The Native Bacterium And Modified Cry Genes Biology Essay

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Bacillus thuringiensis is a naturally occuring bacterial disease,which is the active ingredient in some insecticides and,which are commly used against some leaf and needle-feeding caterpillars.Recent developments in biotechnology have greater use in genetically transformed pest-resistance crops.The major crops used in global agriculture have the great sucess, such as cotton which is highly resistant to lepidopteran larvae and maize resistant to coleopteran larvae Cotton containing two Bt genes began in 1999 for commercial use. Soil bacterium,a source of insecticidal toxins produced in transgenic plants is bacillus thuringensis(Bt) and the growth occurs where nutrients are available.Bt Strains may differ in the acitivity of insects towards pests and constitutes gene coding,produced by the bacterium.The insecticidal activity was first discovered in 1911.Alternative for the control of pests in agriculture has the importance in human public health.

Inclusions formed by different insecticidal crystal proteins (ICP) have a correlation between crystal morphology.Bacterial spore counts do not reflect the insecticidal activity of a Bt strain as the number and amount of ICPs produced per bacterial cell differs.The crystal proteins of Bacillus thuringiensis have pesticidal properties for the production.The 3 domain Cry proteins have the mechanism which involves in proteolytic activation step,occurs after ingestion in the gut of the pest and this process is followed by the 2 domains i.e., Domain I and Domain II with receptors on the surface of the cell.Where the Domain I is responsible for the formation of a channel through cell membrane The three domains is complex to the C-terminal and middle domains of the toxin involved in epithelial cell receptor binding and structural functions, while the N-terminal domain is involved in ion channel and pore formation.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.

Crystal (cry) gene encode ICPs to a specific type either to Lepidoptera (cryI), Diptera and Lepidoptera (cryII),Coleoptera (cryIII), Diptera (cryIV), or Coleoptera and Lepidoptera (cryV).Insect-resistance transgenic plants obtained through the expression of genes cryIA(b),cryIA(a),cryIA(c) and cry IIIA.Toxins cryIA(a) and cry IA(b) are highly sensitive,irreversible than cryIA(c) and CryIC.Cry1F show resistance towards the insect Lepidoptera.CryIE, which is less active invivo than the resistance in invitro.CryIB and CryIIIA, a coleopteran has a specific toxin,which had no significant effect.In case of CryIA(a) or CryIC,the membrane potential is unaffected.

Modified genes expresses 5-to 10 fold higher than the wild-type gene,whereas fully modified gene expresses an increase to 100 fold.Polyadenylation with poor expression of cry genes in plants.The feasibility of cry gene fusions for the transfer and development of r insect to be resistant. Bt subspecies have been isolated from dead insect and isolate toxic activity to the insect from which it is isolated. Organisms have a narrow host range to Coleoptera,Diptera and Lepidoptera and proliferate within the bodies. Bt formulations used in agriculture against coleopteran and lepidopteran pests are directed towards the surface of plants for control of dipteran pests.The characteristics of specific endotoxins determine insects by each product. 

In the experiment, I work on into 5 protocols,the first is to retrieve a sequence from a database,the second protocols is to align the sequence similarity of the two sequences,the third is to translate a sequence,the fourth is to find a similar sequences of the two accession numbers and,the last protocol is to align the multiple sequences and study about the relatedness of different Cry toxins. Bioinformatic tools and a database is tested by analyzing Bacillus thuringiensis insecticidal proteins,using the FASTA algorithm and by searching the identical matches of amino acids.

MATERIALS AND METHODS:

Sequence retrieval from NCBI:

NCBI (national centre for biotechnology information) which is used to find a particular coding sequence of a nucleotide database.

FASTA:

FASTA program achieves a high level of sensitivity for similarity searching at high speed for local alignments using a substitution matrix.

FASTA-protein:

Sequence similarity searching against protein databases using FASTA

FASTA-Nucleotide:

Sequence similarity searching against nucleotide databases using FASTA

The URL http://www.ncbi.nlm.nih.gov/ is 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.

Sequence alignment:

Sequence analysis is the use of various bioinformatic methods to determine the biological function and structure of genes and also the proteins coding. MUSCLE is the similar application of the sequence alignment.

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 reads one or more nucleotide sequences to the corresponding protein sequence translations to file. EMBOSS is a tool which is used to compare the sequences.

The URL http://www.ebi.ac.uk/emboss/transeq is entered into the address bar of the web browser.The 'cds' for AY376665.1 is copied ante attached to the sequence field.The run button is clicked and copied and attached the sequence into the notepad that has (*)character,represents the end codon of the sequence.The same process is 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. The significance of homology is evaluating a non-linear similarity score to the probability of finding the observed level of similarity in the region.BLAST is the tool used in the search of homology.

BLAST:

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:

Nucleotide BLAST:

Search a nucleotide database using a nucleotide query.

Protein BLAST:

Search protein database using a protein query.

BLAST x:

Search protein database using a translated nucleotide query.

tBLASTn:

Search translated nucleotide database using a protein query.

tBLASTx:

Search translated nucleotide database using a translated nucleotide query.

The URL http://www.ncbi.nlm.nih.gov/ is entered into the address bar of the web browser.The BLAST option is clicked which is present at right of the screen.Upon all the tools present BLAST p is choosen for the protein sequence query.The saved sequence is taken from the protocol 1 and is attached to the sequence field for the accession(DQ241675.1).The BLAST button is clicked and wait for the results to appear on the screen,which tells the significan 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:

ClustalW2 is 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/ is entered in the address bar of the web browser.FASTA format is attached for the accession (DQ241675.1).Enter button is clicked.The following FASTA sequences are retrieved from NCBI.The sequences of the accession(M89794,Y09787.1,AY960853.1) are attached to the sequence field box.The screen is scrolled down to view the cladogram,showed the relatedness of the sequences.

RESULTS:

Sequence retrieval from NCBI:

In this result, we use the FASTA tool which is used to observe the sequence of a particular database for he accession

DQ241675.1

>gi|82395048|gb|DQ241675.1| Bacillus thuringiensis isolate BtC008 insecticidal crystal protein Cry1Ab (cry1Ab) gene, complete cds

ATGGATAACAATCCGAACATCAATGAATGCATTCCTTATAATTGTTTAAGTAACCCTGAAGTAGAAGTATTAGGTGGAGAAAGAATAGAAACTGGTTACACCCCAATCGATATTTCCTTGTCGCTAACGCAATTTCTTTTGAGTGAATTTGTTCCCGGTGCTGGATTTGTGTTAGGACTAGTTGATATAATATGGGGAATTTTTGGTCCCTCTCAATGGGACGCATTTCTTGTACAAATTGAACAGTTAATTAACCAAAGAATAGAAGAATTCGCTAGGAACCAAGCCATTTCTAGATTAGAAGGACTAAGCAATCTTTATCAAATTTACGCAGAATCTTTTAGAGAGTGGGAAGCAGATCCTACTAATCCAGCATTAAGAGAAGAGATGCGTATTCAATTCAATGACATGAACAGTGCCCTTACAACCGCTATTCCTCTTTTTGCAGTTCAAAATTATCAAGTTCCTCTTTTATCAGTATATGTTCAAGCTGCAAATTTACATTTATCAGTTTTGAGAGATGTTTCAGTGTTTGGACAAAGGTGGGGATTTGATGCCGCGACTATCAATAGTCGTTATAATGATTTAACTAGGCTTATTGGCAACTATACAGATCATGCTGTACGCTGGTACAATACGGGATTAGAGCGTGTATGGGGACCGGATTCTAGAGATTGGATAAGATATAATCAATTTAGAAGAGAATTAACACTAACTGTATTAGATATCGTTTCTCTATTTCCGAACTATGATAGTAGAACGTATCCAATTCGAACAGTTTCCCAATTAACAAGAGAAATTTATACAAACCCAGTATTAGAAAATTTTGATGGTAGTTTTCGAGGCTCGGCTCAGGGCATAGAAGGAAGTATTAGGAGTCCACATTTGATGGATATACTTAACAGTATAACCATCTATACGGATGCTCATAGAGGAGAATATTATTGGTCAGGGCATCAAATAATGGCTTCTCCTGTAGGGTTTTCGGGGCCAGAATTCACTTTTCCGCTATATGGAACTATGGGAAATGCAGCTCCACAACAACGTATTGTTGCTCAACTAGGTCAGGGCGTGTATAGAACATTATCGTCCACTTTATATAGAAGACCTTTTAATATAGGGATAAATAATCAACAACTATCTGTTCTTGACGGGACAGAATTTGCTTATGGAACCTCCTCAAATTTGCCATCCGCTGTATACAGAAAAAGCGGAACGGTAGATTCGCTGGATGAAATACCGCCACAGAATAACAACGTGCCACCTAGGCAAGGATTTAGTCATCGATTAAGCCATGTTTCAATGTTTCGTTCAGGCTTTAGTAATAGTAGTGTAAGTATAATAAGAGCTCCTATGTTCTCTTGGATACATCGTAGTGCTGAATTTAATAATATAATTCCTTCATCACAAATTACACAAATACCTTTAACAAAATCTACTAATCTTGGCTCTGGAACTTCTGTCGTTAAAGGACCAGGATTTACAGGAGGAGATATTCTTCGAAGAACTTCACCTGGCCAGATTTCAACCTTAAGAGTAAATATTACTGCACCATTATCACAAAGATATCGGGTAAGAATTCGCTACGCTTCTACCACAAATTTACAATTCCATACATCAATTGACGGAAGACCTATTAATCAGGGGAATTTTTCAGCAACTATGAGTAGTGGGAGTAATTTACAGTCCGGAAGCTTTAGGACTGTAGGTTTTACTACTCCGTTTAACTTTTCAAATGGATCAAGTGTATTTACGTTAAGTGCTCATGTCTTCAATTCAGGCAATGAAGTTTATATAGATCGAATTGAATTTGTTCCGGCAGAAGTAACCTTTGAGGCAGAATATGATTTAGAAAGAGCACAAAAGGCGGTGAATGAGCTGTTTACTTCTTCCAATCAAATCGGGTTAAAAACAGATGTGACGGATTATCATATTGATCAAGTATCCAATTTAGTTGAGTGTTTATCTGATGAATTTTGTCTGGATGAAAAAAAAGAATTGTCCGAGAAAGTCAAACATGCGAAGCGACTTAGTGATGAGCGGAATTTACTTCAAGATCCAAACTTTAGAGGGATCAATAGACAACTAGACCGTGGCTGGAGAGGAAGTACGGATATTACCATCCAAGGAGGCGATGACGTATTCAAAGAGAATTACGTTACGCTATTGGGTACCTTTGATGAGTGCTATCCAACGTATTTATATCAAAAAATAGATGAGTCGAAATTAAAAGCCTATACCCGTTACCAATTAAGAGGGTATATCGAAGATAGTCAAGACTTAGAAATCTATTTAATTCGCTACAATGCCAAACACGAAACAGTAAATGTGCCAGGTACGGGTTCCTTATGGCCGCTTTCAGCCCCAAGTCCAATCGGAAAATGTGCCCATCATTCCCATCATTTCTCCTTGGACATTGATGTTGGATGTACAGACTTAAATGAGGACTTAGGTGTATGGGTGATATTCAAGATTAAGACGCAAGATGGCCATGCAAGACTAGGAAATCTAGAATTTCTCGAAGAGAAACCATTAGTAGGAGAAGCACTAGCTCGTGTGAAAAGAGCGGAGAAAAAATGGAGAGACAAACGTGAAAAATTGGAATGGGAAACAAATATTGTTTATAAAGAGGCAAAAGAATCTGTAGATGCTTTATTTGTAAACTCTCAATATGATAGATTACAAGCGGATACCAACATCGCGATGATTCATGCGGCAGATAAACGCGTTCATAGCATTCGAGAAGCTTATCTGCCTGAGCTGTCTGTGATTCCGGGTGTCAATGCGGCTATTTTTGAAGAATTAGAAGGGCGTATTTTCACTGCATTCTCCCTATATGATGCGAGAAATGTCATTAAAAATGGTGATTTTAATAATGGCTTATCCTGCTGGAACGTGAAAGGGCATGTAGATGTAGAAGAACAAAACAACCACCGTTCGGTCCTTGTTGTTCCGGAATGGGAAGCAGAAGTGTCACAAGAAGTTCGTGTCTGTCCGGGTCGTGGCTATATCCTTCGTGTCACAGCGTACAAGGAGGGATATGGAGAAGGTTGCGTAACCATTCATGAGATCGAGAACAATACAGACGAACTGAAGTTTAGCAACTGTGTAGAAGAGGAAGTATATCCAAACAACACGGTAACGTGTAATGATTATACTGCGACTCAAGAAGAATATGAGGGTACGTACACTTCTCGTAATCCAGGATATGACGGAGCCTATGAAAGCAATTCTTCTGTACCAGCTGATTATGCATCAGCCTATGAAGAAAAAGCATATACAGATGGACGAAGAGACAATCCTTGTGAATCTAACAGAGGATATGGGGATTACACACCACTACCAGCTGGCTATGTGACAAAAGAATTAGAGTACTTCCCAGAAACCGATAAGGTATGGATTGAGATCGGAGAAACGGAAGGAACATTCATCGTGGACAGCGTGGAATTACTTCTTATGGAGGAATAA

AY376665.1

>gi|36244768:1-1854 Synthetic construct Cry1Ab1 (cry1Ab1) gene, complete cds

ATGGCTAACAATCCAAACATTAACGAGTGCATCCCATATAATTGCTTGAGCAACCCAGAGGTGGAAGTTCTCGGCGGTGAAAGAATTGAGACCGGCTACACTCCTATCGACATCAGCCTGTCCCTGACTCAGTTTCTGCTTAGCGAGTTCGTTCCCGGTGCTGGATTTGTGTTAGGACTAGTGGACATCATTTGGGGCATCTTCGGCCCCTCCCAATGGGACGCCTTCCTCGTCCAAATCGAGCAGCTGATTAACCAAAGGATCGAAGAGTTCGCTAGGAACCAAGCCATTTCTAGATTAGAAGGACTAAGCAATCTTTATCAAATTTACGCAGAATCTTTTAGAGAGTGGGAAGCAGATCCTACTAATCCAGCATTAAGAGAAGAGATGCGTATTCAATTCAATGACATGAACAGTGCCCTTACAACCGCTATTCCTCTTTTTGCAGTTCAAAATTATCAAGTTCCTCTTTTATCAGTATATGTTCAAGCTGCCAACTTGCACCTCTCAGTTTTGAGGGACGTCTCCGTGTTCGGGCAAAGGTGGGGATTTGATGCCGC

CACCATCAACAGCCGTTATAATGATTTGACCAGGCTCATCGGCAACTACACCGATCATGCTGTACGCTGGTACAACACCGGATTGGAGCGTGTGTGGGGACCAGACTCTAGAGACTGGATCAGGTATAATCAATTTAGAAGGGAATTGACCCTCACTGTGCTTGATATCGTTTCTCTCTTCCCAAACTACGATAGCAGGACCTATCCAATTCGAACAGTTTCCCAATTAACAAGAGAAATTTATACAAACCCAGTATTAGAAAATTTTGATGGTAGTTTTCGAGGCTCGGCTCAGGGCATAGAAGGAAGTATTAGGAGTCCACATTTGATGGATATACTTAACAGTATAACCATCTACACCGACGCCCACCGCGGCGAGTACTATTGGTCCGGGCACCAGATCATGGCCTCCCCAGTCGGGTTTTCCGGGCCAGAATTTACTTTTCCGCTATATGGAACTATGGGAAATGCAGCTCCACAACAACGTATTGTTGCTCAACTAGGTCAGGGCGTGTATAGAACATTGTCTTCCACTTTGTACCGCAGACCTTTTAACATCG

GGATCAACAATCAACAACTATCTGTTCTTGACGGGACAGAATTTGCTTATGGAACCTCCTCAAATTTGCCATCCGCTGTATACAGAAAAAGCGGAACCGTGGACTCCCTGGATGAAATCCCCCCACAGAATAACAACGTGCCACCTAGGCAAGGATTTAGTCATCGATTAAGCCATGTTTCAATGTTTCGTTCAGGCTTCAGTAATAGCTCTGTCAGCATCATTAGAGCTCCAATGTTCTCCTGGATCCACCGCAGTGCTGAATTTAATAATATAATTCCTTCATCACAAATTACACAAATACCTTTAACAAAATCTACTAATCTTGGCTCTGGAACTTCTGTCGTTAAAGGACCAGGATTTACAGGAGGAGATATTCTTCGAAGAACTTCACCTGGCCAGATTTCAACCTTAAGAGTAAATATCACCGCCCCACTCTCACAAAGATATCGGGTGCGCATTCGCTACGCTTCCACCACCAACCTGCAATTCCATACATCAATTGACGGAAGACCTATTAATCAGGGGAATTTTTCAGCAACTATGAGTAGTGGGAGTAAT

TTACAGTCCGGAAGCTTTAGGACTGTAGGTTTTACTACTCCGTTTAACTTTTCAAATGGATCAAGTGTATTTACGTTAAGTGCTCATGTCTTCAATTCAGGCAATGAAGTTTACATCGACCGCATCGAGTTCGTGCCAGCCGAAGTCACCTTTGAGGCCGAGTACGATCTCTAA

Sequence alignment:

In the sequence alignment, I had used tools of a muscle where the both sequences are attached to the sequence field.We use the format format to obtain the results as well the jal view of both the sequences to know the length of the sequences.

>gi|82395048|gb|DQ241675.1| Bacillus thuringiensis isolate BtC008 insecticidal crystal protein Cry1Ab (cry1Ab) gene, complete cds

ATGGATAACAATCCGAACATCAATGAATGCATTCCTTATAATTGTTTAAGTAACCCTGAAGTAGAAGTATTAGGTGGAGAAAGAATAGAAACTGGTTACACCCCAATCGATATTTCCTTGTCGCTAACGCAATTTCTTTTGAGTGAATTTGTTCCCGGTGCTGGATTTGTGTTAGGACTAGTTGATATAATATGGGGAATTTTTGGTCCCTCTCAATGGGACGCATTTCTTGTACAAATT

GAACAGTTAATTAACCAAAGAATAGAAGAATTCGCTAGGAACCAAGCCATTTCTAGATTAGAAGGACTAAGCAATCTTTATCAAATTTACGCAGAATCTTTTAGAGAGTGGGAAGCAGATCCTACTAATCCAGCATTAAGAGAAGAGATGCGTATTCAATTCAATGACATGAACAGTGCCCTTACAACCGCTATTCCTCTTTTTGCAGTTCAAAATTATCAAGTTCCTCTTTTATCAGTA

TATGTTCAAGCTGCAAATTTACATTTATCAGTTTTGAGAGATGTTTCAGTGTTTGGACAAAGGTGGGGATTTGATGCCGCGACTATCAATAGTCGTTATAATGATTTAACTAGGCTTATTGGCAACTATACAGATCATGCTGTACGCTGGTACAATACGGGATTAGAGCGTGTATGGGGACCGGATTCTAGAGATTGGATAAGATATAATCAATTTAGAAGAGAATTAACACTAACTGTA

TTAGATATCGTTTCTCTATTTCCGAACTATGATAGTAGAACGTATCCAATTCGAACAGTTTCCCAATTAACAAGAGAAATTTATACAAACCCAGTATTAGAAAATTTTGATGGTAGTTTTCGAGGCTCGGCTCAGGGCATAGAAGGAAGTATTAGGAGTCCACATTTGATGGATATACTTAACAGTATAACCATCTATACGGATGCTCATAGAGGAGAATATTATTGGTCAGGGCATCAA

ATAATGGCTTCTCCTGTAGGGTTTTCGGGGCCAGAATTCACTTTTCCGCTATATGGAACTATGGGAAATGCAGCTCCACAACAACGTATTGTTGCTCAACTAGGTCAGGGCGTGTATAGAACATTATCGTCCACTTTATATAGAAGACCTTTTAATATAGGGATAAATAATCAACAACTATCTGTTCTTGACGGGACAGAATTTGCTTATGGAACCTCCTCAAATTTGCCATCCGCTGTA

TACAGAAAAAGCGGAACGGTAGATTCGCTGGATGAAATACCGCCACAGAATAACAACGTGCCACCTAGGCAAGGATTTAGTCATCGATTAAGCCATGTTTCAATGTTTCGTTCAGGCTTTAGTAATAGTAGTGTAAGTATAATAAGAGCTCCTATGTTCTCTTGGATACATCGTAGTGCTGAATTTAATAATATAATTCCTTCATCACAAATTACACAAATACCTTTAACAAAATCTACT

AATCTTGGCTCTGGAACTTCTGTCGTTAAAGGACCAGGATTTACAGGAGGAGATATTCTTCGAAGAACTTCACCTGGCCAGATTTCAACCTTAAGAGTAAATATTACTGCACCATTATCACAAAGATATCGGGTAAGAATTCGCTACGCTTCTACCACAAATTTACAATTCCATACATCAATTGACGGAAGACCTATTAATCAGGGGAATTTTTCAGCAACTATGAGTAGTGGGAGTAAT

TTACAGTCCGGAAGCTTTAGGACTGTAGGTTTTACTACTCCGTTTAACTTTTCAAATGGATCAAGTGTATTTACGTTAAGTGCTCATGTCTTCAATTCAGGCAATGAAGTTTATATAGATCGAATTGAATTTGTTCCGGCAGAAGTAACCTTTGAGGCAGAATATGATTTAGAAAGAGCACAAAAGGCGGTGAATGAGCTGTTTACTTCTTCCAATCAAATCGGGTTAAAAACAGATGTG

ACGGATTATCATATTGATCAAGTATCCAATTTAGTTGAGTGTTTATCTGATGAATTTTGTCTGGATGAAAAAAAAGAATTGTCCGAGAAAGTCAAACATGCGAAGCGACTTAGTGATGAGCGGAATTTACTTCAAGATCCAAACTTTAGAGGGATCAATAGACAACTAGACCGTGGCTGGAGAGGAAGTACGGATATTACCATCCAAGGAGGCGATGACGTATTCAAAGAGAATTACGTT

ACGCTATTGGGTACCTTTGATGAGTGCTATCCAACGTATTTATATCAAAAAATAGATGAGTCGAAATTAAAAGCCTATACCCGTTACCAATTAAGAGGGTATATCGAAGATAGTCAAGACTTAGAAATCTATTTAATTCGCTACAATGCCAAACACGAAACAGTAAATGTGCCAGGTACGGGTTCCTTATGGCCGCTTTCAGCCCCAAGTCCAATCGGAAAATGTGCCCATCATTCCCAT

CATTTCTCCTTGGACATTGATGTTGGATGTACAGACTTAAATGAGGACTTAGGTGTATGGGTGATATTCAAGATTAAGACGCAAGATGGCCATGCAAGACTAGGAAATCTAGAATTTCTCGAAGAGAAACCATTAGTAGGAGAAGCACTAGCTCGTGTGAAAAGAGCGGAGAAAAAATGGAGAGACAAACGTGAAAAATTGGAATGGGAAACAAATATTGTTTATAAAGAGGCAAAAGAA

TCTGTAGATGCTTTATTTGTAAACTCTCAATATGATAGATTACAAGCGGATACCAACATCGCGATGATTCATGCGGCAGATAAACGCGTTCATAGCATTCGAGAAGCTTATCTGCCTGAGCTGTCTGTGATTCCGGGTGTCAATGCGGCTATTTTTGAAGAATTAGAAGGGCGTATTTTCACTGCATTCTCCCTATATGATGCGAGAAATGTCATTAAAAATGGTGATTTTAATAATGGC

TTATCCTGCTGGAACGTGAAAGGGCATGTAGATGTAGAAGAACAAAACAACCACCGTTCGGTCCTTGTTGTTCCGGAATGGGAAGCAGAAGTGTCACAAGAAGTTCGTGTCTGTCCGGGTCGTGGCTATATCCTTCGTGTCACAGCGTACAAGGAGGGATATGGAGAAGGTTGCGTAACCATTCATGAGATCGAGAACAATACAGACGAACTGAAGTTTAGCAACTGTGTAGAAGAGGAA

GTATATCCAAACAACACGGTAACGTGTAATGATTATACTGCGACTCAAGAAGAATATGAGGGTACGTACACTTCTCGTAATCCAGGATATGACGGAGCCTATGAAAGCAATTCTTCTGTACCAGCTGATTATGCATCAGCCTATGAAGAAAAAGCATATACAGATGGACGAAGAGACAATCCTTGTGAATCTAACAGAGGATATGGGGATTACACACCACTACCAGCTGGCTATGTGACA

AAAGAATTAGAGTACTTCCCAGAAACCGATAAGGTATGGATTGAGATCGGAGAAACGGAAGGAACATTCATCGTGGACAGCGTGGAATTACTTCTTATGGAGGAATAA

>gi|36244768:1-1854 Synthetic construct Cry1Ab1 (cry1Ab1) gene, complete cds

ATGGCTAACAATCCAAACATTAACGAGTGCATCCCATATAATTGCTTGAGCAACCCAGAGGTGGAAGTTCTCGGCGGTGAAAGAATTGAGACCGGCTACACTCCTATCGACATCAGCCTGTCCCTGACTCAGTTTCTGCTTAGCGAGTTCGTTCCCGGTGCTGGATTTGTGTTAGGACTAGTGGACATCATTTGGGGCATCTTCGGCCCCTCCCAATGGGACGCCTTCCTCGTCCAAATC

GAGCAGCTGATTAACCAAAGGATCGAAGAGTTCGCTAGGAACCAAGCCATTTCTAGATTAGAAGGACTAAGCAATCTTTATCAAATTTACGCAGAATCTTTTAGAGAGTGGGAAGCAGATCCTACTAATCCAGCATTAAGAGAAGAGATGCGTATTCAATTCAATGACATGAACAGTGCCCTTACAACCGCTATTCCTCTTTTTGCAGTTCAAAATTATCAAGTTCCTCTTTTATCAGTA

TATGTTCAAGCTGCCAACTTGCACCTCTCAGTTTTGAGGGACGTCTCCGTGTTCGGGCAAAGGTGGGGATTTGATGCCGCCACCATCAACAGCCGTTATAATGATTTGACCAGGCTCATCGGCAACTACACCGATCATGCTGTACGCTGGTACAACACCGGATTGGAGCGTGTGTGGGGACCAGACTCTAGAGACTGGATCAGGTATAATCAATTTAGAAGGGAATTGACCCTCACTGTG

CTTGATATCGTTTCTCTCTTCCCAAACTACGATAGCAGGACCTATCCAATTCGAACAGTTTCCCAATTAACAAGAGAAATTTATACAAACCCAGTATTAGAAAATTTTGATGGTAGTTTTCGAGGCTCGGCTCAGGGCATAGAAGGAAGTATTAGGAGTCCACATTTGATGGATATACTTAACAGTATAACCATCTACACCGACGCCCACCGCGGCGAGTACTATTGGTCCGGGCACCAG

ATCATGGCCTCCCCAGTCGGGTTTTCCGGGCCAGAATTTACTTTTCCGCTATATGGAACTATGGGAAATGCAGCTCCACAACAACGTATTGTTGCTCAACTAGGTCAGGGCGTGTATAGAACATTGTCTTCCACTTTGTACCGCAGACCTTTTAACATCGGGATCAACAATCAACAACTATCTGTTCTTGACGGGACAGAATTTGCTTATGGAACCTCCTCAAATTTGCCATCCGCTGTA

TACAGAAAAAGCGGAACCGTGGACTCCCTGGATGAAATCCCCCCACAGAATAACAACGTGCCACCTAGGCAAGGATTTAGTCATCGATTAAGCCATGTTTCAATGTTTCGTTCAGGCTTCAGTAATAGCTCTGTCAGCATCATTAGAGCTCCAATGTTCTCCTGGATCCACCGCAGTGCTGAATTTAATAATATAATTCCTTCATCACAAATTACACAAATACCTTTAACAAAATCTACT

AATCTTGGCTCTGGAACTTCTGTCGTTAAAGGACCAGGATTTACAGGAGGAGATATTCTTCGAAGAACTTCACCTGGCCAGATTTCAACCTTAAGAGTAAATATCACCGCCCCACTCTCACAAAGATATCGGGTGCGCATTCGCTACGCTTCCACCACCAACCTGCAATTCCATACATCAATTGACGGAAGACCTATTAATCAGGGGAATTTTTCAGCAACTATGAGTAGTGGGAGTAAT

TTACAGTCCGGAAGCTTTAGGACTGTAGGTTTTACTACTCCGTTTAACTTTTCAAATGGATCAAGTGTATTTACGTTAAGTGCTCATGTCTTCAATTCAGGCAATGAAGTTTACATCGACCGCATCGAGTTCGTGCCAGCCGAAGTCACCTTTGAGGCCGAGTACGATC-----------------------------------------------------------------------

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seq.png

Fig 1: showing the length of the sequences and the start codon for both the sequences(ATG).

stop.png

Fig 2: showing the end codon(TAG) of the sequences in the jalview.

Sequence translation using Transeq:

By using the emboss tools, the sequences are translated where the both accession FASTA formats areattached to a file.

DQ241675.1

>DQ241675.1_1 Bacillus thuringiensis isolate BtC008 insecticidal crystal protein Cry1Ab (cry1Ab) gene, complete cds

MDNNPNINECIPYNCLSNPEVEVLGGERIETGYTPIDISLSLTQFLLSEFVPGAGFVLGLVDIIWGIFGPSQWDAFLVQIEQLINQRIEEFARNQAISRLEGLSNLYQIYAESFREWEADPTNPALREEMRIQFNDMNSALTTAIPLFAVQNYQVPLLSVYVQAANLHLSVLRDVSVFGQRWGFDAATINSRYNDLTRLIGNYTDHAVRWYNTGLERVWGPDSRDWIRYNQFRRELTLTVLDIVSLFPNYDSRTYPIRTVSQLTREIYTNPVLENFDGSFRGSAQGIEGSIRSPHLMDILNSITIYTDAHRGEYYWSGHQIMASPVGFSGPEFTFPLYGTMGNAAPQQRIVAQLGQGVYR

TLSSTLYRRPFNIGINNQQLSVLDGTEFAYGTSSNLPSAVYRKSGTVDSLDEIPPQNNNVPPRQGFSHRLSHVSMFRSGFSNSSVSIIRAPMFSWIHRSAEFNNIIPSSQITQIPLTKSTNLGSGTSVVKGPGFTGGDILRRTSPGQISTLRVNITAPLSQRYRVRIRYASTTNLQFHTSIDGRPINQGNFSATMSSGSNLQSGSFRTVGFTTPFNFSNGSSVFTLSAHVFNSGNEVYIDRIEFVPAEVTFEAEYDLERAQKAVNELFTSSNQIGLKTDVTDYHIDQVSNLVECLSDEFCLDEKKELSEKVKHAKRLSDERNLLQDPNFRGINRQLDRGWRGSTDITIQGGDDVFKENYV

TLLGTFDECYPTYLYQKIDESKLKAYTRYQLRGYIEDSQDLEIYLIRYNAKHETVNVPGTGSLWPLSAPSPIGKCAHHSHHFSLDIDVGCTDLNEDLGVWVIFKIKTQDGHARLGNLEFLEEKPLVGEALARVKRAEKKWRDKREKLEWETNIVYKEAKESVDALFVNSQYDRLQADTNIAMIHAADKRVHSIREAYLPELSVIPGVNAAIFEELEGRIFTAFSLYDARNVIKNGDFNNGLSCWNVKGHVDVEEQNNHRSVLVVPEWEAEVSQEVRVCPGRGYILRVTAYKEGYGEGCVTIHEIENNTDELKFSNCVEEEVYPNNTVTCNDYTATQEEYEGTYTSRNPGYDGAYESNSSV

PADYASAYEEKAYTDGRRDNPCESNRGYGDYTPLPAGYVTKELEYFPETDKVWIEIGETEGTFIVDSVELLLMEE

AY376665.1

>1-1854_1 Synthetic construct Cry1Ab1 (cry1Ab1) gene, complete cds

MANNPNINECIPYNCLSNPEVEVLGGERIETGYTPIDISLSLTQFLLSEFVPGAGFVLGLVDIIWGIFGPSQWDAFLVQIEQLINQRIEEFARNQAISRLEGLSNLYQIYAESFREWEADPTNPALREEMRIQFNDMNSALTTAIPLFAVQNYQVPLLSVYVQAANLHLSVLRDVSVFGQRWGFDAATINSRYNDLTRLIGNYTDHAVRWYNTGLERVWGPDSRDWIRYNQFRRELTLTVLDIVSLFPNYDSRTYPIRTVSQLTREIYTNPVLENFDGSFRGSAQGIEGSIRSPHLMDILNSITIYTDAHRGEYYWSGHQIMASPVGFSGPEFTFPLYGTMGNAAPQQRIVAQLGQGVYRTLSSTLYRRPFNIGINNQQLSVLDGTEFAYGTSSNLPSAVYRKSGTVDSLDEIPPQNNNVPPRQGFSHRLSHVSMFRSGFSNSSVSIIRAPMFSWIHRSAEFNNIIPSSQITQIPLTKSTNLGSGTSVVKGPGFTGGDILRRTSPGQISTLRVNITAPLSQRYRVRIRYASTTNLQFHTSIDGRPINQGNFSATMSSGSNLQSGSFRTVGFTTPFNFSNGSSVFTLSAHVFNSGNEVYIDRIEFVPAEVTFEAEYDL

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Homology searching using BLAST:

The tool used here is BLAST; in the sub tools we take the BLASTP as the protein database with a protein query to know the E value.

swt.png

seq.png

Fig 3: showing the distribution of 100 BLAST hits on the query sequence.

Multiple sequence alignment:

To compare the sequences and observe the relation between them using cladogram.Take the sequences in FASTA format various accessions (M89794, Y09787.1, AY960853.1) used to obtain the results.

clado.png

Fig 4: showing the cladogram about relatedness of the sequences.

DISCUSSION:

The protocols show the retrieved sequence from a database which have the similarity of two sequences to align them.Of this a sequence is translated from DNA to aminoacid,this make a protein o find the similar sequences and align the multiple sequences to see the relations between the sequences using a cladogram.The discussion is about the differences between the native and modified differences of cry genes in transgenic plants, the results show the relation between the sequences that are closely related with the help of the cladogram.The cladogram represents the four sequences which undergo in this protocol,where the sequences have the long pathway,the other two have the small pathway that are closely related. The sequence alignment show the difference between the two accessions,for DQ241675.1 the full length of cry1Ab toxin from Baccillus thuringiensis and, for AY376665.1 is the synthetic cry1Ab.From the jalview,shows the length of the two sequences where the start codon is ATG and the end codons are TAG,TAA,TGA.The accessions of the two 'Cds' are DQ241675.1 and AY376665.1,whereas the lengths of the two accessions are 3468,1854,the main difference between the two sequences.The uncoded sequences show the changes between the two 'cds' in the alignment. The amino acids of cry1Ab are many in number which represents each and every amino acid in a sequence.In translation of the sequence (*) characterizes the termination codon of the sequences, where these all the sequences end up with amino acids.The less the native, more transgenic in plants and, is easy to identify the difference in a sequence. When more native cry 1Ab present, it dominates the modified cry genes. Homology refers to neither insertions or deletions in the sequence with homology. To overcome this BLAST tool is used for cry toxins. They are various cry toxins which helps to know the E value. Lower E value good E value. Cry toxins are closely related with short branched alignments, that shows the good result with the help of the cladogram. It shows the accession AY960853.1 is a vegetative insecticidal protein as it is long branched alignment and more effective with a concentration of the multiple sequence alignment.

CONCLUSION:

From the result, the relatedness of the sequences are known.As per the protocol, FASTA sequence is taken to align the relation between the two sequences that are closely related using a cladogram.The evolutionary make up have the similarity in the sequence using bioinformatic tools for finding the differences in novel and modified cry toxins in transgenic plants.

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