant alignment represented from the accession.
Multiple sequence alignment:
Multiple sequence alignment (MSA) is to extract and represent important but dispersed sequence. The tool used in the multiple sequence aligment is CLUSTALW2.
ClustalW2 was a multiple sequence alignment program for DNA or proteins produces divergent sequences. And calculates the similarity for the selected sequences with the help of the cladograms or phylograms.
The URL http://www.ebi.ac.uk/clustalw2/ was entered in the address bar of the web browser. FASTA format was attached for the accession (DQ241675.1).Enter button was clicked. The following FASTA sequences were retrieved from NCBI. The sequences of the accession (M89794,Y09787.1,AY960853.1) were attached to the sequence field box. The screen was scrolled down to view the cladogram, showed the relatedness of the sequences.
Results and Discussion:
Sequence retrieval from NCBI:
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In this result, we use the FASTA tool which is used to observe the sequence of a particular database for the accession
>gi|82395048|gb|DQ241675.1| Bacillus thuringiensis isolate BtC008 insecticidal crystal protein Cry1Ab (cry1Ab) gene, complete cds
>gi|36244768:1-1854 Synthetic construct Cry1Ab1 (cry1Ab1) gene, complete cds
There are various genes that have different accession number that is used to find the sequence in this software. Here we used that software to find sequence of cry genes. Here the sequences are pasted. The coding sequences are pasted in FASTA format here. Start codons for the sequence is ATG and the stop codons for the sequence is TGA.
>DQ241675.1_1 Bacillus thuringiensis isolate BtC008 insecticidal crystal protein Cry1Ab (cry1Ab) gene, complete cds
RESULT 4 - DQ2416751.1_aa
>1-1854_1 Synthetic construct Cry1Ab1 (cry1Ab1) gene, complete cds
RESULT 5 - 'AY376665.1_aa'
Fig 3: showing the distribution of 100 BLAST hits on the query sequence.
RESULT 6 - BLAST
There are various cry toxins in the list. And there are 14 closest matches for the query sequence.