WO2012131619A1 - A nucleotide sequence, expression cassette, transgenic events, cells and methods thereof - Google Patents

Abstract

The present disclosure is in the area of agricultural biotechnology, wherein two transgenic cotton events, viz., MLS9124 and MLS9878 are disclosed. The present disclosure relates specifically to an expression cassette comprising a nucleotide sequence coding for Cry1C gene in plants of genus Gossypium. The disclosure also provides vectors and host cells tansformed by the said expression cassette and corresponding methods thereof. The disclosure also comprises method of codon optimizing the said gene, and conferring of resistance to plants of genus Gossypium from important pests or insects of cultivated crops causing extensive economic damage.

Description

A NUCLEOTIDE SEQUENCE, EXPRESSION CASSETTE, TRANSGENIC EVENTS, CELLS AND METHODS THEREOF"

TECHNICAL FIELD

The present disclosure is in the area of agricultural biotechnology, wherein two transgenic cotton events, viz., MLS9124 and MLS9878 are disclosed. The present disclosure relates specifically to an expression cassette comprising a nucleotide sequence coding for CrylC gene in plants of genus Gossypium. The disclosure also provides vectors and host cells tansformed by the said expression cassette and corresponding methods thereof. The disclosure also comprises method of codon optimizing the said gene, and conferring of resistance to plants of genus Gossypium from important pests or insects of cultivated crops causing extensive economic damage.

BACKGROUND AND PRIOR ART OF THE DISCLOSURE

Pests cause extensive economic damage of crop plants worldwide and necessitate extensive use of chemical pesticides for control. The losses due to damages by insects cause significant losses in crop yields, a situation that the burgeoning population worldwide can ill-afford. The consequent fallout of this extensive pesticide usage is the damage to the ecosystem caused by leaching into the soils as well as by run-off into the water bodies, thus disturbing the flora and fauna. Pesticides, being generally non-preferential for the target organisms, also affect beneficial insects. The residual pesticides in the crops get into the food chain causing severe damages to the living system, including humans where instances of physical deformities have been reported.

Spodoptera litura, a pest of cotton and tobacco, is also known as the Oriental leafworm moth, Cluster caterpillar, Cotton leafworm, Tobacco cutworm, Tropical armyworm, Taro caterpillar, Tobacco budworm, Rice cutworm, and Cotton Cutworm. This moth is found in Asia, with some specific problematic pest population reports occurring in Cambodia, Hong Kong, India, the Pacific islands, Guam, American Samoa, and Hawaii. In Australia, it is found in northern two thirds of the country. It is established in the U.S., and it is a pest of national, regulatory concern. Besides cotton and tobacco, this insect causes extensive damage in many vegetables and is a major pest in corn as well as a minor pest in rice. One of the most widely used Bacillus thuringiensis strains used in the biological control of insect pests of crops is aizawai which carries the crystal protein CrylC. The very high insecticidal activity of this Cry protein on the diamond-back moth and the army worm {Plutella xylostella, and Spodoptera spp. respectively) combined with the impeccable safety record of the strains carrying this crystal protein has been primarily responsible for this widespread use. Extensive studies on the biosafety of this strain have been done, mostly in the US and details have been published under the joint sponsorship of the UNEP, ILO, WHO and the International Program of Chemical Safety (1).

The US Environmental Protection Agency (EPA) has approved the registration of several formulations, either Bt strains or Pseudomonas fluorescens engineered to express CrylC protein as safe pesticides to be used in all agricultural crops including fruits, vegetables, corn, cotton and nuts; turf; forests; ornamentals; landscape trees; nursery crops. The long history of safe use of these formulations even in crops which are eaten raw like lettuce attest to the high degree of safety of the organism and the CrylC protein that is expressed in them. Many commercial products by some of the leading companies in the world like Abbot (Xentari, Florbac) and Novartis (Certan) contain the expressed CrylC protein and are in wide-spread use in many countries (2).

The gene coding for the CrylC family of proteins has been extensively studied. A search of the GENBANK database shows many sequences of this family of protein deposited. A non-exhaustive list is presented in table 1. In a very significant recent study (3), CrylC expressing broccoli plants were exposed to the laboratory bred resistant strains of diamond back moth, Plutella xylostella in order to understand the effect of the CrylC protein on the parasitoid, Diadegma insular e, which is a natural enemy of Plutella. The results of this study provide the first clear evidence of the lack of hazard to a parasitoid by a Bt plant, compared to traditional insecticides.

Gene Accession No. Reference

designation

CrylC al X 07518 4 CrylC a2 X 13620 5

CrylC a3 M 73251 6

CrylC a4 A 27642 7

CrylC a5 X 96682 8

CrylC bl M 97880 9

Table 1. Selection of CrylC gene sequences deposited in public databases.

An alternative to the use of biological control has been the implementation of transgenic technology. Crop species have been genetically modified to express different Cry genes to control insects. The earliest commercial disclosure of this technology has been Bollgard that expresses the Cry 1 Ac gene in cotton and this was developed by Monsanto (10). Bollgard was developed to resist the bollworm Helicoverpa armigera and commercial cultivation was practised in the United States in 1996. Subsequently, Bollgard II was developed by Monsanto that had the stacked combination of Cry 1 Ac and Cry2Ab and was put into commercial cultivation in 2000 to delay the onset of resistance to the Bt toxin by the pest (10, 11).

Studies on effects of two transgenic cotton lines carrying a CrylA gene on Spodoptera litura, were conducted during 2002-2005 in the cotton planting region of the Yangtze River valley of China. Results showed that the Spodoptera larvae had low susceptibility to Bt cotton (12). There was no significant difference in larval population densities in conventional and Bt cotton fields. However, the larval populations of the insect on conventional plants treated with chemical insecticides for control of target pest of Bt cotton were significantly lower than that in Bt cotton fields. These results indicated that Spodoptera litura had the potential to become a major and alarming pest in Bt cotton fields, and therefore efforts to develop an effective alternative management strategy was needed. The efficacy of CrylC in controlling insects that have developed resistance to CrylA toxins in cauliflower is reported (13).

CrylC was expressed in corn to harbour resistance to Spodoptera frugiperda (15). This was not a codon-optimized gene. Insect resistant transgenic indica rice has been reported where CrylC was expressed (16). Several patents exist for CrylC but the genes protected in these code for the protein found in Bacillus thuringiensis subspecies aizawai (17-26).

STATEMENT OF THE DISCLOSURE

Accordingly, the present disclosure relates to a nucleotide sequence set forth as SEQ ID No. 1 or a nucleotide sequence comprising sequence set forth as SEQ ID No.l; an expression cassette set forth as SEQ ID No.3, comprising CaMV 35S promoter, nucleotide sequence set forth as SEQ ID No. 1 or a nucleotide sequence comprising sequence set forth as SEQ ID No.l and 35 S 3' untranslated region; a vector comprising sequence set forth as SEQ ID No. 5, having an expression cassette as claimed above; a transformed host cell comprising the vector as claimed above; a method of obtaining a transformed host cell comprising an expression cassette set forth as SEQ ID No.3, said method comprising acts of: (a) inserting an expression cassette set forth as SEQ ID No.3 into a vector, and (b) transforming a host cell with said vector to obtain the transformed host cell; a transgenic cell comprising an expression cassette as claimed above; a method of obtaining a transgenic plant member of genus Gossypium, said method comprising acts of: (a) codon optimizing nucleotide sequence of native CrylC gene to obtain nucleotide sequence set forth as SEQ ID No.l, (b) inserting an expression cassette as claimed above into a vector and transforming a host cell with said vector to obtain a transformed host cell, and (c) infecting the plant with the transformed host cell followed by tissue culturing to obtain a transgenic plant member of genus Gossypium; a transgenic transformation event MLS9124 or MLS9878, said event comprising a nucleotide sequence set forth as SEQ ID No. 1 or a nucleotide sequence comprising sequence set forth as SEQ ID No. l in a plant member of genus Gossypium or any part thereof; a method of detecting presence of a trangene comprising an expression cassette set forth as SEQ ID No.3, in a transgenic plant member of genus Gossypium, said method comprising acts of: (a) extracting DNA from the plant source and performing nucleic acid amplification of junction regions of the transgene and the plant to obtain an amplicon, said amplification carried out by primers corresponding to the regions selected from a group comprising left border region of the transgene, right border region of the transgene, left border region of the plant DNA and right border region of the plant DNA or any combination thereof, and (b) detecting and analyzing the amplicon to detect the presence of said transgene in the transgenic plant; primers set forth as SEQ ID No. 1 1, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 15, SEQ ID No. 16, SEQ ID No. 19 and SEQ ID No. 20; and a method of codon optimizing CrylC gene in plant member of genus Gossypium, said method comprising acts of: (a) preparing a codon usage table, for plurality of constitutively expressed genes, on the basis of GC content, TA doublet avoidance in second and third positions of codons, transcription termination signals, splicing signals and polyadenylation signals, from conventionally known constitutively expressed genes in cotton, and (b) using said table for codon optimizing the CrylC gene.

BRIEF DESCRIPTION OF THE ACCOMPANYING FIGURES

In order that the disclosure may be readily understood and put into practical effect, reference will now be made to exemplary embodiments as illustrated with reference to the accompanying figures. The figure together with a detailed description below, are incorporated in and form part of the specification, and serve to further illustrate the embodiments and explain various principles and advantages, in accordance with the present disclosure where

Figure 1 illustrates pairwise alignment of the codon optimized Cr lCGh gene with the 1^st 1890 bp of the CrylC gene (4).

Figure 2 illustrates a map of the CrylCGh expression cassette in the transgenic cotton event MLS9124 and MLS9878.

Figure 3 illustrates a map of the binary vector pMH72.

Figure 4 illustrates a map of the plasmid pMH82 with T -DNA containing two Cry 1C expression cassettes repeated in tandem used for the generation of the cotton transgenic events MLS9124 and MLS9878.

Figure 5 illustrates southern hybridization of event MLS9124 digested with EcoRI and Hindlll.

Figure 6 illustrates southern hybridization of event MLS9878 digested with EcoRI and Hindlll.

Figure 7 illustrates a diagnostic PCR process for the amplification of the left border junction of transgenic cotton event MLS9878.

Figure 8 illustrates a diagnostic PCR process for the amplification of the right border junction of transgenic cotton event MLS9878.

Figure 9 illustrates a diagnostic PCR to locate the unique intermediate region between the two expression cassettes in MLS9124 and LS9878. Figure 10 illustrates a comparative efficacy of CrylC in transgenic cotton events MLS9124 and MLS9878 in Spodoptera litura (A) and Helicoverpa armigera (B). Figure 11 illustrates a diagnostic PCR process for the amplification of the right border junction of transgenic cotton event MLS9124.

DETAILED DESCRIPTION OF THE DISCLOSURE

The present disclosure relates to a nucleotide sequence set forth as SEQ ID No. 1 or a nucleotide sequence comprising sequence set forth as SEQ ID No. l .

The present disclosure further relates to an expression cassette set forth as SEQ ID No.3, comprising CaMV 35 S promoter, nucleotide sequence set forth as SEQ ID No. 1 or a nucleotide sequence comprising sequence set forth as SEQ ID No.l and 35S 3' untranslated region.

In an embodiment of the present disclosure, the nucleotide sequence codes for CrylC gene and corresponding amino acid sequence set forth as SEQ ID No. 2 codes for CrylC protein.

The present disclosure further relates to a vector comprising sequence set forth as SEQ ID No. 5, having an expression cassette as claimed above.

In an embodiment of the present disclosure, the vector is selected from a group comprising an expression vector, replicable vector, transformation vector, binary vector and shuttle vectors or any combination thereof.

In another embodiment of the present disclosure, the vector is preferably binary vector and wherein the expression cassette is either a single copy sequence or tandem repeat sequence separated by an intervening sequence set forth as SEQ ID No.6.

The present disclosure further relates to a transformed host cell comprising the vector as claimed above.

The present disclosure further relates to a method of obtaining a transformed host cell comprising an expression cassette set forth as SEQ ID No.3, said method comprising acts of: (a) inserting an expression cassette set forth as SEQ ID No.3 into a vector, and (b) transforming a host cell with said vector to obtain the transformed host cell. In an embodiment of the present disclosure, the expression cassette comprise CaMV 35 S promoter, nucleotide sequence set forth as SEQ ID No. 1 or a nucleotide sequence comprising sequence set forth as SEQ ID No. l and 35S 3' untranslated region.

In another embodiment of the present disclosure, the nucleotide sequence codes for CrylC gene and corresponding amino acid sequence set forth as SEQ ID No. 2 codes for CrylC protein.

In yet another embodiment of the present disclosure, the host cell is Agrobacterium.

In still another embodiment of the present disclosure, the transforming of the host cell is carried out using techniques selected from a group comprising electroporation, tri- parental mating, microinjection, genegun method, PEG mediated transfer, Calcium phosphate method, liposome mediated transfer and or any combination thereof.

The present disclosure further relates to a transgenic cell comprising an expression cassette as claimed above.

In an embodiment of the present disclosure, the transgenic cell is a transgenic plant cell exhibiting resistance to lepidopteran insect(s) or pest(s) infestation; and wherein the plant is a member of genus Gossypium.

The present disclosure further relates to a method of obtaining a transgenic plant member of genus Gossypium, said method comprising acts of: (a) codon optimizing nucleotide sequence of native CrylC gene to obtain nucleotide sequence set forth as SEQ ID No.l, (b) inserting an expression cassette as claimed above into a vector and transforming a host cell with said vector to obtain a transformed host cell, and (c) infecting the plant with the transformed host cell followed by tissue culturing to obtain a transgenic plant member of genus Gossypium. In an embodiment of the present disclosure, the expression cassette is either a single copy sequence or tandem repeat sequence separated by an intervening sequence set forth as SEQ ID No.6.

In another embodiment of the present disclosure, the host cell is Agrobacterium.

In yet another embodiment of the present disclosure, transforming the host cell is carried out using techniques selected from a group comprising electroporation, tri- parental mating, microinjection, genegun method, PEG mediated transfer, Calcium phosphate method and liposome mediated transfer or any combination thereof.

In still another embodiment of the present disclosure, the infection and tissue culturing comprises incubating cotton leaf with the transformed host cell; transferring and incubating the cotton leaf in bacterial selection medium followed by incubation in plant selection medium; transferring to embryogenesis medium till the occurance of embryogenesis followed by sub-culturing; transferring to suspension medium followed by germination medium; transfering to basal medium after the growth of leaves and root hairs; hardening followed by transfer to soil and greenhouse to obtain the said transgenic plant.

The present disclosure further relates to a transgenic transformation event MLS9124 or MLS9878, said event comprising a nucleotide sequence set forth as SEQ ID No. 1 or a nucleotide sequence comprising sequence set forth as SEQ ID No. l in a plant member of genus Gossypium or any part thereof.

The present disclosure further relates to a method of detecting presence of a trangene comprising an expression cassette set forth as SEQ ID No.3, in a transgenic plant member of genus Gossypium, said method comprising acts of: (a) extracting DNA from the plant source and performing nucleic acid amplification of junction regions of the transgene and the plant to obtain an amplicon, said amplification carried out by primers corresponding to the regions selected from a group comprising left border region of the transgene, right border region of the transgene, left border region of the plant DNA and right border region of the plant DNA or any combination thereof, and (b) detecting and analyzing the amplicon to detect the presence of said transgene in the transgenic plant. In an embodiment of the present disclosure, the primers are selected from a group comprising SEQ ID No. 1 1 corresponding to the left border region of the transgene , SEQ ID No. 13 corresponding to the right border region of the transgene, SEQ ID No. 12 correspdonding to the left border region of the plant DNA and SEQ ID No. 14 corresponding to the right border region of the plant DNA or any combination thereof.

The present disclosure further relates to primers set forth as SEQ ID No. 1 1 , SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 15, SEQ ID No. 16, SEQ ID No. 19 and SEQ ID No. 20.

In an embodiment of the present disclosure, the primers having SEQ ID Nos. 12, 13, 15 and 19 are sense primers and the primers having SEQ ID Nos. 1 1 , 14, 16 and 20 are anti-sense primers.

The present disclosure further relates to a method of codon optimizing Cry l C gene in plant member of genus Gossypium, said method comprising acts of: (a) preparing a codon usage table, for plurality of constitutively expressed genes, on the basis of GC content, TA doublet avoidance in second and third positions of codons, transcription termination signals, splicing signals and polyadenylation signals, from conventionally known constitutively expressed genes in cotton, and (b) using said table for codon optimizing the CrylC gene.

In an embodiment of the present disclosure, the nucleotide sequence is codon optimized for plants of genus Gossypium.

A codon optimized gene is necessary to code for the Cryl C protein that would confer resistance to S. litura in cotton, tobacco, tomato, cabbage, cauliflower, broccoli, rice and maize. Another object of the disclosure is to incorporate it in a stack with other Bt genes to delay the onset of breakdown of resistance to pests. Cry l C is also responsible to confer resistance to the crop plants to other lepidopteran pests. In an embodiment of the present disclosure, the cotton plant is selected from a group comprising Gossypium hirsutum, Gossypium barbadense, Gossypium arboretum and Gossypium herbaceum. Preferably, the cotton plant is Gossypium hirsutum.

CrylC is a potent insecticidal protein found in several subspecies of B. thuringiensis that is effective against a wide range of lepidopteran pests of crops. The range of the crops is very diverse and includes, but is not restricted to, cotton, tobacco, tomato, cauliflower, broccoli, rice and maize. Besides conferring resistance to lepidopteran pests that are not affected by the Cryl A series of insecticidal proteins, this gene, when stacked with any CrylA protein also helps the delay of onset of resistance to CrylA group of proteins expressed in transgenic crops in the target pests.

This disclosure relates to two transgenic cotton events that carry a unique CrylC protein gene that is optimized based on the codon usage for the most constitutively expressed proteins in cotton. This gene, when expressed in tobacco and cotton, demonstrates efficacy against major insect pests hitherto uncontrolled by any CrylA protein.

The N-terminal 630 amino acid sequence of said CrylC gene was codon optimized using a codon usage table for highly constitutively expressed genes based on the codon preference for cotton (27).

The Pairwise alignment of the codon optimized CrylCGh gene with the 1^st 1890 bp of the CrylC gene is provided in figure 1.

Based on the codon preference for cotton (27), a table was created for the most constitutively expressed genes (Table 2):

Codon Gh Actin Gh Rubisco Gh Tubulin Gh Ribosomal Proteins

Frequency Frequency Frequency Frequency per 1000 per 1000 per 1000 per 1000 aa Probability aa Probability aa Probabilit y aa Probability

CGA 1 0.06 5 0.12 1 0.03 0 0.00

CGC 1 0.06 1 0.02 10 .31 0 0.00

CGG 0 0.00 1 0.02 0 0.00 1 0.17

Table: 2 - Codon Optimization usage table.

The designed sequence of the gene is given below as SEQ ID No. 1. The gene was designed using the N-terminal 630 amino acids [SEQ ID No. 2] of the CrylC protein from Bacillus thuringiensis subspecies entomocidus (4, 30), unlike the sequences cited in references 17 - 26, which are for B. thuringiensis subspecies aizawai. Factors that were taken into consideration while designing the gene were GC content, TA doublet avoidance in the second and third positions of codons, transcription termination signal (AGTNNAA), splicing signals (5' splice junction: CRG/GTAAGT; 3' junction: YNAG/N) and polyadenylation signals (AATAAA, AATGAA, AATAAT, GATAAA, AATAAG, AATATT). The gene thus obtained was cloned in a binary vector pMH72 (figure 3, SEQ ID No. 4) which was developed to express the nptll gene for plant selection from pGA643 (28) in pPZP201 (29). The gene was cloned to be driven by the cauliflower mosaic virus (CaMV) double 35 S promoter (D35SP) and the 35 S 3' UTR was used for transcriptional termination. The resultant binary plasmid pMH82 as described in figure 4 and its T-DNA sequence [given in SEQ ID No. 5], was mobilized into Agrobacterium tumefaciens EHA105. The mobilization of pMH82 into Agrobacterium tumefaciens EHA105 can be carried out using various conventionally known techniques of gene transfer which include microinjection, electroporation, tri- parental mating, genegun method, PEG mediated transfer, Calcium phosphate method, liposome mediated transfer. In an embodiment of the present disclosure, electroporation method is used eherein, electrocompetent Agrobacterium tumefaciens cells are prepared and frozen in glycerol at -70°C. The cells are thawed on ice prior to electroporation. Approximately, 50 ng of plasmid DNA is mixed with the electrocompetent cells and transferred to a pre-chilled cuvette. A pulse of 1.8 kV/zero millisecond is applied (feature Eel on BioRad MicroPulser Electroporator). The cells are recovered in 1 ml of Luria Bertani Medium for 45 minutes by shaking at 200 rpm at 26°C. The recovered culture is further diluted 25 fold and plated on a minimal medium plated with kanamycin. This plate is incubated at 26°C for 2 days. The transformed cells are selected and used further.

Hypocotyl bits of cotton variety Coker are infected with the Agrobacterium cultures harbouring theplasmid pMH82. Embryogenic calli are obtained and regenerated following which regenerants are assayed for resistance to S. litura, Earias vitella and Helicoverpa armigera.

The uniqueness of the construct in pMH82 arises from 2 aspects, viz., the presence of dual expression cassettes [each expression cassette set forth as SEQ ID No. 3] and a unique 57 bp region in between the two expression cassettes, as given in SEQ ID No. 6.

Map of the CrylCGh expression cassette in the transgenic cotton event MLS9124 and MLS9878 is provided in figure 2. A Flow chart depicting the course of events for obtaining transgenic cotton plants is provided as below:

Hypocotyl segments of cotton or tobacco leaf discs

i

Incubate with Agrobacterium culture in dark (36 - 48 hours)

Wash with sterile water

Transfer to bacterial selection medium, keep in dark for 5 days i

Transfer to plant selection medium, changing medium periodically every 15 days.

Carry this out for at least 3 passages. i

Transfer to an embryogenesis medium till embryogenesis occurs, sub culturing every

4-6 weeks i

After embryogenesis transfer to suspension medium i

Transfer slightly elongated embryos to a charcoal medium

Transfer to germination medium in vermiculite first in diffused light and then to light

When true leaves and root hairs appear transfer to a basal medium i

Harden the plants in coco peat cum perlite i

Transfer to soil and into the greenhouse

Two independent transgenic cotton events, viz., MLS9124 and MLS9878, were further studied by Southern hybridization (31), analysis of the site of integration using TAIL-PCR (32), ELISA for studying levels of protein expression and bioassays by challenging the events with Spodoptera litura.

For Southern hybridization, 20 μg genomic DNA was digested with either Hindlll or EcoRI overnight and separated on a 0.8% agarose gel in TBE. The gel was transferred to Nylon membrane by downward capillary transfer and hybridized to either a 2.317 kb nptll probe [SEQ ID No. 7] or a 1.954 kb CrylCGh probe[SEQ ID No. 8]. The probes were labeled with a-³²P dCTP using a randomly primed extension with Klenow and hybridized overnight with formamide. Washed gels were exposed to Kodak X-OMAT films.

Southern Hybridization

Southern hybridization of event MLS9124 digested with EcoRI and Hindlll (figure 5); comprises:

(a) Hybridization with probe I (Nptll probe: EcoRI-Hindlll fragment from pMH82-2317 bp)

(b) Hybridization with probe II (CrylCGh probe: Xhol fragment from pMH82- 1954 bp)

Lane Sample Panel A (kb) Panel B (kb) No.

1 9124 Plant #1 Hindlll digest >2.4 kb 2.979kb, 5.729kb

2 9124 Plant #1 EcoRI digest 8.039kb 2.979kb, 8.039kb

3 9124 Plant #5 Hindlll digest >2.4 kb 2.979kb, 5.729kb 4 9124 Plant #5 EcoRI digest 8.039kb 2.979kb, 8.039kb

Table: 3 - Locations of the reference sizes of the λ DNA digested with Hindlll are given between the two panels [event MLS9124]

Southern hybridization of event MLS9878 digested with EcoRI and Hindlll (Figure 6); comprises:

(a) Hybridization with probe I (Nptll probe: EcoRI-Hindlll fragment from pMH82-2317 bp)

(b) Hybridization with probe II (CrylCGh probe: Xhol fragment from pMH82- 1954 bp)

Expectations:

Table: 4 - Expected locations of the reference sizes of the λ DNA digested with Hindlll are given between the two panels [event MLS9878]

The results are as illustrated in Figure 6, wherein the legends for both panels are provided as below:

Table: 5 - Data of samples loaded in the gel (figure 6) The two transgenic events were further analyzed by PCR for the presence of the unique region as described in SEQ ID No. 6 between the 2 expression cassettes. The primer sequences are described in SEQ ID No. 15 (forward primer) and SEQ ID No. 16 (reverse primer). The expected amplicon size was 975 bp.

Diagrammatic representation of diagnostic PCR process for the amplification of the left and right border junction of the transferred DNA [T-DNA] within the transgenic cotton events MLS9878 are illustrated in figures 7 and 8 respectively.

Transgenic cotton events not harbouring the T-DNA of the plasmid pMH82 were also subjected to this PCR and these samples did not amplify.

The results are as illustrated in Figure 9, wherein the legend for the gel loaded with different samples is provided as below:

Table: 6 - Data of samples loaded in the gel (figure 9)

The 975 bp amplicon from both the events MLS9124 and MLS9878 is sequenced and is set forth as SEQ ID NO. 17 which shows the presence of the unique 57 bp region described in SEQ ID No. 6.

Junction Analysis

In an embodiment of the present disclosure, junction analysis studies for both the transgenic cotton events are carried out at a lab scale. The left and right border junction sequence of the transgenic cotton event MLS9878 are set forth as SEQ ID Nos. 9 and 10, respectively.

T-DNA specific primer amplifying the left border junction sequence of the transgenic cotton events MLS9878 is set forth as SEQ ID No. 11, whereas the T-DNA specific primer amplifying the right border junction sequence of the transgenic cotton events MLS9878 is set forth as SEQ ID No. 13.

The corresponding cotton genome specific primer amplifying the left border junction sequence of the transgenic cotton event MLS9878 is set forth as SEQ ID No. 12, whereas the cotton genome specific primer amplifying the right border junction sequence of the transgenic cotton event MLS9878 is set forth as SEQ ID No. 14.

The right border junction sequence of the transgenic cotton event MLS9124 is set forth as SEQ ID No. 18. The corresponding T-DNA specific primer amplifying the right border junction sequence of the transgenic cotton events MLS9124 is set forth as SEQ ID No. 19 and the cotton genome specific primer amplifying the right border junction sequence of the transgenic cotton event MLS9124 is set forth as SEQ ID No. 20.

Diagrammatic representation of diagnostic PCR process for the amplification of the right border junction of transgenic cotton events MLS9124 is illustrated in figure 11, wherein the legend for the gel loaded with different samples is provided as below:

Spodoptera litura and Helicoverpa armigera Bioassays: Transgenic cotton events MLS9124 and MLS9878 were assayed for resistance to S. litura and Helicoverpa armigera up to the T4 generation and mortality up to 100% was observed to be carried through generations.

First fully expanded terminal leaves from unsprayed plots expressing various transgenes are bioassayed for bioactivity against Helicoverpa armigera and Spodoptera litura larvae. These bioassays were carried out at time intervals of 64, 78, 92, 106, 120 and 134 days after planting, in Non transgenic event (control), Event MLS9124 and Event MLS9878.

Length of the sampled leaves is measured approximately 5 cm across and is removed with as much petiole attached as possible. Leaves from each replicate are kept separate in labeled paper sacks and placed in plastic cooler containing cooling packs for transport to the laboratory.

Individual leaves are placed in a Petri dish having wet whatman filter paper and infested with a single day old fed larva (10 dishes/plot) for a total of 40 larvae/trait. Bioassays are kept at a constant temperature (25-30°C and relative humidity greater than 50 %) prior to assessment. After five days after exposure (DAE), larvae are prodded with a camel-hair brush and considered alive if coordinated movement is observed. Larvae from each replicate are scored according to the criteria in Table 8:

Table 8: Scoring criteria for bioassays

L0 - Dead larvae

LI - larvae with morbid growth

L2 - approximately 50% growth of L3

L3 - identical to control larvae (normal and healthy) Percent survival and development of larvae is analyzed using analysis of variance (ANOVA). Insect bioassay results of the two transgenic cotton events MLS9124 and MLS9878 for efficacy against Spodoptera litura are summarized in Figure 10 (A) and 10 (B). The data is captured at different stages of the growth cycle of the transgenic cotton events. In an embodiment of the present disclosure, the bar diagram for each event per insect has one bar for the average worm size. This is calculated as follows: Each category of survival score is assigned a factor which is designated as- Ll - 0.5

L2 - 1

L3 - 2

Further, (number of LI x 0.5) + (number of L2 x 1) + (number of L3 x 2) = total worm size.

Total worm size / Total number of survived larvae = Average worm size

In an embodiment, the total number of survived larvae is 40, wherein LI is 20, L2 is 11 and L3 is 9.

Therefore the total worm size is (20 X 0.5)+(l 1X1)+(9X2) = 39 and the average worm size is-

Total worm size /Total number of survived larvae= 39/40 = 0.975.

In an exemplary embodiment, the average worm size for constructing a bar diagram is calculated as a percentage of the L3 factor, i.e., 2. Therefore, if the value of average worm size is found to be 0.975, it is represented in the bar diagram as (0.975/2) x 100 = 48.75%.

Earias vitella Bioassays:

Squares (flower buds) of approximately 4-6 mm thickness from unsprayed plots were bioassayed for activity against Earias vittella larvae. Sampled squares are removed with as much petiole attached as possible. Squares from each replicate are kept separate in labeled paper sacks and placed in plastic cooler containing cooling packs for transport to the laboratory.

Individual squares are placed into a Petri dish having wet whatman filter paper and infested with a two day old fed larva for a total of 40 larvae/trait. Bioassays are kept at a constant temperature (25-30°C and relative humidity greater than 50%) prior to assessment. After five days after exposure (DAE), larvae are prodded with a camel- hair brush and considered alive if coordinated movement is observed.

Insect bioassay results of the two transgenic cotton events MLS9124 and MLS9878 for efficacy against Earias vitella are summarized in Tables 9 and 10 respectively.

Table-9: Earias vitella bioassay with respect to cotton event MLS9124

Table-10: Earias vitella bioassay with respect to cotton event MLS9878

Protein expression data:

The expression of the CrylC protein as estimated by quantitative ELISA was in the range 0.41 to 4.67 μg/gm fresh weight of leaf. This is explained in Table 11 below.

MLS9124 MLS9878

Average Range Average Range

μg protein per gram fresh weight

Leaf 1.41 1.00 - 1.88 0.41 - 1.93 4.67

Seed - 9.4 -

Table: 11 - ELISA protein expression data.

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11. Gore et al (2001) J. Econ. Entomol, 94, 1445-1451.

12. Wan et al (2008) Environ. Entomol, 37: 1043-1048.

13. Cao et al (2003) J. New Seeds, 5: 193-207.

14. Strizhov, et al (1996) Proc. Natl. Acad. Sci. USA, 93: 15012-15017.

15. Valicenta et al (2005) Proceedings of the 6th Pacific Rim Conference on the Biotechnology of Bacillus thuringiensis and its Environmental Impact (Victoria BC, 2005)

16. Tang et al (2006) Mol. Breeding, 18: 1-10.

17. US Patent 5914318

18. US Patent 5942664

19. US Patent 6033874

20. US Patent 6153814

21. US Patent 6177615

22. US Patent 6313378

23. US Patent 6423828

24. US Patent 6809078

25. US Patent 6825006

26. US Patent 7256017

27. Codon Usage Database (http://www.kazusa.or.jp/codon/) 28. An, G. (1986) Plant Physiol, 81 :86-91.

29. Hajdukiewicz, P., Svab, Z., Maliga, P. (1994) Plant Mol. Biol, 25:989-994.

30. Genbank accession number CAA30396.

31. Sambrook, J., Russell, D.W. (2001) Molecular Cloning - a laboratory manual.

3^rd Edition. CSHL Press, Cold Spring Harbor, New York, USA.

32. Sessions et al (2002) Plant Cell, 14:2985-2994.

SEQUENCE LISTING

<110> METAHELIX LIFESCIENCES PRIVATE LIMITED

<120> A NUCLEOTIDE SEQUENCE, EXPRESSION CASSETTE TRANSGENIC EVENTS,

CELLS AND METHODS THEREOF

<130> IP16850

<160> 20

<170> Patentln version 3 . 5

<210> 1

<211> 1893

<212> DNA

<213> Bacillus thuringiensis

<220>

<221> gene

<222> (1) . . (1893)

<220>

<221> gene

<222> (1) . . (1893)

<223> Synthetic Sequence

<400> 1

atggaagaga acaatcaaaa ccagtgcatt ccatacaact gcctcagtaa tcctgaagag 60 gttcttctcg atggtgaaag gatctccacc ggaaacagtt ccattgacat tagtctttcc 120 ctcgtgcaat tccttgttag taacttcgtc cccggtggtg gattcctcgt tggtcttatc 180 gattttgtgt ggggtattgt tggaccatcc cagtgggatg ctttcctcgt ccaaattgag 240 cagcttatca atgagcgtat tgccgagttc gctaggaacg ctgccattgc taacctcgaa 300 ggtcttggta ataacttcaa catctatgtt gaggctttta aggaatggga ggaagaccct 360 aataaccccg agactcgtac cagggtgatt gatcgtttca ggattctcga tggacttctc 420 gaacgtgaca tcccaagttt caggatttcc ggtttcgagg ttcctcttct cagtgtctac 480 gcccaagctg ctaaccttca cctcgccatt cttcgtgatt ccgttatctt tggtgaaagg 540 tggggactca ccactattaa tgtgaacgag aactataatc gtcttattag gcacatcgat 600 gaatacgctg accactgcgc taacacctat aaccgtggtc tcaataacct tcccaaaagt 660 acctaccagg attggattac ttataacagg ctccgtaggg atcttaccct caccgttctt 720 gacattgccg ctttcttccc aaattacgat aaccgtaggt atcctatcca acccgtcggt 780 cagctcactc gtgaggttta caccgatcca cttattaact tcaatcctca actccagtcc 840 gtggctcaac ttcccacctt taacgttatg gaaagttcca ggattcgtaa cccacacctc 900 ttcgacatcc ttaataacct cactattttc accgattggt tcagtgtcgg aaggaacttt 960 tattggggtg gtcaccgtgt tatttccagt cttatcggag gtggtaacat tacctcccct 1020 atctacggaa gggaggccaa ccaggaaccc ccacgtagtt tcactttcaa cggtcctgtg 1080 ttcaggaccc tctccaatcc cacccttcgt ctccttcaac agccttggcc tgctccccca 1140 tttaacctca ggggtgttga gggagtcgag ttcagtactc ctaccaactc cttcacctat 1200 cgtggtaggg gtactgttga tagtcttacc gagctccccc cagaagacaa ttccgtgcct 1260 ccccgtgagg gatacagtca caggctttgc cacgctacct tcgttcaacg ttccggtact 1320 ccatttctca ccaccggtgt cgttttcagt tggactgata ggtccgccac ccttaccaac 1380 actatcgatc ctgaacgtat taaccagatt cccctcgtga agggattcag ggtttggggt 1440 ggtaccagtg tcatcaccgg accaggtttc actggtggag acattcttcg taggaatacc 1500 tttggtgatt tcgtttccct ccaagtgaac attaacagtc ctatcaccca gcgttatagg 1560 cttcgtttca ggtacgcttc cagtcgtgat gctagggtta ttgtcctcac tggtgccgct 1620 tccaccggag ttggtggtca agtgagtgtt aatatgcccc ttcagaagac tatggagatt 1680 ggagaaaacc tcacttcccg taccttcagg tataccgact ttagtaaccc attctccttc 1740 cgtgctaatc ctgatatcat tggtattagt gagcaacccc ttttcggtgc cggatccatc 1800 agttccggtg aactctacat tgataagatt gagatcattc ttgctgacgc tacttttgaa 1860 gccgagagtg atctcgaaag ggctcagaag tga 1893

<210> 2

<211> 630

<212> PRT

<213> Bacillus thuringiensis <220>

<221> PEPTIDE

<222> (1) . . (630)

<400> 2

Met G^~lu G^~lu Asn Asn Gin Asn Gin Cys lie Pro Tyr Asn Cys Leu Ser

1 5 10 15

Asn Pro Glu Glu val Leu Leu Asp Gly Glu Arg lie Ser Thr Gly Asn

20 25 30

Ser Ser lie Asp lie Ser Leu Ser Leu val Gin Phe Leu val Ser Asn

35 40 45

Phe val Pro Gly Gly Gly Phe Leu val Gly Leu lie Asp Phe val Trp

50 55 60

Gly lie val Gly Pro Ser Gin Trp Asp Ala Phe Leu val Gin lie Glu

65 70 75 80

Gin Leu lie Asn Glu Arg lie Ala Glu Phe Ala Arg Asn Ala Ala lie

85 90 95

Ala Asn Leu Glu Gly Leu Gly Asn Asn Phe Asn lie Tyr val Glu Ala

100 105 110

Phe Lys Glu Trp Glu Glu Asp Pro Asn Asn Pro Glu Thr Arg Thr Arg

115 120 125 val lie Asp Arg Phe Arg lie Leu Asp Gly Leu Leu Glu Arg Asp lie

130 135 140 Pro Ser Phe Arg lie Ser Gly Phe Glu val Pro Leu Leu Ser val Tyr 145 150 155 160

Ala Gin Ala Ala Asn Leu His Leu Ala lie Leu Arg Asp Ser val lie

165 170 175

Phe Gly Glu Arg Trp Gly Leu Thr Thr lie Asn val Asn Glu Asn Tyr

180 185 190

Asn Arg Leu lie Arg His lie Asp Glu Tyr Ala Asp His Cys Ala Asn

195 200 205

Thr Tyr Asn Arg Gly Leu Asn Asn Leu Pro Lys Ser Thr Tyr Gin Asp

210 215 220

Trp lie Thr Tyr Asn Arg Leu Arg Arg Asp Leu Thr Leu Thr val Leu 225 230 235 240

Asp lie Ala Ala Phe Phe Pro Asn Tyr Asp Asn Arg Arg Tyr Pro lie

245 250 255

Gin Pro val Gly Gin Leu Thr Arg Glu val Tyr Thr Asp Pro Leu lie

260 265 270

Asn Phe Asn Pro Gin Leu Gin Ser val Ala Gin Leu Pro Thr Phe Asn

275 280 285 val Met Glu Ser Ser Arg lie Arg Asn Pro His Leu Phe Asp lie Leu

290 295 300

Asn Asn Leu Thr lie Phe Thr Asp Trp Phe Ser val Gly Arg Asn Phe 305 310 315 320

Tyr Trp Gly Gly His Arg val lie Ser Ser Leu lie Gly Gly Gly Asn

325 330 335 lie Thr Ser Pro lie Tyr Gly Arg Glu Ala Asn Gin Glu Pro Pro Arg

340 345 350

Ser Phe Thr Phe Asn Gly Pro val Phe Arg Thr Leu Ser Asn Pro Thr

355 360 365

Leu Arg Leu Leu Gin Gin Pro Trp Pro Ala Pro Pro Phe Asn Leu Arg

370 375 380

Gly val Glu Gly val Glu Phe Ser Thr Pro Thr Asn Ser Phe Thr Tyr

385 390 395 400

Arg Gly Arg Gly Thr val Asp Ser Leu Thr Glu Leu Pro Pro Glu Asp

405 410 415

Asn Ser val Pro Pro Arg Glu Gly Tyr Ser His Arg Leu Cys His Ala

420 425 430

Thr Phe val Gin Arg Ser Gly Thr Pro Phe Leu Thr Thr Gly val val

435 440 445 Phe Ser Trp Thr Asp Arg Ser Ala Thr Leu Thr Asn Thr lie Asp Pro

450 455 460

Glu Arg lie Asn Gin lie Pro Leu val Lys Gly Phe Arg val Trp Gly

465 470 475 480

Gly Thr Ser val lie Thr Gly Pro Gly Phe Thr Gly Gly Asp lie Leu

485 490 495

Arg Arg Asn Thr Phe Gly Asp Phe val Ser Leu Gin val Asn lie Asn

500 505 510

Ser Pro lie Thr Gin Arg Tyr Arg Leu Arg Phe Arg Tyr Ala Ser Ser

515 520 525

Arg Asp Ala Arg val lie val Leu Thr Gly Ala Ala Ser Thr Gly val

530 535 540

Gly Gly Gin val Ser val Asn Met Pro Leu Gin Lys Thr Met Glu lie

545 550 555 560

Gly Glu Asn Leu Thr Ser Arg Thr Phe Arg Tyr Thr Asp Phe Ser Asn

565 570 575

Pro Phe Ser Phe Arg Ala Asn Pro Asp lie lie Gly lie Ser Glu Gin

580 585 590

Pro Leu Phe Gly Ala Gly Ser lie Ser Ser Gly Glu Leu Tyr lie Asp

595 600 605

Lys lie Glu lie lie Leu Ala Asp Ala Thr Phe Glu Ala Glu Ser Asp

610 615 620

Leu Glu Arg Ala Gin Lys

625 630

<210> 3

<211> 2926

<212> DNA

<213> Bacillus thuringiensis

<220>

<221> gene

<222> (1) . . (2926)

<223> Synthetic Sequence

<400> 3

atggtggagc acgacactct cgtctactcc aagaatatca aagatacagt ctcagaagac 60 caaagggcta ttgagacttt tcaacaaagg gtaatatcgg gaaacctcct cggattccat 120 tgcccagcta tctgtcactt catcaaaagg acagtagaaa aggaaggtgg cacctacaaa 180 tgccatcatt gcgataaagg aaaggctatc gttcaagatg cctctgccga cagtggtccc 240 aaagatggac ccccacccac gaggagcatc gtggaaaaag aagacgttcc aaccacgtct 300 tcaaagcaag tggattgatg tgataacatg gtggagcacg acactctcgt ctactccaag 360 aatatcaaag atacagtctc agaagaccaa agggctattg agacttttca acaaagggta 420 atatcgggaa acctcctcgg attccattgc ccagctatct gtcacttcat caaaaggaca 480 gtagaaaagg aaggtggcac ctacaaatgc catcattgcg ataaaggaaa ggctatcgtt 540 caagatgcct ctgccgacag tggtcccaaa gatggacccc cacccacgag gagcatcgtg 600 gaaaaagaag acgttccaac cacgtcttca aagcaagtgg attgatgtga tatctccact 660 gacgtaaggg atgacgcaca atcccactat ccttcgcaag accttcctct atataaggaa 720 gttcatttca tttggagagg acacgctgaa atcaccagtc tctctctaca aatctatctc 780 tctcgagatc tatcgatgca tgccatggaa gagaacaatc aaaaccagtg cattccatac 840 aactgcctca gtaatcctga agaggttctt ctcgatggtg aaaggatctc caccggaaac 900 agttccattg acattagtct ttccctcgtg caattccttg ttagtaactt cgtccccggt 960 ggtggattcc tcgttggtct tatcgatttt gtgtggggta ttgttggacc atcccagtgg 1020 gatgctttcc tcgtccaaat tgagcagctt atcaatgagc gtattgccga gttcgctagg 1080 aacgctgcca ttgctaacct cgaaggtctt ggtaataact tcaacatcta tgttgaggct 1140 tttaaggaat gggaggaaga ccctaataac cccgagactc gtaccagggt gattgatcgt 1200 ttcaggattc tcgatggact tctcgaacgt gacatcccaa gtttcaggat ttccggtttc 1260 gaggttcctc ttctcagtgt ctacgcccaa gctgctaacc ttcacctcgc cattcttcgt 1320 gattccgtta tctttggtga aaggtgggga ctcaccacta ttaatgtgaa cgagaactat 1380 aatcgtctta ttaggcacat cgatgaatac gctgaccact gcgctaacac ctataaccgt 1440 ggtctcaata accttcccaa aagtacctac caggattgga ttacttataa caggctccgt 1500 agggatctta ccctcaccgt tcttgacatt gccgctttct tcccaaatta cgataaccgt 1560 aggtatccta tccaacccgt cggtcagctc actcgtgagg tttacaccga tccacttatt 1620 aacttcaatc ctcaactcca gtccgtggct caacttccca cctttaacgt tatggaaagt 1680 tccaggattc gtaacccaca cctcttcgac atccttaata acctcactat tttcaccgat 1740 tggttcagtg tcggaaggaa cttttattgg ggtggtcacc gtgttatttc cagtcttatc 1800 ggaggtggta acattacctc ccctatctac ggaagggagg ccaaccagga acccccacgt 1860 agtttcactt tcaacggtcc tgtgttcagg accctctcca atcccaccct tcgtctcctt 1920 caacagcctt ggcctgctcc cccatttaac ctcaggggtg ttgagggagt cgagttcagt 1980 actcctacca actccttcac ctatcgtggt aggggtactg ttgatagtct taccgagctc 2040 cccccagaag acaattccgt gcctccccgt gagggataca gtcacaggct ttgccacgct 2100 accttcgttc aacgttccgg tactccattt ctcaccaccg gtgtcgtttt cagttggact 2160 gataggtccg ccacccttac caacactatc gatcctgaac gtattaacca gattcccctc 2220 gtgaagggat tcagggtttg gggtggtacc agtgtcatca ccggaccagg tttcactggt 2280 ggagacattc ttcgtaggaa tacctttggt gatttcgttt ccctccaagt gaacattaac 2340 agtcctatca cccagcgtta taggcttcgt ttcaggtacg cttccagtcg tgatgctagg 2400 gttattgtcc tcactggtgc cgcttccacc ggagttggtg gtcaagtgag tgttaatatg 2460 ccccttcaga agactatgga gattggagaa aacctcactt cccgtacctt caggtatacc 2520 gactttagta acccattctc cttccgtgct aatcctgata tcattggtat tagtgagcaa 2580 ccccttttcg gtgccggatc catcagttcc ggtgaactct acattgataa gattgagatc 2640 attcttgctg acgctacttt tgaagccgag agtgatctcg aaagggctca gaagtgaccc 2700 gggagctcga attcgaagct tctgcagacg cgtccctcga gtttctccat aataatgtgt 2760 gagtagttcc cagataaggg aattagggtt cctatagggt ttcgctcatg tgttgagcat 2820 ataagaaacc cttagtatgt atttgtattt gtaaaatact tctatcaata aaatttctaa 2880 ttcctaaaac caaaatccag tactaaaatc cagatccccc gaatta 2926

<210> 4

<211> 9449

<212> DNA

<213> Bacillus thuringiensis

<220>

<221> gene

<222> (1) . . (9449)

<223> Synthetic Sequence

<400> 4

agtactttga tccaacccct ccgctgctat agtgcagtcg gcttctgacg ttcagtgcag 60 ccgtcttctg aaaacgacat gtcgcacaag tcctaagtta cgcgacaggc tgccgccctg 120 cccttttcct ggcgttttct tgtcgcgtgt tttagtcgca taaagtagaa tacttgcgac 180 tagaaccgga gacattacgc catgaacaag agcgccgccg ctggcctgct gggctatgcc 240 cgcgtcagca ccgacgacca ggacttgacc aaccaacggg ccgaactgca cgcggccggc 300 tgcaccaagc tgttttccga gaagatcacc ggcaccaggc gcgaccgccc ggagctggcc 360 aggatgcttg accacctacg ccctggcgac gttgtgacag tgaccaggct agaccgcctg 420 gcccgcagca cccgcgacct actggacatt gccgagcgca tccaggaggc cggcgcgggc 480 ctgcgtagcc tggcagagcc gtgggccgac accaccacgc cggccggccg catggtgttg 540 accgtgttcg ccggcattgc cgagttcgag cgttccctaa tcatcgaccg cacccggagc 600 gggcgcgagg ccgccaaggc ccgaggcgtg aagtttggcc cccgccctac cctcaccccg 660 gcacagatcg cgcacgcccg cgagctgatc gaccaggaag gccgcaccgt gaaagaggcg 720 gctgcactgc ttggcgtgca tcgctcgacc ctgtaccgcg cacttgagcg cagcgaggaa 780 gtgacgccca ccgaggccag gcggcgcggt gccttccgtg aggacgcatt gaccgaggcc 840 gacgccctgg cggccgccga gaatgaacgc caagaggaac aagcatgaaa ccgcaccagg 900 acggccagga cgaaccgttt ttcattaccg aagagatcga ggcggagatg atcgcggccg 960 ggtacgtgtt cgagccgccc gcgcacgtct caaccgtgcg gctgcatgaa atcctggccg 1020 gtttgtctga tgccaagctg gcggcctggc cggccagctt ggccgctgaa gaaaccgagc 1080 gccgccgtct aaaaaggtga tgtgtatttg agtaaaacag cttgcgtcat gcggtcgctg 1140 cgtatatgat gcgatgagta aataaacaaa tacgcaaggg gaacgcatga aggttatcgc 1200 tgtacttaac cagaaaggcg ggtcaggcaa gacgaccatc gcaacccatc tagcccgcgc 1260 cctgcaactc gccggggccg atgttctgtt agtcgattcc gatccccagg gcagtgcccg 1320 cgattgggcg gccgtgcggg aagatcaacc gctaaccgtt gtcggcatcg accgcccgac 1380 gattgaccgc gacgtgaagg ccatcggccg gcgcgacttc gtagtgatcg acggagcgcc 1440 ccaggcggcg gacttggctg tgtccgcgat caaggcagcc gacttcgtgc tgattccggt 1500 gcagccaagc ccttacgaca tatgggccac cgccgacctg gtggagctgg ttaagcagcg 1560 cattgaggtc acggatggaa ggctacaagc ggcctttgtc gtgtcgcggg cgatcaaagg 1620 cacgcgcatc ggcggtgagg ttgccgaggc gctggccggg tacgagctgc ccattcttga 1680 gtcccgtatc acgcagcgcg tgagctaccc aggcactgcc gccgccggca caaccgttct 1740 tgaatcagaa cccgagggcg acgctgcccg cgaggtccag gcgctggccg ctgaaattaa 1800 atcaaaactc atttgagtta atgaggtaaa gagaaaatga gcaaaagcac aaacacgcta 1860 agtgccggcc gtccgagcgc acgcagcagc aaggctgcaa cgttggccag cctggcagac 1920 acgccagcca tgaagcgggt caactttcag ttgccggcgg aggatcacac caagctgaag 1980 atgtacgcgg tacgccaagg caagaccatt accgagctgc tatctgaata catcgcgcag 2040 ctaccagagt aaatgagcaa atgaataaat gagtagatga attttagcgg ctaaaggagg 2100 cggcatggaa aatcaagaac aaccaggcac cgacgccgtg gaatgcccca tgtgtggagg 2160 aacgggcggt tggccaggcg taagcggctg ggttgtctgc cggccctgca atggcactgg 2220 aacccccaag cccgaggaat cggcgtgacg gtcgcaaacc atccggcccg gtacaaatcg 2280 gcgcggcgct gggtgatgac ctggtggaga agttgaaggc cgcgcaggcc gcccagcggc 2340 aacgcatcga ggcagaagca cgccccggtg aatcgtggca agcggccgct gatcgaatcc 2400 gcaaagaatc ccggcaaccg ccggcagccg gtgcgccgtc gattaggaag ccgcccaagg 2460 gcgacgagca accagatttt ttcgttccga tgctctatga cgtgggcacc cgcgatagtc 2520 gcagcatcat ggacgtggcc gttttccgtc tgtcgaagcg tgaccgacga gctggcgagg 2580 tgatccgcta cgagcttcca gacgggcacg tagaggtttc cgcagggccg gccggcatgg 2640 ccagtgtgtg ggattacgac ctggtactga tggcggtttc ccatctaacc gaatccatga 2700 accgataccg ggaagggaag ggagacaagc ccggccgcgt gttccgtcca cacgttgcgg 2760 acgtactcaa gttctgccgg cgagccgatg gcggaaagca gaaagacgac ctggtagaaa 2820 cctgcattcg gttaaacacc acgcacgttg ccatgcagcg tacgaagaag gccaagaacg 2880 gccgcctggt gacggtatcc gagggtgaag ccttgattag ccgctacaag atcgtaaaga 2940 gcgaaaccgg gcggccggag tacatcgaga tcgagctagc tgattggatg taccgcgaga 3000 tcacagaagg caagaacccg gacgtgctga cggttcaccc cgattacttt ttgatcgatc 3060 ccggcatcgg ccgttttctc taccgcctgg cacgccgcgc cgcaggcaag gcagaagcca 3120 gatggttgtt caagacgatc tacgaacgca gtggcagcgc cggagagttc aagaagttct 3180 gtttcaccgt gcgcaagctg atcgggtcaa atgacctgcc ggagtacgat ttgaaggagg 3240 aggcggggca ggctggcccg atcctagtca tgcgctaccg caacctgatc gagggcgaag 3300 catccgccgg ttcctaatgt acggagcaga tgctagggca aattgcccta gcaggggaaa 3360 aaggtcgaaa aggtctcttt cctgtggata gcacgtacat tgggaaccca aagccgtaca 3420 ttgggaaccg gaacccgtac attgggaacc caaagccgta cattgggaac cggtcacaca 3480 tgtaagtgac tgatataaaa gagaaaaaag gcgatttttc cgcctaaaac tctttaaaac 3540 ttattaaaac tcttaaaacc cgcctggcct gtgcataact gtctggccag cgcacagccg 3600 aagagctgca aaaagcgcct acccttcggt cgctgcgctc cctacgcccc gccgcttcgc 3660 gtcggcctat cgcggccgct ggccgctcaa aaatggctgg cctacggcca ggcaatctac 3720 cagggcgcgg acaagccgcg ccgtcgccac tcgaccgccg gcgcccacat caaggcaccc 3780 tgcctcgcgc gtttcggtga tgacggtgaa aacctctgac acatgcagct cccggagacg 3840 gtcacagctt gtctgtaagc ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg 3900 ggtgttggcg ggtgtcgggg cgcagccatg acccagtcac gtagcgatag cggagtgtat 3960 actggcttaa ctatgcggca tcagagcaga ttgtactgag agtgcaccat atgcggtgtg 4020 aaataccgca cagatgcgta aggagaaaat accgcatcag gcgctcttcc gcttcctcgc 4080 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg 4140 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag 4200 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc 4260 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag 4320 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga 4380 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc 4440 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg 4500 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt 4560 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca 4620 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca 4680 ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag 4740 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca 4800 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg 4860 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg catgatatat 4920 ctcccaattt gtgtagggct tattatgcac gcttaaaaat aataaaagca gacttgacct 4980 gatagtttgg ctgtgagcaa ttatgtgctt agtgcatcta atcgcttgag ttaacgccgg 5040 cgaagcggcg tcggcttgaa cgaatttcta gctagacatt atttgccgac taccttggtg 5100 atctcgcctt tcacgtagtg gacaaattct tccaactgat ctgcgcgcga ggccaagcga 5160 tcttcttctt gtccaagata agcctgtcta gcttcaagta tgacgggctg atactgggcc 5220 ggcaggcgct ccattgccca gtcggcagcg acatccttcg gcgcgatttt gccggttact 5280 gcgctgtacc aaatgcggga caacgtaagc actacatttc gctcatcgcc agcccagtcg 5340 ggcggcgagt tccatagcgt taaggtttca tttagcgcct caaatagatc ctgttcagga 5400 accggatcaa agagttcctc cgccgctgga cctaccaagg caacgctatg ttctcttgct 5460 tttgtcagca agatagccag atcaatgtcg atcgtggctg gctcgaagat acctgcaaga 5520 atgtcattgc gctgccattc tccaaattgc agttcgcgct tagctggata acgccacgga 5580 atgatgtcgt cgtgcacaac aatggtgact tctacagcgc ggagaatctc gctctctcca 5640 ggggaagccg aagtttccaa aaggtcgttg atcaaagctc gccgcgttgt ttcatcaagc 5700 cttacggtca ccgtaaccag caaatcaata tcactgtgtg gcttcaggcc gccatccact 5760 gcggagccgt acaaatgtac ggccagcaac gtcggttcga gatggcgctc gatgacgcca 5820 actacctctg atagttgagt cgatacttcg gcgatcaccg cttcccccat gatgtttaac 5880 tttgttttag ggcgactgcc ctgctgcgta acatcgttgc tgctccataa catcaaacat 5940 cgacccacgg cgtaacgcgc ttgctgcttg gatgcccgag gcatagactg taccccaaaa 6000 aaacatgtca taacaagaag ccatgaaaac cgccactgcg ccgttaccac cgctgcgttc 6060 ggtcaaggtt ctggaccagt tgcgtgacgg cagttacgct acttgcatta cagcttacga 6120 accgaacgag gcttatgtcc actgggttcg tgcccgaatt gatcacaggc agcaacgctc 6180 tgtcatcgtt acaatcaaca tgctaccctc cgcgagatca tccgtgtttc aaacccggca 6240 gcttagttgc cgttcttccg aatagcatcg gtaacatgag caaagtctgc cgccttacaa 6300 cggctctccc gctgacgccg tcccggactg atgggctgcc tgtatcgagt ggtgattttg 6360 tgccgagctg ccggtcgggg agctgttggc tggctggtgg caggatatat tgtggtgtaa 6420 acaaattgac gcttagacaa cttaataaca cattgcggac gtttttaatg tactgaatta 6480 acgccgaatt gctctagcca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt 6540 aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta 6600 atgtgagtta gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta 6660 tgttgtgtgg aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgatt 6720 acgaattcga gctcccaccg cgggtttctg gagtttaatg agctaagcac atacgtcaga 6780 aaccattatt gcgcgttcaa aagtcgccta aggtcactat cagctagcaa atatttcttg 6840 tcaaaaatgc tccactgacg taccataaat tcccctcggt atccaattag agtctcatat 6900 tcactctcaa tccaaataat ctgcaatggc aattacctta tccgcaactt ctttacctat 6960 ttccgcccgg atccgggcag gttctccggc cgcttgggtg gagaggctat tcggctatga 7020 ctgggcacaa cagacaatcg gctgctctga tgccgccgtg ttccggctgt cagcgcaggg 7080 gcgcccggtt ctttttgtca agaccgacct gtccggtgcc ctgaatgaac tgcaggacga 7140 ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct tgcgcagctg tgctcgacgt 7200 tgtcactgaa gcgggaaggg actggctgct attgggcgaa gtgccggggc aggatctcct 7260 gtcatctcac cttgctcctg ccgagaaagt atccatcatg gctgatgcaa tgcggcggct 7320 gcatacgctt gatccggcta cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg 7380 agcacgtact cggatggaag ccggtcttgt cgatcaggat gatctggacg aagagcatca 7440 ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg cgcatgcccg acggcgagga 7500 tctcgtcgtg acccatggcg atgcctgctt gccgaatatc atggtggaaa atggccgctt 7560 ttctggattc atcgactgtg gccggctggg tgtggcggac cgctatcagg acatagcgtt 7620 ggctacccgt gatattgctg aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct 7680 ttacggtatc gccgctcccg attcgcagcg catcgccttc tatcgccttc ttgacgagtt 7740 cttctgagcg ggactctggg gttcgaaatg accgaccaag cgacgcccaa cctgccatca 7800 cgagatttcg attccaccgc cgccttctat gaaaggttgg gcttcggaat cgttttccgg 7860 gacgccggct ggatgatcct ccagcgcggg gatctcatgc tggagttctt cgcccacccc 7920 gatccaacac ttacgtttgc aacgtccaag agcaaataga ccacgaacgc cggaaggttg 7980 ccgcagcgtg tggattgcgt ctcaattctc tcttgcagga atgcaatgat gaatatgata 8040 ctgactatga aactttgagg gaatactgcc tagcaccgtc acctcataac gtgcatcatg 8100 catgccctga caacatggaa catcgctatt tttctgaaga attatgctcg ttggaggatg 8160 tcgcggcaat tgcagctatt gccaaaatcg aaatacccct cacgcatgca ttcatcaata 8220 ttattcatgc ggggaaaggc aagattaatc caactggcaa atcatccagc gtgattggta 8280 acttcagttc cagcgacttg attcgttttg gtgctaccca cgttttcaat aaggacgaga 8340 tggtggagta aagaaggagt gcgtcgaagc agatcgttca aacatttggc aataaagttt 8400 cttaagattg aatcctgttg ccggtcttgc gatgattatc atataatttc tgttgaatta 8460 cgttaagcat gtaataatta acatgtaatg catgacgtta tttatgagat gggtttttat 8520 gattagagtc ccgcaattat acatttaata cgcgatagaa aacaaaatat agcgcgcaaa 8580 ctaggataaa ttatcgcgcg cggtgtcatc tatgttacta gatcgatcaa acttcggtac 8640 tgtgtaatga cgatgagcaa tcgagaggct gactaacaaa aggtatgccc aaaaacaacc 8700 tctccaaact gtttcgaatt ggaagtttct gctcatgccg acaggcataa cttagatatt 8760 cgcgggctat tcccactaat tcgtcctgct ggtttgcgcc aagataaatc agtgcatctc 8820 cttacaagtt cctctgtctt gtgaaatgaa ctgctgactg ccccccaaga aagcctcctc 8880 atctcccagt tggcggcggc tgatacacca tcgaaaaccc acgtccgaac acttgataca 8940 tgtgcctgag aaataggcct acgtccaaga gcaagtcctt tctgtgctcg tcggaaattc 9000 ctctcctgtc agacggtcgt gcgcatgtct tgcgttgatg aagcttatcg ataccgtcgg 9060 ggatcctcta gagtcgacct gcaggcatgc aagcttggca ctggccgtcg ttttacaacg 9120 tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccccttt 9180 cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag 9240 cctgaatggc gaatgagctt gagcttggat cagattgtcg tttcccgcct tcagtttaaa 9300 ctatcagtgt ttgacaggat atattggcgg gtaaacctaa gagaaaagag cgtttattag 9360 aataacggat atttaaaagg gcgtgaaaag gtttatccgt tcgtccattt gtatgtgcat 9420 gccaaccaca gggttcccct cgggatcaa 9449

<210> 5

<211> 11885

<212> DNA

<213> Agrobacteri urn tumefaciens

<220>

<221> gene

<222> (1) . . (11885)

<223> Synthetic Sequence

<400> 5

tggcaggata tattgtggtg taaacaaatt gacgcttaga caacttaata acacattgcg 60 gacgttttta atgtactgaa ttaacgccga attgctctag ccaatacgca aaccgcctct 120 ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc 180 gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact cattaggcac cccaggcttt 240 acactttatg cttccggctc gtatgttgtg tggaattgtg agcggataac aatttcacac 300 aggaaacagc tatgaccatg attacgaatt cgagctccac cgcgggtttc tggagtttaa 360 tgagctaagc acatacgtca gaaaccatta ttgcgcgttc aaaagtcgcc taaggtcact 420 atcagctagc aaatatttct tgtcaaaaat gctccactga cgtaccataa attcccctcg 480 gtatccaatt agagtctcat attcactctc aatccaaata atctgcaatg gcaattacct 540 tatccgcaac ttctttacct atttccgccc ggatccgggc aggttctccg gccgcttggg 600 tggagaggct attcggctat gactgggcac aacagacaat cggctgctct gatgccgccg 660 tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac ctgtccggtg 720 ccctgaatga actgcaggac gaggcagcgc ggctatcgtg gctggccacg acgggcgttc 780 cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg ctattgggcg 840 aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa gtatccatca 900 tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca ttcgaccacc 960 aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt gtcgatcagg 1020 atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc aggctcaagg 1080 cgcgcatgcc cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata 1140 tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg ggtgtggcgg 1200 accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt ggcggcgaat 1260 gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag cgcatcgcct 1320 tctatcgcct tcttgacgag ttcttctgag cgggactctg gggttcgaaa tgaccgacca 1380 agcgacgccc aacctgccat cacgagattt cgattccacc gccgccttct atgaaaggtt 1440 gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg gggatctcat 1500 gctggagttc ttcgcccacc ccgatccaac acttacgttt gcaacgtcca agagcaaata 1560 gaccacgaac gccggaaggt tgccgcagcg tgtggattgc gtctcaattc tctcttgcag 1620 gaatgcaatg atgaatatga tactgactat gaaactttga gggaatactg cctagcaccg 1680 tcacctcata acgtgcatca tgcatgccct gacaacatgg aacatcgcta tttttctgaa 1740 gaattatgct cgttggagga tgtcgcggca attgcagcta ttgccaaaat cgaaataccc 1800 ctcacgcatg cattcatcaa tattattcat gcggggaaag gcaagattaa tccaactggc 1860 aaatcatcca gcgtgattgg taacttcagt tccagcgact tgattcgttt tggtgctacc 1920 cacgttttca ataaggacga gatggtggag taaagaagga gtgcgtcgaa gcagatcgtt 1980 caaacatttg gcaataaagt ttcttaagat tgaatcctgt tgccggtctt gcgatgatta 2040 tcatataatt tctgttgaat tacgttaagc atgtaataat taacatgtaa tgcatgacgt 2100 tatttatgag atgggttttt atgattagag tcccgcaatt atacatttaa tacgcgatag 2160 aaaacaaaat atagcgcgca aactaggata aattatcgcg cgcggtgtca tctatgttac 2220 tagatcgatc aaacttcggt actgtgtaat gacgatgagc aatcgagagg ctgactaaca 2280 aaaggtatgc ccaaaaacaa cctctccaaa ctgtttcgaa ttggaagttt ctgctcatgc 2340 cgacaggcat aacttagata ttcgcgggct attcccacta attcgtcctg ctggtttgcg 2400 ccaagataaa tcagtgcatc tccttacaag ttcctctgtc ttgtgaaatg aactgctgac 2460 tgccccccaa gaaagcctcc tcatctccca gttggcggcg gctgatacac catcgaaaac 2520 ccacgtccga acacttgata catgtgcctg agaaataggc ctacgtccaa gagcaagtcc 2580 tttctgtgct cgtcggaaat tcctctcctg tcagacggtc gtgcgcatgt cttgcgttga 2640 tgaagcttat cgataccgtc ggggatcctc tagtggcgta atcatggtca tagctgtttc 2700 ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgcg gaagcataaa 2760 gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact 2820 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc 2880 ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg 2940 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc 3000 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag 3060 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca 3120 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca 3180 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg 3240 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcaatgct cacgctgtag 3300 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt 3360 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca 3420 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg 3480 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa ggacagtatt 3540 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc 3600 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg 3660 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg 3720 gaacgaaaac tcacgttaag ggattttggt catgagcttg cgccgtcccg tcaagtcagc 3780 gtaatgctct gccagtgtta caaccaatta accaattctg attagaaaaa ctcatcgagc 3840 atcaaatgaa actgcaattt attcatatca ggattatcaa taccatattt ttgaaaaagc 3900 cgtttctgta atgaaggaga aaactcaccg aggcagttcc ataggatggc aagatcctgg 3960 tatcggtctg cgattccgac tcgtccaaca tcaatacaac ctattaattt cccctcgtca 4020 aaaataaggt tatcaagtga gaaatcacca tgagtgacga ctgaatccgg tgagaatggc 4080 aaaagtttat gcatttcttt ccagacttgt tcaacaggcc agccattacg ctcgtcatca 4140 aaatcactcg catcaaccaa accgttattc attcgtgatt gcgcctgagc gagacgaaat 4200 acgcgatcgc tgttaaaagg acaattacaa acaggaatcg aatgcaaccg gcgcaggaac 4260 actgccagcg catcaacaat attttcacct gaatcaggat attcttctaa tacctggaat 4320 gctgtttttc cggggatcgc agtggtgagt aaccatgcat catcaggagt acggataaaa 4380 tgcttgatgg tcggaagagg cataaattcc gtcagccagt ttagtctgac catctcatct 4440 gtaacatcat tggcaacgct acctttgcca tgtttcagaa acaactctgg cgcatcgggc 4500 ttcccataca atcggtagat tgtcgcacct gattgcccga cattatcgcg agcccattta 4560 tacccatata aatcagcatc catgttggaa tttaatcgcg gcctcgtcga gcaagacgtt 4620 tcccgttgaa tatggctcat aacacccctt gtattactgt ttatgtaagc agacagtttt 4680 attgttcatg atgatatatt tttatcttgt gcaatgtaac atcagagatt ttgagacaca 4740 acgtgccccc ccccatgaca ttaacctata aaaataggcg tatcacgagg ccctttcgtc 4800 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 4860 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 4920 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 4980 accataaaat tgtaaacgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca 5040 gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaatagc 5100 ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg 5160 actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccaccc cgatttagag 5220 cttgacgggg aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg 5280 gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc 5340 ttaatgcgcc gctacagggc gcgtactatg gttgctttga cgtatgcggt gtgaaatacc 5400 gcacagatgc gtaaggagaa aataccgcat caggcgccat tcgccattca ggctgcgcaa 5460 ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta cgccagctgg cgaaaggggg 5520 atgtgctgca aggcgattaa gttgggtaac gccagggttt tcccagtcac gacgttgtaa 5580 aacgacggcc agtgaattgt aatacgactc actatagggc gaattgggga tcgatccact 5640 agagcagctt gccaacatgg tggagcacga cactctcgtc tactccaaga atatcaaaga 5700 tacagtctca gaagaccaaa gggctattga gacttttcaa caaagggtaa tatcgggaaa 5760 cctcctcgga ttccattgcc cagctatctg tcacttcatc aaaaggacag tagaaaagga 5820 aggtggcacc tacaaatgcc atcattgcga taaaggaaag gctatcgttc aagatgcctc 5880 tgccgacagt ggtcccaaag atggaccccc acccacgagg agcatcgtgg aaaaagaaga 5940 cgttccaacc acgtcttcaa agcaagtgga ttgatgtgat aacatggtgg agcacgacac 6000 tctcgtctac tccaagaata tcaaagatac agtctcagaa gaccaaaggg ctattgagac 6060 ttttcaacaa agggtaatat cgggaaacct cctcggattc cattgcccag ctatctgtca 6120 cttcatcaaa aggacagtag aaaaggaagg tggcacctac aaatgccatc attgcgataa 6180 aggaaaggct atcgttcaag atgcctctgc cgacagtggt cccaaagatg gacccccacc 6240 cacgaggagc atcgtggaaa aagaagacgt tccaaccacg tcttcaaagc aagtggattg 6300 atgtgatatc tccactgacg taagggatga cgcacaatcc cactatcctt cgcaagacct 6360 tcctctatat aaggaagttc atttcatttg gagaggacac gctgaaatca ccagtctctc 6420 tctacaaatc tatctctctc gagatctatc gatgcatgcc atggaagaga acaatcaaaa 6480 ccagtgcatt ccatacaact gcctcagtaa tcctgaagag gttcttctcg atggtgaaag 6540 gatctccacc ggaaacagtt ccattgacat tagtctttcc ctcgtgcaat tccttgttag 6600 taacttcgtc cccggtggtg gattcctcgt tggtcttatc gattttgtgt ggggtattgt 6660 tggaccatcc cagtgggatg ctttcctcgt ccaaattgag cagcttatca atgagcgtat 6720 tgccgagttc gctaggaacg ctgccattgc taacctcgaa ggtcttggta ataacttcaa 6780 catctatgtt gaggctttta aggaatggga ggaagaccct aataaccccg agactcgtac 6840 cagggtgatt gatcgtttca ggattctcga tggacttctc gaacgtgaca tcccaagttt 6900 caggatttcc ggtttcgagg ttcctcttct cagtgtctac gcccaagctg ctaaccttca 6960 cctcgccatt cttcgtgatt ccgttatctt tggtgaaagg tggggactca ccactattaa 7020 tgtgaacgag aactataatc gtcttattag gcacatcgat gaatacgctg accactgcgc 7080 taacacctat aaccgtggtc tcaataacct tcccaaaagt acctaccagg attggattac 7140 ttataacagg ctccgtaggg atcttaccct caccgttctt gacattgccg ctttcttccc 7200 aaattacgat aaccgtaggt atcctatcca acccgtcggt cagctcactc gtgaggttta 7260 caccgatcca cttattaact tcaatcctca actccagtcc gtggctcaac ttcccacctt 7320 taacgttatg gaaagttcca ggattcgtaa cccacacctc ttcgacatcc ttaataacct 7380 cactattttc accgattggt tcagtgtcgg aaggaacttt tattggggtg gtcaccgtgt 7440 tatttccagt cttatcggag gtggtaacat tacctcccct atctacggaa gggaggccaa 7500 ccaggaaccc ccacgtagtt tcactttcaa cggtcctgtg ttcaggaccc tctccaatcc 7560 cacccttcgt ctccttcaac agccttggcc tgctccccca tttaacctca ggggtgttga 7620 gggagtcgag ttcagtactc ctaccaactc cttcacctat cgtggtaggg gtactgttga 7680 tagtcttacc gagctccccc cagaagacaa ttccgtgcct ccccgtgagg gatacagtca 7740 caggctttgc cacgctacct tcgttcaacg ttccggtact ccatttctca ccaccggtgt 7800 cgttttcagt tggactgata ggtccgccac ccttaccaac actatcgatc ctgaacgtat 7860 taaccagatt cccctcgtga agggattcag ggtttggggt ggtaccagtg tcatcaccgg 7920 accaggtttc actggtggag acattcttcg taggaatacc tttggtgatt tcgtttccct 7980 ccaagtgaac attaacagtc ctatcaccca gcgttatagg cttcgtttca ggtacgcttc 8040 cagtcgtgat gctagggtta ttgtcctcac tggtgccgct tccaccggag ttggtggtca 8100 agtgagtgtt aatatgcccc ttcagaagac tatggagatt ggagaaaacc tcacttcccg 8160 taccttcagg tataccgact ttagtaaccc attctccttc cgtgctaatc ctgatatcat 8220 tggtattagt gagcaacccc ttttcggtgc cggatccatc agttccggtg aactctacat 8280 tgataagatt gagatcattc ttgctgacgc tacttttgaa gccgagagtg atctcgaaag 8340 ggctcagaag tgacccggga gctcgaattc gaagcttctg cagacgcgtc cctcgagttt 8400 ctccataata atgtgtgagt agttcccaga taagggaatt agggttccta tagggtttcg 8460 ctcatgtgtt gagcatataa gaaaccctta gtatgtattt gtatttgtaa aatacttcta 8520 tcaataaaat ttctaattcc taaaaccaaa atccagtact aaaatccaga tcccccgaat 8580 taattcggcg ttaattcaga atcggatccc cctcggacta gagcagcttg ccaacatggt 8640 ggagcacgac actctcgtct actccaagaa tatcaaagat acagtctcag aagaccaaag 8700 ggctattgag acttttcaac aaagggtaat atcgggaaac ctcctcggat tccattgccc 8760 agctatctgt cacttcatca aaaggacagt agaaaaggaa ggtggcacct acaaatgcca 8820 tcattgcgat aaaggaaagg ctatcgttca agatgcctct gccgacagtg gtcccaaaga 8880 tggaccccca cccacgagga gcatcgtgga aaaagaagac gttccaacca cgtcttcaaa 8940 gcaagtggat tgatgtgata acatggtgga gcacgacact ctcgtctact ccaagaatat 9000 caaagataca gtctcagaag accaaagggc tattgagact tttcaacaaa gggtaatatc 9060 gggaaacctc ctcggattcc attgcccagc tatctgtcac ttcatcaaaa ggacagtaga 9120 aaaggaaggt ggcacctaca aatgccatca ttgcgataaa ggaaaggcta tcgttcaaga 9180 tgcctctgcc gacagtggtc ccaaagatgg acccccaccc acgaggagca tcgtggaaaa 9240 agaagacgtt ccaaccacgt cttcaaagca agtggattga tgtgatatct ccactgacgt 9300 aagggatgac gcacaatccc actatccttc gcaagacctt cctctatata aggaagttca 9360 tttcatttgg agaggacacg ctgaaatcac cagtctctct ctacaaatct atctctctcg 9420 agatctatcg atgcatgcca tggaagagaa caatcaaaac cagtgcattc catacaactg 9480 cctcagtaat cctgaagagg ttcttctcga tggtgaaagg atctccaccg gaaacagttc 9540 cattgacatt agtctttccc tcgtgcaatt ccttgttagt aacttcgtcc ccggtggtgg 9600 attcctcgtt ggtcttatcg attttgtgtg gggtattgtt ggaccatccc agtgggatgc 9660 tttcctcgtc caaattgagc agcttatcaa tgagcgtatt gccgagttcg ctaggaacgc 9720 tgccattgct aacctcgaag gtcttggtaa taacttcaac atctatgttg aggcttttaa 9780 ggaatgggag gaagacccta ataaccccga gactcgtacc agggtgattg atcgtttcag 9840 gattctcgat ggacttctcg aacgtgacat cccaagtttc aggatttccg gtttcgaggt 9900 tcctcttctc agtgtctacg cccaagctgc taaccttcac ctcgccattc ttcgtgattc 9960 cgttatcttt ggtgaaaggt ggggactcac cactattaat gtgaacgaga actataatcg 10020 tcttattagg cacatcgatg aatacgctga ccactgcgct aacacctata accgtggtct 10080 caataacctt cccaaaagta cctaccagga ttggattact tataacaggc tccgtaggga 10140 tcttaccctc accgttcttg acattgccgc tttcttccca aattacgata accgtaggta 10200 tcctatccaa cccgtcggtc agctcactcg tgaggtttac accgatccac ttattaactt 10260 caatcctcaa ctccagtccg tggctcaact tcccaccttt aacgttatgg aaagttccag 10320 gattcgtaac ccacacctct tcgacatcct taataacctc actattttca ccgattggtt 10380 cagtgtcgga aggaactttt attggggtgg tcaccgtgtt atttccagtc ttatcggagg 10440 tggtaacatt acctccccta tctacggaag ggaggccaac caggaacccc cacgtagttt 10500 cactttcaac ggtcctgtgt tcaggaccct ctccaatccc acccttcgtc tccttcaaca 10560 gccttggcct gctcccccat ttaacctcag gggtgttgag ggagtcgagt tcagtactcc 10620 taccaactcc ttcacctatc gtggtagggg tactgttgat agtcttaccg agctcccccc 10680 agaagacaat tccgtgcctc cccgtgaggg atacagtcac aggctttgcc acgctacctt 10740 cgttcaacgt tccggtactc catttctcac caccggtgtc gttttcagtt ggactgatag 10800 gtccgccacc cttaccaaca ctatcgatcc tgaacgtatt aaccagattc ccctcgtgaa 10860 gggattcagg gtttggggtg gtaccagtgt catcaccgga ccaggtttca ctggtggaga 10920 cattcttcgt aggaatacct ttggtgattt cgtttccctc caagtgaaca ttaacagtcc 10980 tatcacccag cgttataggc ttcgtttcag gtacgcttcc agtcgtgatg ctagggttat 11040 tgtcctcact ggtgccgctt ccaccggagt tggtggtcaa gtgagtgtta atatgcccct 11100 tcagaagact atggagattg gagaaaacct cacttcccgt accttcaggt ataccgactt 11160 tagtaaccca ttctccttcc gtgctaatcc tgatatcatt ggtattagtg agcaacccct 11220 tttcggtgcc ggatccatca gttccggtga actctacatt gataagattg agatcattct 11280 tgctgacgct acttttgaag ccgagagtga tctcgaaagg gctcagaagt gacccgggag 11340 ctcgaattcg aagcttctgc agacgcgtcc ctcgagtttc tccataataa tgtgtgagta 11400 gttcccagat aagggaatta gggttcctat agggtttcgc tcatgtgttg agcatataag 11460 aaacccttag tatgtatttg tatttgtaaa atacttctat caataaaatt tctaattcct 11520 aaaaccaaaa tccagtacta aaatccagat cccccgaatt aattcggcgt taattcagaa 11580 tcggatcccc ctcggactag agtcgacctg caggcatgca agcttggcac tggccgtcgt 11640 tttacaacgt cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca 11700 tccccctttc gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca 11760 gttgcgcagc ctgaatggcg aatgagcttg agcttggatc agattgtcgt ttcccgcctt 11820 cagtttaaac tatcagtgtt tgacaggata tattggcggg taaacctaag agaaaagagc 11880 gttta 11885

<210> 6

<211> 57

<212> DNA

<213> Bacillus thuringiensis

<220>

<221> mi sc_feature

<222> (1) . . (52)

<220>

<221> mi sc_feature

<222> (1) . . (57)

<400> 6

ttcggcgtta attcagaatc ggatccccct cggactagat tagagcagct tgccaac 57

<210> 7

<211> 2317

<212> DNA

<213> Bacillus thuringiensis <220>

<221> prim_transcript

<222> (1) .. (2317)

<400> 7

aattcgagct ccaccgcggg tttctggagt ttaatgagct aagcacatac gtcagaaacc 60 attattgcgc gttcaaaagt cgcctaaggt cactatcagc tagcaaatat ttcttgtcaa 120 aaatgctcca ctgacgtacc ataaattccc ctcggtatcc aattagagtc tcatattcac 180 tctcaatcca aataatctgc aatggcaatt accttatccg caacttcttt acctatttcc 240 gcccggatcc gggcaggttc tccggccgct tgggtggaga ggctattcgg ctatgactgg 300 gcacaacaga caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc 360 ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga atgaactgca ggacgaggca 420 gcgcggctat cgtggctggc cacgacgggc gttccttgcg cagctgtgct cgacgttgtc 480 actgaagcgg gaagggactg gctgctattg ggcgaagtgc cggggcagga tctcctgtca 540 tctcaccttg ctcctgccga gaaagtatcc atcatggctg atgcaatgcg gcggctgcat 600 acgcttgatc cggctacctg cccattcgac caccaagcga aacatcgcat cgagcgagca 660 cgtactcgga tggaagccgg tcttgtcgat caggatgatc tggacgaaga gcatcagggg 720 ctcgcgccag ccgaactgtt cgccaggctc aaggcgcgca tgcccgacgg cgaggatctc 780 gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct 840 ggattcatcg actgtggccg gctgggtgtg gcggaccgct atcaggacat agcgttggct 900 acccgtgata ttgctgaaga gcttggcggc gaatgggctg accgcttcct cgtgctttac 960 ggtatcgccg ctcccgattc gcagcgcatc gccttctatc gccttcttga cgagttcttc 1020 tgagcgggac tctggggttc gaaatgaccg accaagcgac gcccaacctg ccatcacgag 1080 atttcgattc caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg 1140 ccggctggat gatcctccag cgcggggatc tcatgctgga gttcttcgcc caccccgatc 1200 caacacttac gtttgcaacg tccaagagca aatagaccac gaacgccgga aggttgccgc 1260 agcgtgtgga ttgcgtctca attctctctt gcaggaatgc aatgatgaat atgatactga 1320 ctatgaaact ttgagggaat actgcctagc accgtcacct cataacgtgc atcatgcatg 1380 ccctgacaac atggaacatc gctatttttc tgaagaatta tgctcgttgg aggatgtcgc 1440 ggcaattgca gctattgcca aaatcgaaat acccctcacg catgcattca tcaatattat 1500 tcatgcgggg aaaggcaaga ttaatccaac tggcaaatca tccagcgtga ttggtaactt 1560 cagttccagc gacttgattc gttttggtgc tacccacgtt ttcaataagg acgagatggt 1620 ggagtaaaga aggagtgcgt cgaagcagat cgttcaaaca tttggcaata aagtttctta 1680 agattgaatc ctgttgccgg tcttgcgatg attatcatat aatttctgtt gaattacgtt 1740 aagcatgtaa taattaacat gtaatgcatg acgttattta tgagatgggt ttttatgatt 1800 agagtcccgc aattatacat ttaatacgcg atagaaaaca aaatatagcg cgcaaactag 1860 gataaattat cgcgcgcggt gtcatctatg ttactagatc gatcaaactt cggtactgtg 1920 taatgacgat gagcaatcga gaggctgact aacaaaaggt atgcccaaaa acaacctctc 1980 caaactgttt cgaattggaa gtttctgctc atgccgacag gcataactta gatattcgcg 2040 ggctattccc actaattcgt cctgctggtt tgcgccaaga taaatcagtg catctcctta 2100 caagttcctc tgtcttgtga aatgaactgc tgactgcccc ccaagaaagc ctcctcatct 2160 cccagttggc ggcggctgat acaccatcga aaacccacgt ccgaacactt gatacatgtg 2220 cctgagaaat aggcctacgt ccaagagcaa gtcctttctg tgctcgtcgg aaattcctct 2280 cctgtcagac ggtcgtgcgc atgtcttgcg ttgatga 2317

<210> 8

<211> 1954

<212> DNA

<213> Bacillus thuringiensis

<220>

<221> prim_transcript

<222> (1) .. (1954)

<400> 8

tcgagatcta tcgatgcatg ccatggaaga gaacaatcaa aaccagtgca ttccatacaa 60 ctgcctcagt aatcctgaag aggttcttct cgatggtgaa aggatctcca ccggaaacag 120 ttccattgac attagtcttt ccctcgtgca attccttgtt agtaacttcg tccccggtgg 180 tggattcctc gttggtctta tcgattttgt gtggggtatt gttggaccat cccagtggga 240 tgctttcctc gtccaaattg agcagcttat caatgagcgt attgccgagt tcgctaggaa 300 cgctgccatt gctaacctcg aaggtcttgg taataacttc aacatctatg ttgaggcttt 360 taaggaatgg gaggaagacc ctaataaccc cgagactcgt accagggtga ttgatcgttt 420 caggattctc gatggacttc tcgaacgtga catcccaagt ttcaggattt ccggtttcga 480 ggttcctctt ctcagtgtct acgcccaagc tgctaacctt cacctcgcca ttcttcgtga 540 ttccgttatc tttggtgaaa ggtggggact caccactatt aatgtgaacg agaactataa 600 tcgtcttatt aggcacatcg atgaatacgc tgaccactgc gctaacacct ataaccgtgg 660 tctcaataac cttcccaaaa gtacctacca ggattggatt acttataaca ggctccgtag 720 ggatcttacc ctcaccgttc ttgacattgc cgctttcttc ccaaattacg ataaccgtag 780 gtatcctatc caacccgtcg gtcagctcac tcgtgaggtt tacaccgatc cacttattaa 840 cttcaatcct caactccagt ccgtggctca acttcccacc tttaacgtta tggaaagttc 900 caggattcgt aacccacacc tcttcgacat ccttaataac ctcactattt tcaccgattg 960 gttcagtgtc ggaaggaact tttattgggg tggtcaccgt gttatttcca gtcttatcgg 1020 aggtggtaac attacctccc ctatctacgg aagggaggcc aaccaggaac ccccacgtag 1080 tttcactttc aacggtcctg tgttcaggac cctctccaat cccacccttc gtctccttca 1140 acagccttgg cctgctcccc catttaacct caggggtgtt gagggagtcg agttcagtac 1200 tcctaccaac tccttcacct atcgtggtag gggtactgtt gatagtctta ccgagctccc 1260 cccagaagac aattccgtgc ctccccgtga gggatacagt cacaggcttt gccacgctac 1320 cttcgttcaa cgttccggta ctccatttct caccaccggt gtcgttttca gttggactga 1380 taggtccgcc acccttacca acactatcga tcctgaacgt attaaccaga ttcccctcgt 1440 gaagggattc agggtttggg gtggtaccag tgtcatcacc ggaccaggtt tcactggtgg 1500 agacattctt cgtaggaata cctttggtga tttcgtttcc ctccaagtga acattaacag 1560 tcctatcacc cagcgttata ggcttcgttt caggtacgct tccagtcgtg atgctagggt 1620 tattgtcctc actggtgccg cttccaccgg agttggtggt caagtgagtg ttaatatgcc 1680 ccttcagaag actatggaga ttggagaaaa cctcacttcc cgtaccttca ggtataccga 1740 ctttagtaac ccattctcct tccgtgctaa tcctgatatc attggtatta gtgagcaacc 1800 ccttttcggt gccggatcca tcagttccgg tgaactctac attgataaga ttgagatcat 1860 tcttgctgac gctacttttg aagccgagag tgatctcgaa agggctcaga agtgacccgg 1920 gagctcgaat tcgaagcttc tgcagacgcg tccc 1954

<210> 9

<211> 848

<212> DNA

<213> Gossypium hirsutum

<220>

<221> gene

<222> (1) .. (848)

<400> 9

ccagcaggat cagctggtac tcgcgaagca tctagattca ttccacacaa catacgagcc 60 ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaacacac attaattgcg 120 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc 180 ggccaacgcg cggggagagg cggtttgcgt attggctaga gcaattcggc gttaattcag 240 tacattaatt caattctgca cggagatgag gaaggtggag tggagttgat gtggacgggg 300 tctaaccttc ttcgctcatt tattttttat aatggtcttc atgacatggg atctcaggga 360 cctatgttca cttggaatcg ggttaacctt tttagcaacc cgatcgggca atttgtaatg 420 atctatggaa tgtgatcgct cctgaaagta acgtaggcac cttcctagac ccaagtctga 480 tcgtcaaccg attttattgt ctgtcttcaa ggccgcacat tactactaat cgaccaattt 540 gtttcttagc tagttggtta ttgcaccctc gattccgagg tcttgtacaa gaaactcgga 600 actggactca ggatattgtt actaacttgg atgattttac tttgaaagtc taagagtggg 660 ataaatgtgt ttatggtaac atctttgcaa ttgggttggg ttcagaaagc tctcaaatat 720 tttgacacca atcgactcca aggacaggaa caagtaccta aagttgagct tgagagcatc 780 ttgagtcagg aagagatcct ccggtaccag aaaccgagaa gcgattggct tcttaatggc 840 gataggaa 848

<210> 10

<211> 853

<212> DNA

<213> Gossypium hirsutum <220>

<221> gene

<222> (D..C853)

<400> 10

cgatgaagat cgccagctct atacgactca ctatagggaa agcttgcatg caggcctctg 60 cagtcgacgg gcccgggatc cgattgcact ggccgtcgtt ttacaacgcg tgactgggaa 120 aaccctggcg ttacacaact taatcgcctt gcagcacatc cccctttcgc cagctggcgt 180 aatagcgaag aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa 240 tgagcttgag cttggatcag attgtcgttt cccgccttca gtttaaacta tcagtgtttg 300 attgtctccc ttcccttccc ggtatcggtt catggattcg gttagatggg aaaccgccat 360 cagtaccagg tcgtaatccc acacactggc catgccggcg ggccctgcgg aaacctctac 420 gtgcccgtct ggaagctcgt agcggatcac ctcgccagct cgtcgctcac gcttcgacag 480 acggaaaacg gccacgtcca tgatgctgcg actaccgcgg gtgcccacgt catagagcat 540 cggaacgaaa aaatctggtt gctcgtcgcc cttgggcggc ttcctaatcg acggcgcacc 600 ggctgccggc ggttgccggg attctttgcg gattcgatca gcggccgctt gccacgattc 660 accggggcgt gcttctgcct cgatgcgttg tcgctgggcg gcctgcgcgg ccttcaactt 720 ctccaccagg tcatcaccca gcgccgcgcc gatttgtacc gggccggatg gtttgcgacc 780 gctcacgccg attcctcggg ctcgggggtt ccagtgccat tgcagggccg gcagacaacc 840 cagccgctta cgc 853

<210> 11

<211> 26

<212> DNA

<213> Gossypium hi rsututn

<220>

<221> primer_bind

<222> (1) .. (26)

<400> 11

gaccttaggc gacttttgaa cgcgca 26

<210> 12

<211> 23

<212> DNA

<213> Gossypium hi rsututn

<220>

<221> primer_bind

<222> (D..C23)

<400> 12

agtagacgag gagttgattg cga 23

<210> 13

<211> 25

<212> DNA

<213> Gossypium hi rsutum

<220>

<221> primer_bind

<222> (1) .. (25)

<400> 13

aacagttgcg cagcctgaat ggcga 25

<210> 14

<211> 24

<212> DNA

<213> Gossypium hi rsutum

<220>

<221> primer_bind

<222> (1).. (24)

<400> 14

atcaagaaca accaggcacc gacg 24

<210> 15

<211> 24

<212> DNA

<213> Gossypium hi rsutum <220>

<221> primer_bind

<222> (D . . C24)

<400> 15

gggtttcgct catgtgttga gcat

<210> 16

<211> 28

<212> DNA

<213> Gossypiutn hi rsututn

<220>

<221> primer_bind

<222> (1) . . (28)

<400> 16

aactgcagtg cgaaagctcg agagagat

<210> 17

<211> 975

<212> DNA

<213> Gossypiutn hi rsututn

<220>

<221> misc feature

<222> (1) . . (975)

<400> 17

gggtttcgct catgtgttga gcatataaga aacccttagt atgtatttgt atttgtaaaa 60 tacttctatc aataaaattt ctaattccta aaaccaaaat ccagtactaa aatccagatc 120 ccccgaatta attcggcgtt aattcagaat cggatccccc tcggactaga ttagagcagc 180 ttgccaacat ggtggagcac gacactctcg tctactccaa gaatatcaaa gatacagtct 240 cagaagacca aagggctatt gagacttttc aacaaagggt aatatcggga aacctcctcg 300 gattccattg cccagctatc tgtcacttca tcaaaaggac agtagaaaag gaaggtggca 360 cctacaaatg ccatcattgc gataaaggaa aggctatcgt tcaagatgcc tctgccgaca 420 gtggtcccaa agatggaccc ccacccacga ggagcatcgt ggaaaaagaa gacgttccaa 480 ccacgtcttc aaagcaagtg gattgatgtg ataacatggt ggagcacgac actctcgtct 540 actccaagaa tatcaaagat acagtctcag aagaccaaag ggctattgag acttttcaac 600 aaagggtaat atcgggaaac ctcctcggat tccattgccc agctatctgt cacttcatca 660 aaaggacagt agaaaaggaa ggtggcacct acaaatgcca tcattgcgat aaaggaaagg 720 ctatcgttca agatgcctct gccgacagtg gtcccaaaga tggaccccca cccacgagga 780 gcatcgtgga aaaagaagac gttccaacca cgtcttcaaa gcaagtggat tgatgtgata 840 tctccactga cgtaagggat gacgcacaat cccactatcc ttcgcaagac cttcctctat 900 ataaggaagt tcatttcatt tggagaggac acgctgaaat caccagtctc tctctacaaa 960 tctatctctc tcgag 975

<210> 18

<211> 1373

<212> DNA

<213> Gossypiutn hi rsututn

<220>

<221> gene

<222> (I) . . (1373) <400> 18

gcactggccg tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat 60 cgccttgcag cacatccccc tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat 120 cgcccttccc aacagttgcg cagcctgaat ggcgaatgag cttgagcttg gatcagattg 180 tcgtttctcc gccgtcattc attaatgcac tggcacatga ttgtgaaccg aaaaacctag 240 cccaggttta ttaggccctt gaagaaagct gcagacactg agaaaatatt aatatttgat 300 attgtaattt taatggtttt catccttttc ttgtatttaa atttgaaatt tattttttat 360 agtttgtttg gtataacttg gtctctttat tttttaacta ttattaaact attccgatta 420 aatgcatatg caaatttttc ttaaaaatat tccttttaat ttatatgtac tctttttcag 480 gttgaaataa gtgtttttaa caattaatgg tattataaga gtagaaatat caaattatgc 540 aaaattaaaa taaataaaaa acaactaaat accaaattta aacacagtag atacttaata 600 tatattcatg tgatcataat atataaaatt ttgtaattta agaataaaag aagttgtaaa 660 tgttggagat gcaaacatag ctccttcatt agccacattc ctttctccaa ttgtttacct 720 acctgcaaag acaattgcaa tatcggatca tctcaatcat ataccattgt tcttcaacaa 780 atcttttact gccatcatgg tgttatatct ttcacgaaaa ataatattgt tctctccaat 840 tattaacaag aatccccact ccaaagttgg atatcgttcc taacttacac acttgtctcc 900 tttttagaat gtctttccaa atccacgagt ctgatgcctc aagggtttct ttatttttgt 960 actattttgc tttgaaagcc ttggtgatta gtagatttgg gttggttgtg agacgccaca 1020 cctgttttgc taaaagtgct ttattcatca aagcaatagg tctaatacct agacctcctt 1080 caactttagg actacagatt gtagtctaat tacacaaatg catgcatgta ccatgactcc 1140 actagaaagc tctcaaatct ggtcaatctc atcacaaata tagcgacagg ctttgaaaac 1200 agaacatgta aagaaagtgg atttaatgag ggtgagtcta cctcattatg aaagacactt 1260 cgcctttcaa tgtgccactt tagcttgaat cttgtcaatg atgttttaga atagctcttt 1320 tatcactgat ctcgcaaaca ccaaactcga tatcttgctg aaaaactcga gcc 1373

<210> 19

<211> 26

<212> DNA

<213> Gossypium hirsutum

<220>

<221> primer_bind

<222> (1) .. (26)

<400> 19

cgttacccaa cttaatcgcc ttgcag

<210> 20

<211> 30

<212> DNA

<213> Gossypium hi rsututn

<220>

<221> primer_bind

<222> (D..C30)

<400> 20

agaaaccctt gaggcatcag actcgtggat 30

Claims

We Claim:

1. A nucleotide sequence set forth as SEQ ID No. 1 or a nucleotide sequence comprising sequence set forth as SEQ ID No. l .

2. An expression cassette set forth as SEQ ID No.3, comprising CaMV 35S promoter, nucleotide sequence set forth as SEQ ID No. 1 or a nucleotide sequence comprising sequence set forth as SEQ ID No. l and 35 S 3' untranslated region.

3. The nucleotide sequence as claimed in claim 1 and the expression cassette as claimed in claim 2, wherein the nucleotide sequence codes for CrylC gene and corresponding amino acid sequence set forth as SEQ ID No. 2 codes for CrylC protein.

4. A vector comprising sequence set forth as SEQ ID No. 5, having an expression cassette as claimed in claim 2.

5. The vector as claimed in claim 4, wherein the vector is selected from a group comprising an expression vector, replicable vector, transformation vector, binary vector and shuttle vectors or any combination thereof.

6. The vector as claimed in claims 4 and 5, wherein the vector is preferably binary vector and wherein the expression cassette is either a single copy sequence or tandem repeat sequence separated by an intervening sequence set forth as SEQ ID No.6.

7. A transformed host cell comprising the vector as claimed in claim 4.

8. A method of obtaining a transformed host cell comprising an expression cassette set forth as SEQ ID No.3, said method comprising acts of:

a) inserting an expression cassette set forth as SEQ ID No.3 into a vector; and

b) transforming a host cell with said vector to obtain the transformed host cell.

9. The method as claimed in claim 8, wherein the expression cassette comprise CaMV 35 S promoter, nucleotide sequence set forth as SEQ ID No. 1 or a nucleotide sequence comprising sequence set forth as SEQ ID No. l and 35 S 3' untranslated region.

10. The method as claimed in claim 9, wherein said nucleotide sequence codes for CrylC gene and corresponding amino acid sequence set forth as SEQ ID No. 2 codes for CrylC protein.

11. The method as claimed in claim 8, wherein the host cell is Agrobacterium.

12. The method as claimed in claim 8, wherein the transforming of the host cell is carried out using techniques selected from a group comprising electroporation, microinjection, genegun method, PEG mediated transfer, Calcium phosphate method, liposome mediated transfer and tri-parental mating or any combination thereof.

13. A transgenic cell comprising an expression cassette as claimed in claim 2.

14. The transgenic cell as claimed in claim 13, wherein the transgenic cell is a transgenic plant cell exhibiting resistance to lepidopteran insect(s) or pest(s) infestation; and wherein the plant is a member of genus Gossypium.

15. A method of obtaining a transgenic plant member of genus Gossypium, said method comprising acts of:

a) codon optimizing nucleotide sequence of native CrylC gene to obtain nucleotide sequence set forth as SEQ ID No. l;

b) inserting an expression cassette as claimed in claim 2 into a vector and transforming a host cell with said vector to obtain a transformed host cell; and

c) infecting the plant with the transformed host cell followed by tissue culturing to obtain a transgenic plant member of genus Gossypium.

16. The method as claimed in claim 15(b), wherein the expression cassette is either a single copy sequence or tandem repeat sequence separated by an intervening sequence set forth as SEQ ID No.6.

17. The method as claimed in claim 15, wherein the host cell is Agrobacterium.

18. The method as claimed in claim 15, wherein the transforming the host cell is carried out using techniques selected from a group comprising electroporation, microinjection, genegun method, PEG mediated transfer, Calcium phosphate method and liposome mediated transfer or any combination thereof.

19. The method as claimed in claim 15 (c), wherein the infection and tissue culturing comprises incubating cotton leaf with the transformed host cell; transferring and incubating the cotton leaf in bacterial selection medium followed by incubation in plant selection medium; transferring to embryogenesis medium till the occurence of embryogenesis followed by sub- culturing; transferring to suspension medium followed by germination medium; transfering to basal medium after the growth of leaves and root hairs; hardening followed by transfer to soil and greenhouse to obtain the said transgenic plant.

20. A transgenic transformation event MLS9124 or MLS9878, said event comprising a nucleotide sequence set forth as SEQ ID No. 1 or a nucleotide sequence comprising sequence set forth as SEQ ID No. l in a plant member of genus Gossypium or any part thereof.

21. A method of detecting presence of a trangene comprising an expression cassette set forth as SEQ ID No.3, in a transgenic plant member of genus Gossypium, said method comprising acts of:

a. extracting DNA from the plant source and performing nucleic acid amplification of junction regions of the transgene and the plant to obtain an amplicon, said amplification carried out by primers corresponding to the regions selected from a group comprising left border region of the transgene, right border region of the transgene, left border region of the plant DNA and right border region of the plant DNA or any combination thereof;

b. detecting and analyzing the amplicon to detect the presence of said transgene in the transgenic plant.

22. The method as claimed in claim 21, wherein the primers are selected from a group comprising SEQ ID No. 1 1 corresponding to the left border region of the transgene , SEQ ID No. 13 corresponding to the right border region of the transgene, SEQ ID No. 12 correspdonding to the left border region of the plant DNA and SEQ ID No. 14 corresponding to the right border region of the plant DNA or any combination thereof.

23. Primers set forth as SEQ ID No. 1 1 , SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 15, SEQ ID No. 16, SEQ ID No. 19 and SEQ ID No. 20.

24. The primers as claimed in claim 23, wherein the primers having SEQ ID Nos.12, 13, 15 and 19 are sense primers and the primers having SEQ ID Nos.l 1, 14, 16 and 20 are anti-sense primers.

25. A method of codon optimizing CrylC gene in plant member of genus Gossypium, said method comprising acts of: a) preparing a codon usage table as described in Table 2, for plurality of constitutively expressed genes, on the basis of GC content, TA doublet avoidance in second and third positions of codons, transcription termination signals, splicing signals and polyadenylation signals, from conventionally known constitutively expressed genes in cotton; and b) using said table for codon optimizing the CrylC gene.

26. The nucleotide sequence as claimed in claim 1, the expression cassette as claimed in claim 2 and the method as claimed in claim 9, wherein the nucleotide sequence is codon optimized for plants of genus Gossypium.