WO2017210764A1

WO2017210764A1 - Plant resistant to pest insect

Info

Publication number: WO2017210764A1
Application number: PCT/BR2017/050146
Authority: WO
Inventors: Francisco José LIMA ARAGÃO; Abdulrazak BABA IBRAHIM
Original assignee: Embrapa - Empresa Brasileira De Pesquisa Agropecuaria
Priority date: 2016-06-07
Filing date: 2017-06-07
Publication date: 2017-12-14
Also published as: AR108760A1; BR102016013023A2

Abstract

The present invention belongs to the field of plant genetics and describes a method of producing plants resistant to pest insects. The invention further provides molecules, nucleic acid constructs and other elements that refer to the induction of greater resistance of the plant to pest insect. A plant that incorporating such elements, as well as a method of controlling pest by using molecules in different presentation forms are also provided.

Description

PLANT RESISTANT TO PEST INSECT

FIELD OF THE INVENTION

[1] The present invention relates to nucleic acid molecules, constructs and other agents associated to manipulation of essential genes. In particular, the elements of the present invention are related to the suppression of the expression of genes that code for ATPases in insects, particularly Whitefly. It also relates to the genetic manipulation of plants to silence genes in whitefly that can interact with these plants. It also relates to systems for handling populations of white flies by using genetically modified plants.

DESCRIPTION OF THE PRIOR ART

[2] Prior to the discovery of the RNAi mechanisms, researchers faced great obstacles in the study of the function of insect-specific ge- nes. (Garbutt, J.S., 2011. RNA interference in insects: persistence and uptake of doublestranded RNA and activation of RNAi genes). With the availability of more genomic data and advances in DNA sequencing technologies with reduced cost of the application of RNAi in insect re- search, there was an increase in the number of studies based on the analyses of the function of the gene in various non-model insects (Mito, T, Bando, T., Ohuchi, H., Noji, S.. 2011. The advent of RNA interference in Entomology. Entomol. Sci. doi: 10.1111 /j.1479- 8298.2010.00408.).

[3] In the case of Whitefly, there are few reports involving RNAi.

With the availability of a number of transcriptome data on this insect in the database NCBI, the RNAi machinery of the whitefly will be illustra- ted soon. Important proteins of this machinery like Dicers2, R2D2, Ar- gonuata2 and Sid1 have already been identified in the whitefly, as well as their expressions in different phases of development (Upadhyay, SK Dixit, S., Sharma, S., Singh, H., Kumar, J., Verma, P.C.Chandrashekar, K., 2013. SiRNA machinery in whitefly (Bemisia tabaci). PioS One 8. doi:10.1371/joumal. pone.0083692).

[4] The whitefly (Bemisia tabaci) is an insect belonging to the

Family Aleyrodidea, which has more than 1.550 identified species (Byrne, D., 1991. Whitefly Bioiogy. Annu. Rev. Entomol. doi:10.1146/annurev.ento.36.1.431). The insect is responsible for cau- sing damage to agriculture, since it is adapted to various hosts and de- velops rapidly. This insect feed on the abaxial part of the leaves, sucking the phloem, introducing its toxic saliva into the plant tissue, thus reducing the turgescence 50% (Byrne, D., 1991. Whitefly Biology. Annu. Rev. Entomol. doi:10.1146/annurev.ento.36.1.431). Damage caused by nymphs and adults destroy the attacked plants altogether. Whiteflies can transmit at least 111 species of virus and colonize more than 600 species of plants (Li, S.J., Xue, X., Ahmed, M.Z., Ren, S.X., Du, Y.Z., Wu, J.H., Cuthbertson, A.G.S., Qiu, B.L., 2011. Host plants and natural enemies of Bemisia tabaci (Hemiptera: Aleyrodidae) in China. Insect Sci. 18, 101-120. doi:10.1111/j.1744- 7917.2010.01395.x). Among the viral agents transmitted by this vector, 90% belong to the genus B eg omo virus, 6% to the genus Crini virus, and the remaining 4% belong to the genera Closterovirus, Ipormovirus and Carlavirus. As an example of virus transmitted by B. tabaci one cites African cassava mosaic virus, bean golden mosaic virus, bean dwarf mosaic virus, tomato yellow leaf curl virus, tomato mottle virus, and others (De Barro, P.J., Liu, S.-S., Boykin, L.M., Dinsdale, A.B., 2011. Bemisia tabaci: a statement of species status. Annu. Rev. Ento- mol. 56, 1-19. doi:10.1146/annurev-ento-112408-085504; Li, S.J., Xue, X., Ahmed, M.Z., Ren, S.X., Du, Y.Z., Wu, J.H., Cuthbertson, A.G.S., Qiu, B.L., 2011. Host plants and natural enemies of Bemisia tabaci (Hemiptera: Aleyrodidae) in China. Insect Sci. 18, 101-120. doi:10.1111/j.1744-7917.2010.01395; Oliveira, M.R. V, Henneberry, T.J., Anderson, P., 2001. History, current status, and collaborative research projects for Bemisia tabaci, in: Crop Protection, pp. 709-723. doi: 10.1016/S0261 -2194(01 )00108-9).

[5] Damage caused by the whitefly may be minimized through crop handling and biological control. The initial application of pesticides has been controlling the insect, but the development of resistance is a limiting factor. However, it should be pointed out that the most widely used pesticides have chemical agents that may have effects harmful to the beneficial insect. The invading nature of the whitefly and its high reproduction rate cause the insect to be highly resistant to various in- secticides. In Brazil, the application of insecticides by producers of dif- ferent crops, which used to be carried out 16 times per crop cycle, may reach 40 applications today. The presence of 1 to 3 insects in a field is enough to cause total loss of the crop, due to virus transmission by the white fly. The damage level caused to agriculture by the whitefly and the appearance of mechanisms of this insect resistant to insecticides evidence the need for the development of more effective strategies of control of this factor.

[6] The control of the pH in the intracellular compartments in eukaryotic cells is a phenomenon that is highly coordinated by means of an enzyme called v-ATPase. The activity of this enzyme affects vari- ous cellular processes, such as the transportation through the mem- brane, processing and transportation of neurotransmitters, as well as the regulation of the entry of microorganisms like (Beyenbach, K.W., Wieczorek, H., 2006. The V-type H+ ATPase: molecular structure and function, physiological roles and regulation. J. Exp. Biol. 209, 577-589. doi:10.1242/jeb.02014). Although different specialists describe v- ATPase from different points of view, all of them have a single structure composed by great domains designated as V₀ e V₁, which have 13 su- bunits (Wieczorek, H., Grber, G., Harvey, W.R., Huss, M., Merzendor- fer, H., Zeiske, W., 2000. Structure and regulation of insect plasma membrane H(+)V-ATPase. J. Exp. Biol. 203, 127-135.). X-ray studies enabled characterization of the structure of ATPase in Manduca sexta and Chlostridium fervidus. ATPases are present in the membranes of intracellular compartments, such as liposomes, vacuoles, coated vesi- cles, secreting granules and in the complex Golgi. Their enzymatic function is mediated by pumping H ⁺ (together with the hydrolysis of ATP) for the lumen of the organelles, causing acidification thereof. V- ATPases are also found in the plasma membranes of many types of animal cells and are involved in the homeostasis of the pH and mem- brane energization. The enzyme has also been reported in many epi- thelial cells for transportation of ions. The subunit of the domain V₁ is the catalytic site, responsible for the hydrolysis of ATP. The suppressi- on of this subunit is lethal (Baum, J.A., Bogaert, T., Clinton, W., Heck, G.R., Feldmann, P., I lagan, O., Johnson, S., Plaetinck, G., Munyikwa, T., Pleau, M., Vaughn, T., Roberts, J., 2007. Control of coleopteran insect pests through RNA interference. Nat. Biotechnol. 25, 1322-1326. doi:10.1038/nbt1359; Upadhyay, S.K., Chandrashekar, K., Thakur, N., Verma, P.C., Borgio, J.F., Singh, P.K., Tuli, R., 2011. RNA interference for the control of whiteflies (Bemisia tabaci) by oral route. J. Biosci. 36, 153-161. doi: 10.1007/s12038-011 -9009-1 ).

[7] In the present invention, one presents genetically transfor- med plants for expression of molecules that promote the suppression of the genus of the vacuolar ATPase of whitefly (Bemisia tabaci). Such plants exhibit grater resistance to infestation by the insect. From that results from the researched literature, no documents were found to an- ticipate the teachings of the present invention, so that the solution pro- posed herein has novelty and inventive step over the prior art. SUMMARY OF THE INVENTION:

[8] Said method of producing a plant aims at obtaining plants with resistance to insect by inserting into said plant a DNA molecule whose expression results in a RNA molecule, in which the sequence comprises at least one segment of 18 or more contiguous nucleotides complementary to a fragment of SEQ ID 1 or SEQ ID 2.

[9] The invention further relates to a plant produced by the me- thod described in this invention, with resistance to pest insect; fruit, seed or parts that propagate, characterized by being of the plant of claim 6.

[10] The invention further relates to a nucleic acid molecule comprising a nucleic acid sequence with at least 90% similarity with the sequence described in SEQ ID No1 or in SEQ ID No2. Besides, the invention relates to a nucleic acid molecule that hybridizes with a nu- cleic acid sequence with similarity of at least 90% with the sequence described in SEQ ID No1 or in SEQ ID No2. Another objective of the present invention is to provide a fragment of at least 20 continuous nu- cleotides of a nucleic acid sequence that exhibits at least 90% similarity with the sequence described in SEQ ID No1 or in SEQ ID No2.

[11] The present invention also relates to a chimeric gene that comprises

a) a polynucleotide that presents itself as i) a nucleic acid mo- lecule, said molecule comprising a nucleic acid sequence with at least 90, 95 or 99% similarity with the sequence described in SEQ ID No1 or in SEQ ID No2, or ii) a nucleic acid molecule that hybridizes with a nu- cleic acid sequence with similarity of at least 90, 95 or 99% similarity with the sequence described in SEQ ID No1 or in SEQ ID No2, or iii) fragment of at least 20 continuous nucleotides of a nucleic acid se- quence exhibiting at least 90, 95 or 99% similarity with the sequence described in SEQ ID No1 or in SEQ ID No2; and b) an active promoter, operatively linked to the polynucleotide defined in (a).

[12] The present invention further relates to a genie construct that comprises one or more chimeric genes, such chimeric genes com- prising:

a) a polynucleotide that presents itself as i) a nucleic acid mo- lecule, said molecule comprising a nucleic acid sequence with at least 90, 95 or 99% similarity with the sequence described in SEQ ID No1 or in SEQ ID No2, or ii) a nucleic acid molecule that hybridizes with a nu- cleic acid sequence with similarity of at least 90, 95 or 99% similarity with the sequence described in SEQ ID No1 or in SEQ ID No2, or iii) fragment of at least 20 continuous nucleotides of a nucleic acid se- quence exhibiting at least 90, 95 or 99% similarity with the sequence described in SEQ ID No1 or in SEQ ID No2; and

b) an active promoter, operatively linked to the polynucleotide defined in (a).

[13] In a preferred embodiment, the invention relates to a genie construct that comprises: a first region with a nucleotide sequence of 20 to 564 consecutive nucleotides having at least 90, 95 or 99% simila- rrty with a sequence of 20 to 564 consecutive sense nucleotides, as defined in SEQ ID No1 , and a second region with a sequence of nucle- otides of 20 to 564 consecutive nucleotides having at least 90, 95, or 99% similarity with the complement of 20 to 564 nucleotides of the nu- cleotide sequence, as defined in SEQ ID No1.

[14] It is also an objective of the invention to provide a vector that comprises a nucleic acid molecule, such a molecule comprising a nucleic acid sequence with at least 90, 95 or 99% similarity with the sequence described in SEQ ID No1 or in SEQ ID No2, or nucleic acid molecule that hybridizes with a nucleic acid sequence with similarity of at least 90, 95, or 99% with the sequence described in SEQ ID No1 or in SEQ ID No2, or still a fragment of at least 20 contiguous nucleotides of a nucleic acid sequence that presents at least 90, 95, or 99% simila- rity with the sequence described in SEQ ID No1 or in SEQ ID No2. Pre- ferably, such a vector is capable of promoting the expression of the molecule of interest or a fragment thereof.

[15] Another objective of the invention is to provide a double- stranded ribonucleotide characterized by being produced from the ex- pression of the vector that comprises a nucleic acid molecule that comprises a nucleic acid sequence with at least 90, 95, or 99% simila- rity with the sequence described om SEQ ID No1 or in SEQ ID No2, or a nucleic acid molecule that hybridizes with a nucleic acid sequence with similarity of at least 90, 95 or 99% with the sequence described in SEQ ID No1 or in SEQ ID No2, or still a fragment of at least 20 conti- nuous nucleotides of a nucleic acid sequence that presents at least 90, 95 or 99% similarity with the sequence described in SEQ ID No1 or in SEQ ID No2.

[16] It is also an objective of the invention to provide a transfor- med cell that comprises nucleic acid molecule that comprises a nucleic acid sequence with at least 90, 95 or 99% similarity with the sequence described in SEQ ID No1 or in SEQ ID No2, or nucleic acid molecule that hybridizes with a nucleic acid sequence with similarity of at least 90, 95 or 99% with the sequence described in SEQ ID No1 or in SEQ ID No2, or still a fragment of at least 20 continuous nucleotide that pre- sent at least 90, 95 or 99% similarity with the sequence described in SEQ ID No1 or in SEQ ID No2. Preferably, such a cell is an eukaryotic cell. More preferably, such a cell is a plant cell.

[17] Another objective of the invention is to provide a transfor- med plant that comprises nucleic acid molecule that comprises a nu- cleic acid sequence with at least 90, 95 or 99% similarity with the se- quence described in SEQ ID No1 or in SEQ ID No2, or nucleic acid molecule that hybridizes with a nucleic acid sequence with similarity of at least 90, 95 or 99% with the sequence described in SEQ ID No1 or in SEQ ID No2, or still a fragment of at least 20 continuous nucleotides of a nucleic acid sequence that presents at least 90, 95 or 99% simila- rrty with the sequence described in SEQ ID No1 or in SEQ ID No2.

[18] It is also an objective of the invention to provide a transfor- med plant that comprises one of the above-mentioned genie construct. Another objective of the invention is to provide a transformed plant, such a plant comprising one of the above-mentioned genie construc- tions, such genie constructs exhibiting a first and a second region ca- pable of forming a double-stranded RNA region, and still containing, in addition to the total length of the first and of the second region, a spa- cer region.

[19] Another objective of the invention is to provide a transgenic seed or a propagating part of the plant described above.

[20] The invention also addresses a method for controlling in- sect, which consists in exposing such an insect that infests plant to a dsRNA molecule, the sequence of which comprises at least one seg- ment of 18 or more nucleotides of SEQ ID 1 or SEQ ID 2.

[21] Additionally, the invention also includes an insecticidal com- position containing a nucleic acid molecule that comprises a nucleic acid sequence with at least 90, 95 or 99% similarity with the sequence described in SEQ ID No1 or in SEQ ID No2.

[22] Finally, the invention contemplates a plant that is treated with the above-mentioned composition.

BRIEF DESCRIPTION OF THE FIGURES

[23] Figure 1 : Vetor pBtATPaseC3300. Figure generated through the software SnapGene.

[24] Figure 2: Production of transgenic lettuce (Lactuca sativa) transformed by expressing the siRNA of the v-ATPase. Regeneration in vitro and routing in a selective medium (A) enabled acclimatization of the plants (B), which were subsequently transferred to vases for the production of seeds (C).

[25] Figure 3: Test for detection of the bar gene in transformed lettuce plants. More than 25 transgenic lines were generated. Numbers on the left of the panel denote the lineage number as presented in Ta- ble 2.

[26] Figure 4: Analysis of PCR of transformed transgenic lettuce with the vector pBtATPaseC3300. PCR of the lines TO 1 , 3, 4, 5, 6, 9, 12, 15,18,19, 24, 25, 31 , 33, 34 and 36 using the primer pair BarF/BarR (SEQ ID 9 and SEQ ID 10) enabled detection of a fragment of 408pb corresponding to the region of the bar gene bar (A), while the pair of primer ATPSK/ATPXS (SEQ ID 5 and SEQ ID 6) led to the de- tection of a fragment of 576pb corresponding to the region of the gene v-ATPase (B).

[27] Figure 5: Analysis of Northern blot of siRNAs isolated from transformed lettuce plants. Blots of RNA were hybridized with probes amplified from the pair of primers ATPXS/ATPSK (SEQ ID 5 and SEQ ID 6) to detect the presence of siRNA molecules corresponding to the fragments of v-ATPase. The control is extracted from a non-transgenic plant (Nt).

[28] Figure 6: Monitoring of the population of whitefly fed with transgenic and non-transgenic (NT) lettuce plants (T). The figure shows a higher number of whiteflies at NT than at T on day 5 after ino- culation of 20 whiteflies Bar = 1 cm.

[29] Figure 7: Toxicity of transgenic lettuce plants producing siRNA of v-ATPase against whiteflies during 11 days. All the transgenic plants with v-ATPase (lineages 1 , 3, 4, 6, 19, 25 and 31) induced signi- ficantly higher mortality (P < 0,05) than the internal controls (plants ex- pressing bar gene) and negative controls (non-transgenic plants), within 5 days of infestation. The analysis was carried out by using the Tukey test with the software Prism version 5.0 with P < 0,05 from ave- rages of 12 repetitions.

[30] Figure 8: Opposition of 20 whiteflies fed with transgenic plants producing siRNA of v-ATPase (lineages 1 , 3, 4, 6, 19, 25 and 31), control (expressing bar gene) and negative control plants. The count of eggs was initiated 12 days after inoculation (regarded, in this figure, as day 1). In all the test plants one observed a lower number of eggs than in control plants. The analysis was carried out by using the Tukey test with software Prism version 5.0 with P<0,05 with averages of 12 repetitions.

[31] Figure 9: Appearance of larvae (nymphs) of eggs of 20 whi- teflies fed with transgenic lettuce plants producing siRNA of v-ATPase (lineages 1 , 3, 4, 6, 19, 25 and 31), control (expressing bar gene) and negative control plants). The count of larvae was initiated 12 days after inoculation (regarded, in this figure, as day 1). In all the test plants one observed a lower number of larvae than in control plants. The analysis was carried out by using the Tukey test with software Prism version 5.0 with P < 0,05 with averages of 12 repetitions.

[32] Figure 10. Appearance of egg pupas of 20 whiteflies fed with transgenic lettuce plants producing siRNA de v-ATPase (lineages 1 , 3, 4, 6, 19, 25 and 31), control (expressing bar gene) and negative control plants. The count of larvae was initiated 12 days after inocula- tion (regarded, in this figure, as day 1). In all the test plants one obser- ved a lower number of pupas than in control plants. The analysis was carried out by using the Tu8key test with software Prism version 5.0 com P < 0,05 with averages of 12 repetitions.

[33] Figure 11. Appearance of adults from the eggs of 20 white- flies fed with transgenic lettuce plants producing siRNA de v-ATPase (lineage 1 , 3, 4, 6, 19, 25 and 31), control (expressing bar gene) and negative control plants. Adults began to emerge 12 days after inocula- tion (regarded, in this figure, as day 1). In all the test plants one obser- ved a lower number of flies than in control plants. The analysis was carried out by using the Tukey test with software Prism version 5.0 with P < 0,05 with averages of 12 repetition.

DETAILED DESCRIPTION OF THE INVENTION

[34] The present invention describes a novel and inventive me- thod of producing a plant resistant to pest insect. In the context of this description, numberless terms are used, and so they will be better de- tailed hereinafter.

[35] The term "nucleic acid" refers to a molecule which may be single-stranded or double-stranded molecule, composed of monomers (nucleotides) containing sugar, a phosphate and a purine or pyrimidine base. A "nucleic acid fragment" is a fraction of a given nucleic acid mo- lecule. "Complementariness" refers to the specific pairing of purine and pyrimidine bases that consists of nucleic acids: pairs of adenine with thymine and pairs of guanine with cytosine. Then, the "complement" of a first nucleic acid fragment refers to the second nucleic acid fragment whose nucleotide sequence is complementary to the first nucleotide sequence.

[36] In more complex organisms, deoxyribonucleic acid (DNA) is a nucleic acid that containing the information of the genetic material of a given organism, while the ribonucleic acid (RNA) is a nucleic acid that is involved in the transfer of the information of the DNA in proteins. A "genome" is all main part of the genetic material contained in each cell of an organism. The term "nucleotide sequence" refers to the se- quences of nucleotide polymers, forming a DNA or RNA strand, which may be single-stranded or double-stranded, optionally synthetic, non- natural or with altered nucleotide bases, capable of incorporation into DNA or RNA polymers. The term "oligomer" refers to the short nucleo- tide sequences, usually up to 100 bases in length. The term "homolog" refers to the ancestral linking between the nucleotide sequences of two nucleic acid molecules or between the amino acid sequences of two protein molecules. The estimate of such homology is provided through hybridization of DNA-DNA or RNA-RNA under stringency conditions, as defined in the prior art (as mentioned in document US20030074685, Hames, B. D. and Higgins, S. J. (1985) Nucleic Acid Hybridization: A Practical Approach, IRL Press Oxford, U.K); or by comparison of simi- larity of sequence between two nucleic acid molecules or protein (as mentioned in document US20030074685, Needleman et al., J. Mol. Biol. (1970) 48:443-453).

[37] "Gene" refers to the nucleotide fragment that expresses a specific protein, including, including preceding regulatory (untranslated 5' region) and following regulatory (untranslated 5' region) sequences) with respect to the coding region. "Native gene" refers to an isolated gene with its own regulatory sequence found in nature. "Chimeric ge- ne" refers to the gene that comprises coding, regulatory and heteroge- neous sequences not found in nature. The chimeric gene of the pre- sent invention comprises molecules isolated from nucleic acid, in the sense or antisense orientation, linked operatively to active promoters. Genie constructs of the present invention may contain one or more chimeric genes and may or may not exhibit introns. "Endogenous ge- ne" refers to the native gene usually found in its natural location in the genome and is not isolated. An "exogenous gene" refers to a gene that is not usually found in the host organism, but rather is introduced by gene transfer. "Pseudogene" refers to a nucleotide sequence that does not encode a functional enzyme.

[38] "Encoding sequence" refers to the DNA sequence that en- codes a specific protein and excludes the non-encoding sequence. An "interrupted encoding sequence" means a sequence that acts as a se- parator (for example, one or more introns linked through junctions). An "intron" is a nucleotide sequence that is transcribed and is present in the pre-mRNA, bus tis removed through cleavage and the re-linking of the mRNA within the cell, generating a mature mRNA that may be translated to a protein. Examples of introns include, but are not limited to intron pdk, intron pdk2, intron catalase of castor plant, intron Delta 12 desnaturase of cotton plant, Delta 12 desnaturase of Arabidopsis thaliana, intron ubiquitin of maize plant, intron of SV40, introns of the gene of malate synthase.

[39] "RNA transcript" refers to the product resulting from the transcription catalyzed by RNA polimerase of a DNA sequence. When the RNA transcript is a perfect copy of the DNA sequence, it is referred to as a primary transcript or it may be an RNA sequence derived from a post-transctiptional process of the primary transcript and is then refer- red to as a mature transcript. "Messenger RNA (mRNA)" refers to the RNA without introns. "Sense RNA" refers to an RNA transcript that in- cludes the mRNA. "Antisense RNA" refers to an RNA transcript that is complementary to all the parts of a primary transcript or mRNA and that may block the expression off a target gene through interference in the processing, transportation and/or translation of its primary transcript or mRNA. The complementariness of an antisense RNA may be with any part of the specific genie transcript, that is, untranslated 5' region, introns or encoding sequence. Besides, the antisense RNA may con- tain ribozyme sequence regions that increase the efficacy of the anti- sense RNA to block the gene expression.

[40] "Ribozyme" refers to the catalytic RNA and includes specific sequences of endoribonucleases.

[41] "dsRNA" or "double-strand RNA" refers to a structure for- med between the of the mRNA or sense RNA, the sequence of a spa- cer sequence and the antisense RNA sequence, wherein "Spacer re- gion" refers to the nucleotide sequence that is not related to the target gene sequence, as an intron sequence. .

[42] The term "vector" refers to replicon, as a plasmid, fagus or virus, in which other gene sequences or elements (be they of DNA or RNA) may be linked. In this way, the genes may be replicated together with the vector. Such vectors may be obtained commercially, including those supplied by Clontech Laboratories, Inc (Palo Alto, Calif.), Strata- gene (La Jolla, Calif), Invitrogen (Carlsbad, Calif.), New England Bio- labs (Beverly, Mass.) and Promega (Madison, Wis.). A few non-limiting examples of vectors, are the vectors pKannibal (Wesley SV, Heliwell CA, Smith NA, Wang M, Rouse DT, Liu Q, Gooding PS, Singh SP, Abbott D, Stoutjesdijk PA, Robinson SP, Gleave AP, Green A G, Wate- rhouse PM (2001) Construct design for efficient, effective and highthroughput Gene silencing in plants. Plant J. 27:581590) pGLYP, pAC321 , series pCambia (BioForge Co.), pBI121 (Chen, Po-Yen; Wang, Chen-Kuen; Soong, Shaw-Ching; To, Kin-Yina. Complete se- quence of the binary vector DBI121 and its application in cloning T- DNA insertion from transgenic plants. Molecular Breeding vol. 11 issue 4 May 2003. p. 287-293), pBSK (Addgene Co.), pGEM-T easy (Pro- mega Corporation), pET101/ D-TOPO (Invitrogen). The obtainment of recombinant vectors comprising promoters linked to nucleic acids is known from the prior art and may befound in Sambrook et al. (Sambro- ok, J., Russell, D. W., Molecular Cloning, A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press. 1989).

[43] A way of producing dsRNA is by inserting into the DNA mo- lecule nucleotide sequence of the target gene in the sense orientation, and a nucleotide sequence in the antisense orientation, wherein there may or may not be a spacer region between the sense and antisense nucleotide sequences. The nucleotide sequences mentioned above may be constituted by about 19nt to 2000nt or still about 5000 nucleo- tide or more.

[44] In one of the aspects of the invention, the dsRNA molecule may comprise one or more regions having at least 90 to 100% sequen- ce for the regions with at least 20 consecutive nucleotides of the sense nucleotides of the sequence described in SEQ ID No 1 and/or in SEQ ID No 2, one or more regions having similarity of at least 90 to 100% sequence for the regions with at least 20 consecutive nucleotides of the complement of the sense nucleotides of the target gene, where these regions may have pairs of bases separating them from each other.

[45] Consequently, the dsRNA (double-stranded RNA) as des- cribed may be introduced into the host cells by introduction and possi- ble integration of a gene construct containing the nucleic acid molecu- les of the present invention, their transcription for production of the dsRNA. Therefore, in another embodiment, the invention is also sup- ported by a gene construct that is capable of being expressed in cells of eukaryotic organisms of interest, operatively linked to a DNA mole- cule that, when transcribed, produces a dsRNA molecule.

[46] "Promoter" refers to the DNA sequence in a gene, usually located upstream of the encoding sequence, which controls the ex- pression of the encoding sequence, promoting recognizance by the DNA and other factors required for the transcription itself. In an artificial DNA construct, promoters may also be used for transcribing dsRNA. Promoters may also contain DNA sequences that are involved in the linking of protein factors, which control the effect of the beginning of transcription in response to the physiological or development condi- tions. "Constitutive promoters" refer to those that guide the gene ex- pression in all the tissues of the organism and all the time. 'Tissue- specific" or "development-specific" promoters are those that guide the gene expression almost exclusively in specific tissues, such as leaves, routs, stems, flowers, fruits or seeds, or in specific development stages in a tissue, as in the beginning or at the end of the embryogenesis.

[47] The promoter may contain elements "enhancers". An enhancer is a DNA sequence that may stimulate promoter activity. It may be an innate element of the promoter or a heterologous element to raise the level and/the tissue-specificity of a promoter.

[48] The term "expression" Refers to the transcription of a given DNA sequence. More specifically in this invention, it refers to the trans- cription and stable accumulation of the dsRNA derived from the nucleic acid fragments of the invention.

[49] "Inhibition by interference" refers to the production of dsR- NA transcripts capable of preventing the expression of the target pro- tein.

[50] 'Transformation" refers to the transfer of the exogenous gene into the genome of the host organism and its consequent inclusi- on in the genetically stable inheritance.

[51] "Plants" refer to multicellular, eukaryotic, autotrophic and photosynthetic organism belonging to the Kingdom Plantae.

[52] In one of the aspects of the invention, the promoter is a promoter expressed in plants. As used herein, the term "promoter ex- pressed in plants" means a DNA sequence that is capable of initiating and/or controlling the transcription in a plant cell. This includes any promoter of vegetable origin that is capable of guiding the expression. In a plant cell, for example promoters of viral or bacterial origin such as CaMV35S (as mentioned in patent application the US20030175783, Hapster et al, 1988 Mol. Gen. Genet. 212, 182-190) and promoters of the gene present in T-DNA de Agrobactehum; tissue-specific promo- ters or organ specific promoters, including, but not limited to seed- specific promoters (WO8903887), primordia-specific promoters (as mentioned in patent application US20030175783, An et al., 1996 The Plant Cell 8, 15-30), stem-specific promoters (as mentioned in patent application US20030175783, Keller et al., 1988 EMBO J. 7: 3625- 3633), leaf-specific promoters (as mentioned in patent application US20030175783, Hudspeth et al., 1989 Plant Mol Biol 12:579-589), mesophile-specific promoter, root-specific promoters (as mentioned in patent application US20030175783, Keller et al., 1989 Genes Devel. 3:1639-1646), tuber-specific promoters (as mentioned in patent appli- cation US20030175783, Keil et al., 1989 EMBO J. 8: 1323:1330), vas- cular-tissue-specific promoters (as mentioned in patent application US20030175783, Peleman et al., 1989 Gene 84: 359-369), stamen- specific promoters (WO8910396, W09213956), dehiscence-zone spe- cific promoters W09713865); and the like.

[53] The termination signal of the transcription and the polyadenylation region of the present invention includes, but is not limi- ted to the termination signal of SV40, adenylation signal of HSV TK, termination signal of the gene of nopaline synthetase of Agrobacterium tumefasciens, termination signal of the gene RNA 35S do CaMV, ter- mination signal of the virus that attacks Trifolium subterranean (SCSV), termination signal of the gene trpC of Aspergillus nidulans, and other similar ones.

[54] The present invention also includes promoting dsRNA mo- lecules, which may be obtained by transcription of the gene constructs, and that are useful to the methods according to the invention.

[55] Another objective of the present to provide eukaryotic cells and eukaryotic organisms containing dsRNA molecules of the inven- tion, or containing the chimeric genes or the gene constructs capable of producing dsRNA molecules of the invention. The gene constructs may be stably integrated in the genome of the cells of eukaryotic orga- nisms.

[56] The gene constructs or chimeric genes of the invention may be introduced into the genome of the desired host plant by a variety of conventional techniques. For instance, the construct may be introduced directly into the plant tissue by using ballistic methods, such as bom- bing particles covered with DNA, as described in Rech EL, Vianna GR, Aragao FJL (2007) High transformation efficiency by biolistics of soybean, bean and cotton transgenic plants, Nature Protocols, 3 (3): 410-418. They may also be introduced directly into the genome DNA of the plant cell, by using techniques such as electroporation and microin- jection of protoplasts of plant cells.

[57] Transformed plant cells that are derived from any of the transformation techniques descried above may be grown to generate a whole plant that has the transformed genotype and, as a result, the ex- pected phenotype. Such regeneration techniques rely on manipulation of certain phytohormones in tissu re-culture growth medium, typically containing a biocide and/or herbicide marker that should be introduced together with the desired nucleotide sequence. Regeneration of plants from protoplast culture is described in Evans et al., Protoplasts Isola- tion and Culture, Handbook of Plant Cell Culture, pp. 124-176, MacMil- lilan Publishing Company, New York, 1983; and Binding, Regeneration of Plants, Plant Protoplasts, pp. 21-73, CRC Press, Boca Raton, 1985 (as mentioned in patent application US20020152501). The regenera- tion may also be obtained from plant calli, explants, organs, or a part thereof. Such regeneration techniques are described generally in Klee et al., Ann. Ver. Of Plant Phys. 38:467-486, 1987 1985 (as mentioned in patent application US20020152501 ).

[58] Said method of producing a plant aims at obtaining plants with resistance to insect by inserting into said plant a DNA molecule whose expression results in an RNA molecule, wherein the sequence comprises at least one segment of 18 or more continuous nucleotides complementary to a fragment of SEQ ID 1 or SEQ ID 2. Preferably, the RNA molecule expressed by said plant either causes death or prevents the development of the insect that ingests it. In another preferred em- bodiment, said nucleotide segment is transcribed in sense and anti- sense orientations, resulting in the RNA, at least partly double- stranded.

[59] Said method of producing a plant resistant to pest may have as its target an insect belonging to the order Hemiptera. Preferably, such an insect belongs to the genus Bemisia sp. More preferably, the insect belongs to the species Bemisia tabaci.

[60] The invention further relates to a plant produced by the me- thod described in this invention, wherein the plant may be selected from the species of soybean, caupi bean plant, common bean plant, tomato plant, cotton plant, vegetables and related crops. Fruit, seed or propagatable parts characterized by of the plant of claim 6.

[61] The present invention relates to an isolated nucleic acid mo- lecule with the nucleic acid sequence with at least 90% similarity with the sequence described in SEQ ID No1 or in SEQ ID No2. In a prefer- red embodiment, the nucleic acid molecule exhibits at least 95% simila- rity with the sequence described in SEQ ID No1 or in SEQ ID No2. In a more preferred embodiment, the nucleic acid molecule exhibits at least 99% similarity with the sequence described in SEQ ID No1 or in SEQ ID No2. In an even more preferred embodiment, said nucleic acid mo- lecule exhibits a nucleic acid sequence according to the sequence described in SEQ ID No1 or in SEQ ID No2.

[62] The invention also relates to an isolated nucleic acid mole- cule with a nucleic acid sequence with at least 90% similarity with the complement of the sequence described in SEQ ID No1 or in SEQ ID No2. Preferably, said the nucleic acid molecule exhibits at least 95% similarity with the complement of the sequence described in SEQ ID No1 or in SEQ ID No2. More preferably, the nucleic acid molecule exhibits at least 99% similarity with the complement of the sequence described in SEQ ID No 1 or in SEQ ID No2. Even more preferably, said nucleic acid molecule exhibits a nucleic acid sequence comple- mentary to the sequence described in SEQ ID No1 or in SEQ ID No2.

[63] The invention further relates to a nucleic acid molecule that hybridizes with a nucleic acid sequence with similarity of at least 90% with the sequence described in SEQ ID No1 or in SEQ ID No2. Prefe- rably, the nucleic acid molecule hybridizes with a nucleic acid sequen- ce with similarity of at least 95% with the sequence described in SEQ ID 1 or in SEQ ID No2. More preferably, the nucleic acid molecule hybridizes with a nucleic acid sequence with similarity with at least 99% with the sequence described in SEQ ID 1 or in SEQ ID No2. Even mo- re preferably, said nucleic acid molecule hybridizes with a nucleic acid sequence as described in SEQ ID 1 or in SEQ ID No2.

[64] Another embodiment of the invention is a fragment of at least 20 contiguous nucleotides of a nucleic acid sequence that exhi- bits at least 90% similarity with the sequence described in SEQ ID No1 or in SEQ ID No2; Preferably, the fragment of at least 20 contiguous nucleotides of a nucleic acid sequence exhibits at least 95% similarity with the sequence described in a SEQ ID No1 or in SEQ ID No2. More preferably, the fragment of at least 20 contiguous nucleotides of a nu- cleic acid sequence exhibits at least 99% similarity with the sequence described in SEQ ID No1 or in SEQ ID No2. Even more preferably, said fragment of 20 contiguous nucleotides of a nucleic acid sequence exhibits a sequence as described in SEQ ID No1 or in SEQ ID No2.

[65] Another objective of the present invention relates to a chi- meric gene characterized by comprising:

a) a polynucleotide according to any one of claims 10 to 25; and

b) an active promoter, operatively linked to the polynucleotide defined in a (a).

[66] The present invention also relates to a gene construct cha- racterized by comprising one or more chimeric genes constructed as mentioned above.

[67] Additionally, the present invention provides a gene construct characterized by comprising:

a) a first region with a nucleotide sequence of 20 to 564 con- secutive nucleotides having at least 90, 95 or 99% similarity with a se- quence of 20 to 564 consecutive nucleotides of the sense nucleotide sequence as described in SEQ ID No1 or SEQ ID No2;

b) a second region with a nucleotide sequence of 20 to 564 consecutive nucleotides having at least 90, 95, or 99% similarity with the complement of 20 to 564 consecutive nucleotides of the nucleotide sequence as described in SEQ ID No1 or SEQ ID No2.

[68] In a preferred embodiment, in said gene construct, the first and the second regions are capable of forming a double-stranded RNA region, which may have, in addition to the total length of the first and of the second region, a spacer region. Such a spacer region between the first and the second regions may exhibit a length of 100 to 800 nucleo- tides. In a more preferred embodiment, said spacer sequence is an in- tron.

[69] It is also an objective of the invention to provide a vector that comprises a nucleic acid molecule, such a molecule comprising a nucleic acid sequence with at least 90, 95, 99% similarity with the se- quence described in SEQ ID No1 or in SEQ ID No2, or a nucleic acid molecule that hybridizes with a nucleic acid sequence with similarity of at least 90, 95, or 99% with the sequence described in SEQ ID No1 or in SEQ ID No2, or still a fragment of at least 20 contiguous nucleotides of a nucleic acid sequence that exhibits at least 90, 95, or 99% simila- rity with the sequence described in SEQ ID No1 or in SEQ ID No2. Pre- ferably, such a vector is capable of promoting the expression of the molecule of interest or a fragment thereof.

[70] Another objective of the invention is to provide a double- stranded ribonucleotyde sequence characterized by being produced from the expression of5f the vector that comprises a nucleic acid mole- cule that comprises a nucleic acid sequence with at least 90, 95, or 99% similarity with the sequence describe in SEQ ID No1 or in SEQ ID No2, or a nucleic acid molecule that hybridizes with nucleic acid se- quence with similarity of at least 90, 95, or %% with the sequence des- cribed in SEQ ID No1 or in SEQ ID No2, or still a fragment of at least 20 contiguous nucleotides of a nucleic acid sequence that exhibits at least 90, 95, or 99% similarity with the sequence described SEQ ID No1 or in SEQ ID No2.

[71] Preferably, another objective of the invention is a double- stranded ribonucleotide, characterized by being produced from the ex- pression. Of the vector that comprises a nucleic acid molecule that comprises a nucleic acid sequence that corresponds to the sequence described in ID No1 or in SEQ ID No2, or a nucleic acid molecule that hybridizes with a nucleic acid sequence that corresponds to the se- quence described in SEQ ID No1 or in SEQ ID No2, or still a fragment of at least 20 contiguous nucleotides of a nucleic acid sequence that corresponds to the sequence described in SEQ ID No1 or in SEQ ID No2.

[72] It is also an objective of the invention to provide a transfor- med cell that comprises a nucleic acid molecule that comprises a nu- cleic acid sequence with at least 90, 95, or 99% similarity with the se- quence described in SEQ ID No1 or in EQ ID No2, or a nucleic acid molecule that hybridizes with a nucleic acid sequence with similarity of at least 90, 95, or 99% with the sequence described in SEQ ID No1 or in SEQ ID No2, or still a fragment of at least 20 contiguous nucleotides that exhibit at least 90, 95, or 99% similarity with the sequence descri- bed in SEQ ID No1 or in SEQ ID No2. Preferably, such a cell is an eu- karyotic cell. Even more preferably, such a cell is a plant cell.

[73] Preferably, it is an objective of the invention to provide a transformed cell that comprises a nucleic acid molecule that comprises a nucleic acid sequence that corresponds to the sequence described in SEQ ID No1 or in SEQ ID No2, or a nucleic acid molecule that hybridi- zes with a nucleic acid sequence that corresponds to the sequence described in SEQ ID No1 or in SEQ ID No2, or still a fragment of at least 20 contiguous nucleotides of a nucleic acid sequence that corres- ponds to the sequence described in SEQ ID No1 or in SEQ ID No2. Preferably, such a cell is an eukaryotic cell. Even more preferably, such a cell is a plant cell.

[74] Another objective of the invention is to provide a transfor- med plant that comprises a nucleic acid molecule that comprises a nu- cleic acid sequence with at least 90, 95, or 99% similarity with the se- quence described in SEQ ID No1 or in SEQ ID No2, or nucleic acid molecule that hybridizes with a nucleic acid sequence with similarity of at least 90, 95, or 99% with the sequence described in SEQ ID No1 or in SEQ ID No2, or still a fragment of at least 20 contiguous nucleotides of a nucleic acid sequence that exhibits 90, 95, or 99% similarity with the sequence described in SEQ ID No1 or in SEQ ID No2.

[75] Preferably, another objective of the invention is to provide a transformed plant that comprises nucleic acid molecule that comprises a nucleic acid sequence that corresponds to the sequence described in SEQ ID No1 or in SEQ ID No2, or a nucleic acid molecule that hybridi- zes with a nucleic acid sequence that corresponds to the sequence described in SEQ ID No1 or in SEQ ID No2, or still a fragment of at least 20 contiguous nucleotides of a nucleic acid sequence that corres- ponds to the sequence described in SEQ ID No1 or in SEQ ID No2. [76] It is also an objective of the invention to provide a transfor- med plant that comprises one of the above-mentioned gene constructs. Another objective of the invention is to provide a transformed plant, such a plant comprising one of the above-mentioned gene constructs, such gene constructs being its first and second regions capable of for- ming a double-stranded RNA region, and still containing, in addition to the total length of the first and second region, a spacer region.

[77] Additionally, the present invention provides a transgenic seed or a propagative part belonging to the transformed plant mentio- ned in the invention.

[78] In an embodiment of the invention, one describes a method of controlling insect, which consists in exposing the insect that infests the plant to dsRNA molecule, whose sequence comprises at least a segment of 18 or more nucleotides of SEQ ID 1 or SEQ ID 2. Prefera- bly, in said method, the insect is selected from the order Hemiptera. More preferably, the insect is selected from the genus Bemisia sp. Even more preferably, the insect belongs to the species Bemisia taba- ci.

[79] Preferably, another objective of the invention consists of the method of controlling the insects by exposing the insect that infests the plant to the dsRNA molecule, whose sequence comprises at least one segment of 18 or more nucleotides of SEQ ID 1 or SEQ ID 2, in which the dsRNA molecule is synthesized chemically or produced by expres- sion in microorganism or by expression in plant cell.

[80] In another preferred embodiment, said method approaches the exposure of the insect that infests the plant to the dsRNA molecule by applying a composition containing said dsRNA onto the insect or onto the plant surface infested by the insect. More preferably, said composition comprises a treatment as follows: solid, liquid, powder, emulsion, spray, encapsulated, with microbeads, with particulate carri- ers, in film, in matrix or seed.

[81] In another preferred embodiment, said method exposes the insect to the dsRNA molecule in a composition that comprises one or more components selected from the group consisting of a carrying agent, a surfactant, an organosilicone, a herbicidal polynucleotide mo- lecule, a n on- poly nucleotide herbicidal molecule.

[82] The present invention further refers to an insecticidal com- position that contains molecule with at least 90, 95, or 99% similarity with SEQ ID 1 or SEQ ID 2. Preferably, said insecticidal composition contains molecule similar to SEQ ID 1 or SEQ ID 2. Additionally, the present invention also contemplates a plant treated with said composi- tion.

[83] Experiments carried out and results obtained:

[84] dsRNA vector constructs

[85] The initiating sequences ATPaF1/ATPaR1 (SEQ ID 3 and SEQ ID 4) were used for amplifying the fragment of v-ATPase from ge- ne DNA of Bemisia tabaci. The fragment was cloned in Eschereha coli, then it was subjected to a sequencing and analysed using the nucleoti- de of the NCBI, to identify the gene. The cloning of the gene in the vec- tor pGEM®-T Easy vector (Promega) enabled addition of the restriction sites for Xba I, Sac I, Spe I and Kpn I, generating a new fragment with 576 pb.

[86] A new pair of initiators was designed, containing the new restriction sites, and used to clone the interfering fragments of the gene in the vector pSIU (Tinoco, M.L.P., Dias, B.B.A., Dall'Astta, R.C., Pam- phile, J.A., Aragao, F.J.L., 2010. In vivo trans-specific gene silencing in fungal cells by in plant expression of a double-stranded RNA. BMC Bi- ol. 8, 27. doi:10.1186/1741-7007-8-27), separated by an intron, so as to guarantee the formation of a hairpin after its expression, which resul- ted in the vector called pBtATPase. For transformation with Agrobacte- hum tumefaciens, the vector pBtATPaseC3300 was constructed through digestion of the vector pBtATPase and of the GS54365- 5pCAMBIA33000-Construct5 with Hindi! I and EcoRI, releasing the cassette v-ATPase and a bigger fragment, respectively. The resulting fragments were linked to form pBtATPaseC3300, which was used for transforming A. tumefaciens.

[87] DNA sequencing, manipulation of sequences, cloning and design of Initiators

[88] The sequence data were analyzed using the tools BLAST (Basic Local Alignment Search Tool) and MEGA5 (http://www.megasoftware.net). The initiating sequences were desig- ned by using the tool PrimerQuest (http://www.idtdna.com/site).

[89] Transformation of bacteria:

[90] Competent cells of Eschereha coli were used for preparing mini-concentration of vector pBtATPaseC3300. The bacteria was grown in Petri dish (15 x 90mm) containing 25m L of LB medium su- pplemented with kanamycin at 100 mg/L, and incubated at 37 ± 2Ό for 48 h. Twelve individual colonies were selected and used for reaction of PCR using the pair of initiators ATPXS/ATPSK (SEQ ID 5 and SEQ ID 6). An individual colony was selected and the plasmid isolated from it was used to transfect lineage EHA105 of Agrobactehum tumefaciens. The bacteria were stored at -80°C in glycerol 50% and LB medium.

[91] Culture of plant tissue and gene transformation:

[92] A total of 6400 cotyledons were subjected to transformation via Agrobactehum, using modified protocol of Dias, B.B.A., Cunha, W.G., Morais, L.S., Vianna, G.R., Rech, E.L., Capdeville, G., Aragao, F.J.L., 2006. Expression of an oxalate decarboxylase gene from Flammulina sp. in transgenic lettuce (Lactuca sativa) plants and resistance to Sclerotinia sclerotiorum. Plant Pathol. 55, 187-193. doi:10.1111/j.1365- 3059.2006.01342. Lettuce seeds (cultivar Veroni- ca) had their surface sterilized by immersion into 0.01% of Tween 20 followed by NaOCI at 2,5% for 7 minutes. After this, the seeds were rinsed with distilled water three times and cultivated on Petri dish con- taining 20mL of medium ½ MS (Murashige and Skoog, 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15, 473-497. doi:10.1021/jf9040386) and 0,8% of agar. The Petri dishes containing the seeds were then sealed with plastic and cultivated for 2-3 days in the dark at 25 ± 2 °C, until germination.

[93] Paralelly, the frozen lineage of Agrobactehum was activated in LB medium containing kanamycin and rifampicin for 48 h. For the co- culture, colonies were inserted in medium MS salts containing ace- tosyringone 0.5M and glucose 3% pH 5.2, which was previously sterili- zed and filtered.

[94] The bacterial suspension was homogenized to a OD.600 de 0.5-1.0 measured on spectrophotometer (Spectrometry Genesys 8) and then used for co-culture with pieces of lettuce cotyledon for 15minutes. Autoclaved paperfilter was used for drying the explants, which were incubated in medium MS containing 3%, 0.05mg/L na- phthaleneacetic acid (NAA) and 0.2mg/L 6-benzilaminopurine (BAP) at 25 ± 2°C for 48 h. The bacteria were washed from the explants with ste- rilized distilled water containing 250mg/L thymidine, which were dried with sterilized filter paper. The explants were then subcultivated in the same medium with addition of 4 mg/L ammonium glyphosate. The calli in proliferation that emerged were subcultivated in the same medium every week. The new spruces were individualized and grown in me- dium MS containing 3% sucrose, 0.5 mg/L zeatine and 4 mg/L ammo- nium glyphosate. The explants were subcultivated weekly until they reached a maturity degree, so that they were transferred to the rooting medium containing MS and 4mg/L ammonium glyphosate. One obtai- ned twenty-five (25) independent lineages of genetically modified lettu- ce (Table 2) which, with the development of roots, had their explants placed in plastic glasses containing soil and vermiculite in the ratio 50:50 and acclimatized in a moisture chamber made of plastic and elastic. With the growth of the stem, the plants were transferred to bi- gger pots. And with the appearance of seeds, they were collected and planted in plastic glasses for future evaluations. (Figure 2 and Table 2). A few lineages died during the acclimatization, aborted their seeds du- ring the flowing, suffered laboratory accidents were lost in the green house (Table 2).

[95] Detection of the gene bar.

[96] The regenerated plants were used in preliminary immu- nochromatography analysis by the method of TraitChek™ (Romer Labs), following directions from the manufacturer. Pieces cut out of le- aves were kneaded in 1.5ml_ tubes containing 300μΙ_ buffer AgraStrip, as described by the maker. Then, strips were inserted for detection of the expression of the protein PAT (Figure 3).

[97] Extraction of DNA and PCR

[98] The total DNA was extracted from approximately 100 in- sects using a slightly modified version of the method described by Cal- deron-Cortes, N., Quesada, M., Cano-Camacho, H., Zavala-Paramo, G., 2010. A simple and rapid method for DNA isolation from xylopha- gous insects. Int. J. Mol. Sci. 11 , 5056-5064. doi:10.3390/ijms11125056. The insects were kneaded with liquid nitro- gen and transferred to a tube of 1.5ml_-tube to be homogenized 1ml_ of pre-heated extraction buffer containing 20ml_ of EDTA (pH 8), 100 mM Tris-HCI (pH 7.5), 1.4 M NaCI, 2% CTAB, 4% PVP and 2% β- mercaptoethanol. The material was then incubated at 60°C for 30 mi- nutes with occasional agitation. Then, 2 μΙ_ of 1mg/ml_ RNase were added and the mixture emulsified by mild inversion. This mixture was subjected to centrifugation at 13000xg for 15 minutes and the superna- tant was collected, precipitated and incubated for 20 minutes. The re- sulting DNA pellets were washed in 70% ethanol and re-suspended in water. This DNA was used for cloning and identification of the fragment of v-ATPase.

[99] In order to isolate the DNA from leavesk a modified version of the method of Doyle and Doyle, 1987. A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochem. Bull. 19, 11- 15. doi: 10.2307/4119796 was adopted. Discs cut out of the leaves we- re collected and homogenized in CTAB containing 1% of PVP at 65°C, then treated with pure chloroform and centrifuged at 13000xg for 5 mi- nutes. The supernatant was collected in tubes and precipitated with cold ethanol. The pellet was then washed with ethanol, 70%, and sus- pended in water. Before the PCR, samples of lettuce DNA were heated at 95°C for 5 minutes. All the nucleic acids were quantified by using the apparatus ND-1000 NanoDrop Spectrophotometer (BioRad).

[100] The reactions of PCR were carried out by using the enzyme Taq DNA Polimerase (Invitrogen), except for another indication, with annealing temperature of 58°C. Typically, the reaction of PCR contai- ned the components listed in Table 1.

[101] The initiators ATPSK/ATPXS (SEQ ID 5 and SEQ ID 6) we- re used in the reactions of PCR that detected the presence of the fra- gment of v-ATPase in the samples of T₀ e T^ The same thing was con- firmed with the use of the pair of initiators BarF/BarR (SEQ ID 9 and SEQ ID 10), which enabled detection of a fragment of 408pb, which detected the presence of the gene bar (Figure 4).

[102] One observed inheritance of the characteristic in the men- delian pattern in most of the first lineages of the generation TF Ti pro- ducing seeds. These were subjected to PCR analysis, and the indivi- duals with positive result of the lineages 1 , 3, 4, 6, 19, 25 e 31 were selected for bioassay and other molecular analyses.

Table 2: Summary of 25 lineages of transgenic lettuce generated by transformation with A. tumefaciens.

Table 3: Analysis of segregation of self-pollinated lettuce plants in ge- neration T

b: P is probability that the rates observed reflect the expected segrega- tion rate;

'correction of Yates for continuity was used for analysing the segrega- tion pattern.

[104] Seed of the first generation (T₁ of self-pollinated plants we- re germinated and analysed for the presence of v-ATPase-LEG by PCR, as described before. The Chi-square of Pearson (χ2) was used for determining if the segregation rate observed was consistent with the Mendelian pattern Mendelian (3:1), with reliability degree of 95%. When necessary, the Yate correction fact was used for the same pur- pose. The results demonstrated that the inheritance took place within the Mendelian pattern.

[105] Analysis of siRNAs:

[106] The total lettuce RNA was isolated by using the minikit miRNeasy (Qiagen - 217004), following the directions of the manufac- turer. The analysis of siRNA was carried out according to Bonfim, K., Faria, J.C., Nogueira, E.O.P.L., Mendes, E.A., Aragao, F.J.L., 2007. RNAi-mediated resistance to Bean golden mosaic virus in genetically engineered common bean (Phaseolus vulgaris). Mol. Plant. Microbe. Interact. 20, 717-726. doi:10.1094/MPMI-20-6-0717. The hybridization was carried out by using a DNA probe corresponding to the fragment of v-ATPase amplified by PCR with the initiators ATPSK/ATPXS (SEQ ID 5 e SEQ ID 6), marked with dCTP a32P using the kit random primer DNA labeling (Amersham Pharmacia Biotech), following the directions of the manufacturer. The hybridization and post-hybridization washings were conducted as described in Yoo, B.-C, Kragler, F., Varkonyi- Gasic, E., Haywood, V., Archer-Evans, S., Lee, Y.M., Lough, T.J., Lu- cas, W.J. ,2004. A systemic small RNA signaling system in plants. Plant Cell 16, 1979-2000. doi:10.1105/tpc.104.023614. Three oligonucleoti- des (18, 24 and 39 nucleotides) were used as molecular size markers. The RNA extracted from leaves of seven transgenic plants (T1) were used in Northern blot analyses for detection of siRNA derived from transgene, besides the RNA of a non-transgenic plant (control - Nt) (figure 5). The analysis disclosed siRNA bands of expected size in all the transgenic plants with greater signal intensity observed in the line- ages 1 , 4, 19 and 31 , as compared with the other lineages.

[107] Essay for toxicity on whitefly:

[108] The text for toxicity was carried out using 20 subjects of whi- tefly exposed to plants with 4-week age, grown in plastic glasses, which were transferred to covered plastic jars. The covering of the jars was perforated and each perforation was sealed with cotton to enable circulation of air. The population of whiteflies was then monitored daily. The number of whiteflies per plant was recorded for a period of two weeks, until all the flies were dead. During the 3^rd post-release week, eggs, nymphs, pupas and adults were viewed leaves of each plant with stereomicroscope. The transgenic lineage 1 , 3, 4, 6, 19, 25 and 31 we- re used for the analysis. Two control types were used: transgenic plants expressing the gene bar and non-transgenic plants. The appea- ranee of eggs, nymphs of all the instar, pupas and adults was monito- red during a 28-32-day cycle. The experiment was kept in a photoperi- od of 16h to 25±2°C in the system D, as described in Figure 2. The count of eggs, larvae, pupas and adults was carried out every 4 days, from the 11^th day after release of the insects on the plant. The day 11 was regarded as day 1 for the analysis of the development of egg for adult. For each lineage, 12 biologic repetitions were used and their averages were used for Tukey (Prism software, version 5.0) analysis.

[109] The feed essay, in which the transgenic lettuce plants ex- pressing siRNA of v-ATPase were challenged with newly emerged adults of whiteflies and their mortality was monitored for a period of 32 days. Within the first three days, three was a clear decrease in the number of whiteflies that fed on all the transgenic lineages (Figure 7). This drop was significantly slower in the two control experiments carri- ed out. On day 5, one observed a large number of flies on the control plants, a picture quite different from that observed on transgenic linea- ges (Figure 6. Whiteflies fed with transgenic plants exhibited statisti- cally higher mortality rates as compared with insects fed with non- transgenic plants and transgenic plants expressing a marking (P<0,05). The death by ingestion of siRNA from leaves apparently began in the first three days of feeding, when over 50% of the adults originally intro- duced were dead, whereas only 5% of the population was reduced on control plants, representing a mortality rate of 75% (figure 7). For exa- mple, in day tree, when there were between 15.25 and 17 insects (cor- responding to 76.25 to 85% of the population) on control plants, there were only between 6 and 10.92 (30 - 54.6% of the population) of in- sects on test plants Table 4). This may be translated as a mortality between 56.4 and 70% on transgenic plants within the first three days. On day five, this numeric relationship kept at 11.25 to 11.33 insects (56.25 - 53.65%) on control plants and 1.92 to 3.25 (9.6 - 16.25%) on transgenic plants, corresponding to a mortality rate between 83.75 to 98.08% on test plants, respectively. On about the 10 day, the number of whiteflies on all the transgenic plants expressing the siRNA of v- ATPase was close to zero, while on control plants one still observed a little less than 5% of the original population.

Table 4. Number of whiteflies feeding on lettuce plants for 11 days. The numbers are averages of 12 repetitions.

[110] Molecules of siRNAs of v-ATPase on transgenic lettuce plants promote low oviposition and delay In the formation of pu- pas on whiteflies:

[111] The feeding cycle of whiteflies on lettuce plants was evalua- ted to the effect of monitoring the appearance of eggs, larvae, pupas and adults. Generally, one observed a smaller number at all the stages off lies that were fed with test plants, as compared to those fed with control plants.

[112] The monitoring of the oviposition of 20 whiteflies fed with transgenic lettuce plants demonstrated that the flies of the test plants produced a smaller number of eggs than both controls (P < 0.05). On the 12^th post-inoculation day (day 1 of the count), the 20 flies deposited between 227 and 231 eggs (Figure 8) and produced between 107 and 125 larvae (Figure 9) on control plants, while the flies of the test plants produced between 24 and 66 eggs and from 13 to 52 larvae. The num- ber of eggs dropped in all the lineages and controls along the 32 days of analysis. However, on control plants, this drop seemed more mar- king within the first nine days of egg counting. After this, it decelerated until on the 17^th day the drop became more significant again, and then suddenly the number of eggs began to rise. This increase is related to the number of adults that emerge (Figure 11). However, on test plants the drop was slower (Figure 8). Although the lineages 3 and 31 seem to stabilize the number of eggs during the first week, all of them tend to converge with the other lineages until day 9, when they demonstrate a sudden drop until day 13. Additionally, many eggs seem to have been aborted. Although this abortion is observed in both experiments with transgenic and non-transgenic plants, it was not clear whether it was more frequent in the first case.

[113] The pattern of appearance of larvae followed in a way simi- lar to that of egg deposition, with the number of larvae being much lo- wer (Figure 9). The difference between the control and the test plants in terms of population of nymphs became quite clear when one consi- dered the fact that day 1 is the peak day for its production. While the control lineages recorded between 107.41 and 124.91 larvae, the number of larvae generated in the transgenic lineages on this day was of 48 (lineage 4) with the lowest, of lineage 6, being of 13.92. Both con- trols produced pupae earlier and the highest rates than the test plants (Figure 10). On day 5, between 4.9 and 6.2 pupae emerged on control plants, while the transgenic lineages did not demonstrate any evidence of new pupas. The peak day for appearance of pupas on both control plants and test plants was day 13. However, while on the control plants the number of pupas that appeared was of 23.75 to 24.42, on the test plants one observed a maximum number of new pupas of 15.05, in li- neage 4, with the minimum number of 2, observed in lineages 6 and 19.

[114] New adults began to emerge from the 18^th day after infesta- tion on all the control plants. However, transgenic plants began to exhibit new adults 2 days after the control plants (Figure 11). While the peak for the appearance of adults for control plants took place on the 17th day, with 11.33 to 13.33 flies on the day, on the test plants the peak took place on the 13th day, with the largest number of adults at 1.94. Since then, the appearance of adults decreased. It is not clear whether the siRNA molecules act differently at each stage of deve- lopment of the flies, but it was evident that the death of emerging flies generally occurred after the beginning of the feeding with the transge- nic plant.

Claims

1. A method of producing a plant resistant to insect, cha- racterized by inserting into said plant a DNA molecule whose expressi- on results in an RNA molecule in which the sequence comprises at least one segment 18 or more contiguous nucleotides complementary to a fragment of SEQ ID 1 or SEQ ID 2.

2. The method of claim 1 , characterized in that the expres- sed RNA molecule causes death or impairs the development of the in- sect that ingests it.

3. The method of claim 1 , wherein said nucleotide segment is transcribed in sense and antisense orientation, resulting in an, at least partially, double-stranded RNA.

4. The method of claim 1 , characterized in that the insect belongs to the order Hemiptera.

5. The method of claim 2, characterized in that the insect belongs to the genus Bemisia sp.

6. The method of claim 3, characterized in that the insect belongs to the species Bemisia tabaci.

7. A plant characterized by being produced by the method of claim 1.

8. The plant of claim 7, characterized by being selected from the species of soybean plant, tomato plant, cau pi-bean plant and common-bean plant, cotton plant, vegetables and related crops.

9. A fruit, seed or propagative parts, characterized by being of the plant of claim 6.

10. An isolated nucleic acid molecule characterized by com- prising a nucleic acid sequence with at least 90% similarity with the se- quence described in SEQ ID No1 or in SEQ ID No2.

11. The nucleic acid molecule according to claim 10, cha- racterized by exhibiting at least 95% similarity with the sequence des- cribed in SEQ ID No1 or in SEQ ID No2.

12. The nucleic acid molecule according to claim 11 , cha- racterized by exhibiting at least 99% similarity with the sequence des- cribed in SEQ ID No1 or in SEQ ID No2.

13. The nucleic acid molecule according to claim 12, cha- racterized by exhibiting a nucleic acid sequence according to the se- quence described in SEQ ID No1 or in SEQ ID No2.

14. An isolated nucleic acid molecule characterized by com- prising a nucleic acid sequence with at least 90% similarity with the complement of the sequence described in SEQ ID No1 or in SEQ ID No2.

15. The nucleic acid molecule according to claim 14, cha- racterized by exhibiting at least 95% similarity with the complement of the sequence described in SEQ ID No1 or in SEQ ID No2.

16. The nucleic acid molecule according to claim 15, cha- racterized by exhibiting at least 99% similarity with the complement of the sequence described in SEQ ID No 1 or in SEQ ID No2.

17. The nucleic acid molecule according to claim 16, cha- racterized by exhibiting a nucleic acid sequence complementary to the sequence described in SEQ ID No1 or in SEQ ID No2.

18. A nucleic acid molecule characterized by hybridizing with the nucleic acid sequence with similarity of at least 90% with the sequence described in SEQ ID No1 or in SEQ ID No2.

19. The nucleic acid molecule according to claim 18, cha- racterized by hybridizing with a nucleic acid sequence with similarity of at least 95% with the sequence described in SEQ ID 1 or in SEQ ID No2.

20. The nucleic acid molecule according to claim 19, cha- racterized by hybridizing with a nucleic acid sequence with similarity of at least 99% with the sequence described in SEQ ID 1 or in SEQ ID Νθ2.

21. The nucleic acid molecule according to claim 20, cha- racterized by hybridizing with a nucleic acid sequence according to the sequence described in SEQ ID 1 or in SEQ ID No2.

22. A fragment of at least 20 contiguous nucleotides, cha- racterized by exhibiting at least 90% similarity with the sequence des- cribed in SEQ ID No1 or in SEQ ID No2;

23. The fragment according to claim 22 of at least 20 conti- guous nucleotides of a nucleic acid sequence, characterized by exhibi- ting at least 95% similarity with the sequence described in SEQ ID No1 or in SEQ ID No2.

24. The fragment according to claim 23 of at least 20 conti- guous nucleotide of a nucleic acid sequence, characterized by exhibi- ting at least 99% similarity with the sequence described in SEQ ID No1 or in SEQ ID No2.

25. The fragment according to claim 24 of at least 20 conti- guous nucleotides of a nucleic acid sequence, characterized by exhibi- ting a sequence like the sequence described in SEQ ID No1 or in SEQ ID No2.

26. A chimeric gene characterized by comprising: a) a polynucleotide according to any one of claims 10 to 25; and

b) an active promoter, operatively defined in (a).

27. A gene construct characterized by comprising one or more chimeric genes constructed according to claim 25.

28. A gene construct characterized by comprising:

I. a first region with a nucleotide sequence of 20 to 564 consecutive nucleotides having at least 90% similarity with a sequence of 20 to 564 consecutive nucleotides of the sense nucleotide sequence described in SEQ ID No1 or SEQ ID No2; II. a second region with a nucleotide sequence of 20 to 564 consecutive nucleotides having at least 90% similarity with the com- plement of 20 to 564 consecutive nucleotides of the nucleotide se- quence described in SEQ ID No1 or SEQ ID No2.

29. A gene construct characterized by comprising: a. a first region with a nucleotide sequence of 20 to 564 consecutive nucleotides having at least 95% similarity with a sequence of 20 to 564 consecutive nucleotides of the sense nucleotide sequence as described in SEQ ID No1 or SEQ ID No2;

b. a second region with a nucleotide sequence of 20 to 564 consecutive nucleotide having at least 95% similarity with the comple- ment of 20 to 564 consecutive nucleotides of the nucleotide sequence as described in SEQ ID No1 or SEQ ID No2.

30. The gene construct according to claim 29, characterized by comprising:

a. a first region with a nucleotide sequence of 20 to 564 consecutive nucleotides having at least 99% similarity with the sequen- ce of 20 to 564 consecutive nucleotides of the sense nucleotide se- quence described in SEQ ID No1 or SEQ ID No2;

b. a second region with a nucleotide sequence of 20 to 564 consecutive nucleotides having at least 99% sequence similarity with the complement of 20 to 564 consecutive nucleotides as described in SEQ ID No1 or SEQ ID No2.

31. The gene construct according to claim 30, characterized by:

a. a first region with a nucleotide sequence of 20 to 564 consecutive nucleotides as described in SEQ ID No1 or SEQ ID No2;

b. a second region with a nucleotide sequence of 20 to 564 consecutive nucleotides complement of 20 to 564 consecutive nucleo- tides of the sequence as described in SEQ ID No1 or SEQ ID No2.

32. The gene construct according to claim 27, 28, 29, 30 or 31 , characterized in that the first and the second regions are capable of forming a double-stranded RNA region, which may have, in addition to the total length of the first and of the second region, a spacer region.

33. The gene construct according to claim 32, characteri- zed in that the spacer region between the first and the second region exhibits an extent of 100 to 800 nucleotides.

34. The gene construct according to claim 32, or 33, charac- terized in that the spacer sequence is an intron.

35. A vector characterized by comprising the isolated nu- cleic acid molecule according to claim 10, 11 , 12, 13, 14, 15, 16 or 17.

36. The vector according to claim 35, characterized in that said vector is capable of promoting the expression of the molecule of interest or a fragment thereof.

37. A double-stranded ribonucleotide sequence characteri- zed by being produced from the expression of a nucleic acid molecule according to claim 36.

38. A transformed cell characterized by comprising the nu- cleic acid molecule according to claim 10 to 25.

39. The cell according to claim 38, characterized by being an eukaryotic cell.

40. The cell according to claim 39, characterized by being a plant cell.

41. A transformed plant characterized by comprising a nu- cleic acid molecule defined in any of claims 10 to 25.

42. A transformed plant characterized by comprising the nu- cleic acid molecule defined in claim 32.

43. A transgenic seed or propagative part characterized by being of a plant according to claim 41 or 42.

44. A method of controlling insect, characterized by expo- sing insect that infests plant to the dsRNA molecule, whose sequence comprises at least one segment of 18 or more nucleotides of SEQ ID 1 or SEQ ID 2.

45. The method according to claim 43, the insect being se- lected from the order Hemiptera.

46. The method according to claim 44, the insect being se- lected from the genus Bemisia sp.

47. The method according to claim 45, the insect belonging to the species Bemisia tabaci.

48. The method according to claim 43, characterized in that the dsRNA molecule is synthesized chemically or produced by expres- sion in a microorganism or by expression in plant cell.

49. The method according to claim 43, characterized by ex- posing the insect that infests plant to dsRNA molecule through applica- tion of a composition containing said dsRNA onto the insect or onto a surface of the plant infested by the insect.

50. The method according to claim 48, characterized in that the composition comprises a solid treatment, liquid, in powder, in sus- pension, in emulsion, in spray, with microbeads, with particulate carri- ers, in film in matrix or seed.

51. The method according to claim 43, characterized by ex- posing the insect to the dsRNA molecule in a composition that compri- ses one or more components selected from the group consisting of a carrying agent, a surfactant, an organosilicone, a herbicidal polyncleo- tide molecule, a herbicidal non-polynucleotide molecule.

52. An insecticidal composition characterized by containing a molecule with at least 90% similarity with SEQ ID 1 or SEQ ID 2.

53. An insecticidal composition characterized by containing a molecule with at least 95% similarity with SEQ ID 1 or SEQ ID 2.

54. An insecticidal composition characterized by containing a molecule with 100% similarity with SEQ ID 1 or SEQ ID 2.

55. A plant characterized by being treated with the composi- tion of claims 52 to 54.