Phân lập đoạn gen CP từ soybean mosaic virus và phát triển vector chuyển gen mang cấu trúc RNAi phục vụ tạo cây chuyển gen kháng bệnh

B GIO DC V O TO I HC THI NGUYấN Lề TH MAI THU PHÂN LậP ĐOạN GEN CP Từ SOYBEAN MOSAIC VIRUS Và PHáT TRIểN VECTOR CHUYểN GEN MANG CấU TRúC RNAi PHụC Vụ TạO CÂY ĐậU TƯƠNG CHUYểN GEN KHáNG BệNH Chuyờn ngnh: Di truyn hc Mó s: 62 42 01 21 LUN N TIN S SINH HC Ngi hng dn khoa hc: GS.TS Chu Hong Mu PGS.TS Chu Hong H THI NGUYấN - 2014 S húa bi Trung tõm Hc liu http://www.lrc-tnu.edu.vn/ i LI CAM OAN Tụi xin cam oan õy l cụng trỡnh nghiờn cu ca tụi Cỏc kt qu trỡnh by lun ỏn l trung thc, mt phn ó c cụng b trờn cỏc Tp khoa hc vi s ng ý v cho phộp ca cỏc ng tỏc gi, phn cũn li cha c cụng b bt k cụng trỡnh no khỏc Tỏc gi S húa bi Trung tõm Hc liu http://www.lrc-tnu.edu.vn/ ii LI CM N Tụi xin by t lũng bit n ti GS.TS Chu Hong Mu, PGS.TS Chu Hong H ó tn tỡnh hng dn, to iu kin thun li cho tụi sut quỏ trỡnh hc tp, nghiờn cu v hon thnh lun ỏn Tụi xin cm n Phũng Thớ nghim trng im Cụng ngh gen, cm n TS Lờ Vn Sn cựng ton th cỏn b , Phũng Cụng ngh t bo thc vt thuc Vin Cụng ngh Sinh hc ó tn tỡnh giỳp , truyn t nhng kinh nghim quý bỏu cho tụi quỏ trỡnh nghiờn cu v thc hin ti lun ỏn Xin cm n s giỳp ThS , ThS Tụi xin chõn thnh cm n PGS.TS Nguyn Th Tõm cựng th cỏn b B mụn Di truyn & Sinh hc hin i ó giỳp , to iu kin thun li cho tụi sut quỏ trỡnh hc v nghiờn cu ti lun ỏn Tụi xin cm n cỏc thy cụ Khoa Sinh-K thut nụng nghip v B phn qun lý nghiờn cu sinh, Phũng o to, trng i hc S phm i hc Thỏi Nguyờn, xin cm n Lónh o trng i hc S phm v Lónh o i hc Thỏi Nguyờn Tụi xin cm n s giỳp v to iu kin ca Ban ch nhim khoa Sinh Tụi xin cm n gia ỡnh v bn bố ó luụn ng viờn, giỳp tụi sut quỏ trỡnh hc v thc hin thnh cụng lun ỏn ny Nghiờn cu sinh Mai Thu S húa bi Trung tõm Hc liu http://www.lrc-tnu.edu.vn/ iii MC LC Trang Li cam oan i Li cm n ii Mc lc iii vi viii x M U 1 t Mc tiờu nghiờn cu . Ni dung nghiờn cu Nhng úng gúp mi ca lun ỏn í ngha khoa hc v thc tin Chng TNG QUAN TI LIU 1.1 BNH KHM DO VIRUS CY U TNG . nh virus cõy u tng 1.1.2 S xõm nhp ca SMV v BYMV vo t bo u tng . 1.1.3 H gen ca SMV v BYMV . 10 1.2 NGHIấN CU CHUYN GEN CY U TNG 16 1.2.1 16 1.1.1 1.2.2 nh hng ng dng v mt s thnh tu v chuyn gen cõy u tng S húa bi Trung tõm Hc liu http://www.lrc-tnu.edu.vn/ 23 iv 1.2.3 Tỡnh hỡnh nghiờn cu to cõy u tng bin i gen Vit Nam 28 1.3 K THUT RNAi TRONG TO CY CHUYN GEN KHNG VIRUS 29 1.3.1 Cỏc c ch RNAi c ch gen thc vt 30 1.3.2 ng dng k thut RNAi nghiờn cu to cõy chuyn gen khỏng virus 36 Chng VT LIU V PHNG PHP NGHIấN CU . 40 2.1 VT LIU, THIT B, HểA CHT V A IM NGHIấN CU 40 2.1.1 Vt liu thc vt 40 2.1.2 Vector v chng vi khun . 42 2.1.3 Húa cht v thit b 42 2.1.4 a im nghiờn cu . 42 2.2 PHNG PHP NGHIấN CU . 43 2.2.1 Nhúm cỏc phng phỏp phõn lp gen 43 2.2.2 Nhúm cỏc phng phỏp thit k vector chuyn gen mang cu trỳc RNAi 48 2.2.3 Phng phỏp chuyn gen thụng qua A tumefaciens 52 2.3.4 Nhúm cỏc phng phỏp phõn tớch cõy chuyn gen 58 Chng KT QU NGHIấN CU V THO LUN 60 3.1 TCH DềNG V PHN TCH TRèNH T ON GEN CP CA SMV 60 3.1.1 Tỏch dũng v xỏc nh trỡnh t nucleotide on gen CP t SMV. 60 3.1.2 Phõn tớch s a dng ca trỡnh t gen CP ca cỏc dũng SMV 67 S húa bi Trung tõm Hc liu http://www.lrc-tnu.edu.vn/ v 3.2 THIT K VECTOR CHUYN GEN MANG CU TRC RNAi 74 3.2.1 Thit k vector chuyn gen mang cu trỳc RNAi khỏng n loi SMV 74 3.2.2 Thit k vector chuyn gen mang cu trỳc RNAi khỏng hai loi SMV v BYMV 81 3.3 KT QU TO CY THUC L CHUYN GEN KHNG SMV V BYMV 89 3.3.1 Kt qu chuyn cu trỳc CPi (SMV-BYMV) vo cõy thuc lỏ 89 . 92 RNAi 96 96 3.4 K ut cỏc dũng cõy u - 99 KT LUN V NGH . 106 KT LUN 106 NGH . 107 108 TI LIU THAM KHO 109 S húa bi Trung tõm Hc liu http://www.lrc-tnu.edu.vn/ vi DANH MC CH VIT TT A tumefaciens Agrobacterium tumefaciens AS Acetosyrigone BAP 6-benzyladenine purin BGM Bean golden mosaic virus bp Base pair BYMV Bean yellow mosaic virus CCM Co-cultivation medium CAMV Cauliflower mosaic virus CMV Cucumber mosaic virus ) CP ) ) CPi SMV on bo th c thit k t gen CP ca SMV CPi(SMV-BYMV) on bo th c thit k t gen CP ca c SMV v BYMV DNA Deoxyribo nucleic acid dNTP Deoxynucleoside triphosphate tg ng tỏc gi E coli Escherichia coli EDTA Ethylene diamine tetraacetic acid GA3 Gibberellic acid GM Germination medium (mụi trng ny mm) gus Gen mó húa enzyme -Glucuronidase IAA Indoleacetic acid IBA Indole-3-butyric acid kb Kilo base LB Luria and Bertani S húa bi Trung tõm Hc liu http://www.lrc-tnu.edu.vn/ vii MS Murashige & Skoog (1962) -Mụi trng c bn NAA -Naphthaleneacetic acid nptII Neomycin phosphotransferase gene OD Optical density Mt quang PCR Polymerase Chain Reaction Phn ng chui Polymerase PPT Phosphinothricin PRSV Papaya ringspot virus pK7GW-CPi-SMV: Vector chuyn gen mang cu trỳc CPi-SMV pK7GW-CPi (SMV-BYMV) Vector chuyn gen mang cu trỳc CPi (SMVBYMV) RM Rooting medium (mụi trng r) RNAi RNA interference SDS Sodium dodecylsulfat SEM Shoot elongation medium (mụi trng kộo di chi) SIM Shoot induction medium (mụi trng to chi) SL1 SMV dũng Sn La - Vit Nam SL2 SMV dũng Sn La - Vit Nam SMV Soybean mosaic virus Taq Thermus aquaticus T-DNA Vựng DNA plasmid chuyn vo thc vt Ti- plasmid Plasmid to u TMV Tobacco mosaic virus TYLCV Tomato yellow leaf curl virus v/p vũng/phỳt Vir Virulence Region X-gluc 5-bromo-4-chloro-3-indolyl glucuronide YEP Yeast extract peptone S húa bi Trung tõm Hc liu ) ) http://www.lrc-tnu.edu.vn/ viii DANH MC BNG Trang Bng 1.1 2014 Bng 2.1 Thnh phn phn ng tng hp cDNA 45 Bng 2.2 Thnh phn phn ng PCR 46 Bng 2.3 Thnh phn phn ng ghộp ni gen vo vector pBT 47 Bng 2.4 Thnh phn phn ng ghộp ni gen vo vector pENTRTM/D-TOPO 50 Bng 2.5 Thnh phn phn ng LR 51 Bng 2.6 Thnh phn phn ng ct bng enzyme hn ch 52 Bng 2.7 Thnh phn cỏc loi mụi trng tỏi sinh in vitro 53 Bng 2.8 Thnh phn dung dch m tỏch DNA tng s 58 Bng 3.1 Trỡnh t cp mi PCR thit k s dng nhõn bn on gen CP 60 Bng 3.2 Cỏc v trớ sai khỏc gia trỡnh t on gen CP ca SMV dũng SL2 so vi dũng SL1 v trỡnh t cú mó s X63771 65 Bng 3.3 Cỏc v trớ sai khỏc gia trỡnh t amino acid ca protein suy din dũng SL2 so vi dũng SL1 v trỡnh t cú mó s CAA45307 66 Bng 3.4 Ngõn hng gen quc t ng phõn tớch S húa bi Trung tõm Hc liu http://www.lrc-tnu.edu.vn/ 68 ix Bng 3.5 69 Bng 3.6 Trỡnh t cp mi SMV-CPi-Fi/SMV-CPi-Ri 76 Bng 3.7 Trỡnh t cp mi SMV-CPi-Fi/BYMV-CPi-Ri 83 Bng 3.8 Kt qu bin np cu trỳc CPi (SMV-BYMV) vo mnh lỏ thuc lỏ C9-1 91 gen 95 Bng 3.9 Bng 3.10 Kt qu bin np cu trỳc 2008 S húa bi Trung tõm Hc liu http://www.lrc-tnu.edu.vn/ 99 112 28 Afanasiev M M., Morris H E (1952), Bean Virus (yellow) on Great Northern bean in Montana, Phytopathology, 42, pp 101-104 29 Ambros V., Bartel B., Bartel D P., Burge, C B., Carrington J C., Chen X., Dreyfuss G., Eddy S R., Griffiths J.S., Marshall M (2003), A uniform system for microRNA annotation, RNA 9, pp 277 - 279 30 Aragóo F J L., Sarokin L., Vianna G R., Rech E L (2000), Selection of transgenic meristematic cells utilizing a herbicidal molecule results in the recovery of fertile transgenic soybean [Glycine max (L.) Merril] plants at a high frequency, Theor Appl Genet., 101, pp 31 Asad S., Haris W A A., Bashir A., Zafar Y., Malik K A., Malik N N., Lichtenstein C P (2003), Transgenic tobacco expressing geminiviral RNAs are resistant to the serious viral pathogen causing cotton leaf curl disease, Arch Virol 148, pp 2341-2352 32 Barnett O W (1991), Potyviridae, a proposed family of plant viruses, Arch Virol 118, pp 139 - 141 33 Bartel B., Bartel D P (2003), MicroRNAs: At the root of plant development Plant Physiol 132, pp 709 717 34 Bartel D P (2004), MicroRNAs: genomics, biogenesis, mechanism and function, Cell., 116, pp 281297 35 Bateson M., Lines R E., Revill P., Challprom W., Ha C., Gibbs A., Dale J L (2002), On the evolution and molecular epidem biology of the potyvirus Papaya ringspot virus, J Gen Virol., 83, pp 2575 - 2585 36 Baulcombe D (2004), RNA silencing in plants, Nature, 431, pp 356 - 363 37 Beijersbergen A., Dulk R A D., Schilperoort R A., Hooykaas P J J (1992), Conjugative transfer by the virulence system of Agrobacterium tumefaciens, Science 256, pp 1324 1327 38 Berger P H., Wyatt S., Shiel P J., Silbernagel M J., Druffel K., Mink G I (1997), Phylogenetic analysis of the Potyviridae with emphasis on legumeinfecting potyviruses, Arch Virol., 142, pp 1979 1999 39 Bonfim K., Faria J C., Nogueira, E O., Mendes E A., Aragóo F J (2007), RNAi-mediated resistance to Bean golden mosaic virus in genetically engineered common bean (Phaseolus vulgaris), Mol Plant Microbe Interact, 20, pp 717 726 S húa bi Trung tõm Hc liu http://www.lrc-tnu.edu.vn/ 113 40 Bousalem M., Douzery E J P., Fargette D (2000), High genetic diversity, distant phylogenetic relationships and intraspecies recom bination events among natural populations of Yam mosaic virus: a contribution to understanding potyvirus evolution, J Gen Virol., 81, pp 243-255 41 Bowers G R., Goodman R M (1979), Soybean mosaic virus: Infection of soybean seed parts and seed transmission, Phytopathology, 69 (6), pp 569 - 572 42 Bramley P M., Elmadfa I., Kafatos A., Kelly F J., Manios Y., Roxborough H E., Schuch W., Sheehy P J A., Wagner K H (2000), Vitamin E, J Sci Food Agric., 80, pp 13938 43 Brasileiro A C M., Aragao F J L (2001), Marker genes for in vitro selection of transgenic plants, Plant Biotech, 3, pp.113121 44 Brunt A A., Crabtree K., Dallwitz M J., Gibbs A J., Watson L., Zurcher E J (2003), Plant Viruses Online, Descriptions and Lists from the VIDE Database Version: 20th August 1996 45 Bubner B., Gase K., Baldwin I T (2004), Two - fold differences are the detection limit for determining transgene copy numbers in plants by real time PCR, BMC Biotechnology, 4, pp 4-14 46 Cahoon E B., Ripp K G., Hal S E., McGonigle B (2002), Transgenic production of epoxy fatty acids by expression of a cytochrome P450 enzyme from Euphorbia lagascae seed, Plant Physiol., 128, pp 615624 47 Carrington J C., Jensen P E., Schaad M C (1998), Genetic evidence for an essential role for potyvirus CI protein in cell-to-cell movement, Plant J., 14, pp 393 - 400 48 Chiera J M., Finer J J., Grabau E A (2004), Ectopic expression of a soybean phytase in developing seeds of Glycine max to improve phosphorus availability, Plant Mol Biol., 56, pp 895904 49 Cho E K and Goodman R M (1982), Evaluation of resistance in soybeans to soybean mosaic virus strains, Crop Sci., 22, pp 1133-1136 50 Christou P., McCabe D E., Swai W F (1988), Stable transformation of soybean callus by DNA-coated gold particles, Plant Physiol., 87, pp 671674 S húa bi Trung tõm Hc liu http://www.lrc-tnu.edu.vn/ 114 51 Chu Hoang Lan, Nguyen Tuan Anh, Nguyen Vu Thanh Thanh, Nguyen Hiep Hoa, Chu Hoang Mau (2010), Characterization of the GmDREB5 gene isolated from the soybean cultivar Xanh Tiendai, Vietnam, IEEE, pp 354-358 52 Chu Hoang Mau, Nguyen Thuy Huong, Chu Hoang Lan, Nguyen Tuan Anh, Le Van Son, Chu Hoang Ha (2011), Characteristics of the gene encoding pyrroline-5-carboxylate synthase (P5CS) in Vietnam soybean cultivars (Glycine max L Merrill), IACSIT Press., pp 319-323 53 Chu Hoang Mau, Nguyen Tuan Anh, Pham Thi Thanh Nhan, Dinh Thi Ngoc, Bui Thi Tuyet (2010), The characteristics of chaperonin gene isolated local soybean cultivars (Glycine max L Merrill) grown in Tay Nguyen region, Viet Nam, CCEA, pp 452-456 54 Chyi Y., Jorgense R A., Goldstein D., Tanksley S D., Figueroa L F (1986), Locations and stability of Agrobacterim-mediated T-DNA insertions in the Lycopersicon genome, Mol Gen Genet., 204, pp 64 69 55 Cunha N B., Murad A M., Cipriano T M., Araỳjo A C G., Aragóo F J L., Leite A., Vianna G R., McPhee T R., Souza G H M F., Waters M J.(2011), Expression of f unctional recombinant human growth hormone in transgenic soybean seeds, Transgenic Res., 20, pp 811826 56 Dahmer M L., Collins G B., and Hildebrand D F (1991), Lipid content and composition of soybean somatic embryos, Crop Sci., 31, pp 741746 57 Dahmer M L., Hildebrand D F., Collin, G B (1992), Comparative protein accumulation patterns in soybean somatic and zygotic embryos, In Vitro Cell Dev Biol, 28, pp 106114 58 DeCleene M and DeLey J (1976), The host range of crown gall, Bot Rev., 42, pp 389466 59 Denli A M., Tops B B J., Plasterk R H A., Ketting R F., Hannon G J (2004), Processing of primary microRNAs by the Microprocessor complex, Nature 432, pp 231235 60 DeRonde J A., Laurie R N., Caetano T., Greyling M M., Kerepesi I (2004), Comparative study between transgenic and non-transgenic soybean lines proved transgenic lines to be more drought tolerant, Euphytica,138, pp 123132 S húa bi Trung tõm Hc liu http://www.lrc-tnu.edu.vn/ 115 61 Dougherty W., Parks, D (1991), Post-translational processing of the tobacco etch virus 49-kDa small nuclear inclusion polyprotein: Identification of an internal cleavage site and delimitation of VPg and proteinase domains, Virology, 183, pp 449-456 62 Dugas D V., Bartel, B (2004), MicroRNA regulation of gene expression in plants, Curr Opin Plant Biol., 7, pp 512520 63 Elbashir S M., Lendeckel W., Tuschl T (2001), RNA interference is mediated by 21- and 22- nucleotide RNAs, Genes Dev., 15, pp 188200 64 Eun A J C., Seoh M L., Wong S M (2000), Sim ultaneous quantitation of two orchid viruses by the TaqMan real-time RT-PCR, J Virol Meth., 87, pp 151-160 65 FAO/WHO (1990), Expert consultation on protein quality evaluation Food and Agriculture Organization of the United Nations, Rome 66 Finer J J., Nagasawa A (1988), Development of an embryogenic suspension culture of soybean (Glycine max Merrill.), Plant Cell Tissue and Organ Cult., 15, pp 125136 67 Fire A., Xu S., Montgomery M K., Kostas S A., Driver S E., Mello C C (1998), Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans, Nature, 391, pp 806-811 68 Fondong V N., Pita J S., Rey M E C., de Kochko A., Beachy R N., Fauquet C M (2000) Evidence of synergism between African cassava mosaic virus and a new double-recombinant geminivirus infecting cassava in Cameroon, J Gen Virol., 81, pp 287297 69 Fuentes A., Ramos P.L., Fiallo E., Callard D., Sỏnchez Y., Peral R., Rodrớguez R., Pujol, M (2006), Intron-hairpin RNA derived from replication associated protein C1 gene confers immunity to Tomato yellow leaf curl virus infection in transgenic tomato plants, Transgenic Res.,15, pp 291-304 70 Furutani N., Yamagishi N., Hidaka S., Shizukawa Y., Kanematsu S., Kosaka Y (2007), Soybean mosaic virus resistance in transgenic soybean caused by post-transcriptional gene silencing, Breed Sci., 57, pp 123128 S húa bi Trung tõm Hc liu http://www.lrc-tnu.edu.vn/ 116 71 Furutani N., Hidaka S., Kosaka Y., Shizukawa Y., Kanematsu S., (2006), Coat protein gene - mediated resistance to soybean mosaic virus in transgenic soybean, Breed Sci., 56, pp 119124 72 Gelvin S B (2010), Plant proteins involved in Agrobacterium - mediated genetic transformation, Annu Rev Phytopathol., 48, pp 4568 73 Gibbs A., Harrison B (1976), Plant virology the principles, Edward Amold 74 Gillings M., Broadbent P., Indsto J., Lee, R (1993), Characterisation of isolates and strains of citrus tristeza clostero virus using restriction analysis of the coat protein gene amplified by the polymerase chain reaction, J Virol Meth., 44, pp 305-317 75 Gough K H., Shukla D D (1992), Major sequence variations in the N terminal region of the capsid protein of a severe strain of passion fruit woodiness potyvirus, Arch Virol., 124, pp 389-396 76 Hamilton J H., Baulcombe D C (1999), A species of small antisense RNA in post - transcriptional gene silencing in plants, Science, 286, pp 950-952 77 Hammond R W., Crosslin J M., Pasini R., Howell W E., Mink G I (1999), Differentiation of closely related but biologically distinct cherry isolates for prunus necrotic ringspot virus by polymerase chain reaction, J Virol Meth., 80, pp 203-212 78 Hammond S M., Bernstein E., Beach D., Hannon G J (2000), An RNAdirected nuclease mediates post - transcriptional gene silencing in Drosophila cells, Nature, 404, pp 293 - 296 79 Hansen G., Wright M S (1999), Recent advances in the transformation of plants, Trends Plant Sci., 4, pp 226231 80 Hartman G L., Sinclair J B., Rupe J C (1999), Compendium of Soybean Diseases, Fourth Edition, The American Phytopathological Society Press, Minnesota, USA 81 Hartman G L., West E D., Herman, T K (2011), Crops that feed the World Soybean-worldwide production, use and constraints caused by pathogens and pests, Food Sec., 3, pp 517 S húa bi Trung tõm Hc liu http://www.lrc-tnu.edu.vn/ 117 82 Herbers K., Meuwly P., Frommer W B., Metraux J P., Sonnewald U (1996), Systemic acquired resistance mediated by the ectopic expression of invertase: Possible hexose sensing in the secretory pathway, Plant Cell, (5), pp 793803 83 Herbers K (2003), Vitamin production in transgenic plants, J Plant Physiol., 160, pp 821829 84 Herman E M., Helm R M., Jung R., Kinney A J (2003), Genetic modification removes an immunodominant allergen from soybean, Plant Physiol., 132, pp 3643 85 Hill J., Bailey T B., Benner H I., Tachibana H., Durand D P (1987), Soybean mosaic virus: Effects of primary disease incidence on yield and seed quality, Plant Disease, 71, pp 237-239 86 Hill J H (1999), Soybean mosaic virus In Compendium of Soybean Diseases, 4th edn, pp 7071, Edited by G L Hartman J B Sinclair & J C Rupe St Paul, MN: American Phytopathological Society 87 Hinchee M A W., Conner W D V., Newell C A., McDonnell R E., Sato S J, Gasser C S., Fischhoff D A., Re D B., Fraley R T., Horsch R B (1988), Production of transgenic soybean plants using Agrobacterium-mediated DNA transfer, Nat Biotechnol., 6, pp 915922 88 Homrich M S., Strohm B W., Weber R L M., Zanettini M H B (2012), Soybean genetic transformation: A valuable tool for the functional study of genes and the production of agronomically improved plants, Genet Mol Biol., 35, pp 9981010 89 Hoppe P P., Krennrich G (2000), Bioavailability and potency of naturalsource and all-racemic -tocopherol in the human: a dis-pute, Eur J Nutr., 39, pp 183193 90 Hunst P L., Tolin S A (1983), Ultrastructural cytology of soybean infected with mild and severe strains of soybean mosaic virus, Phytopathology, 73, pp 615-619 91 James B (1982), Compendium of Soybean Diseases, Fourth Edition, The American Phytopathological Society Press, Minnesota, USA S húa bi Trung tõm Hc liu http://www.lrc-tnu.edu.vn/ 118 92 James C (2007), Global status of commercialized biotech/GM crops, Briefs 37, ISAAA 93 Jayaram C., Hill J H., Miller W A (1992), Complete nucleotide sequences of two soybean mosaic virus strains differentiated by response of soybean containing the Rsv resistance gene, J Gen Virol., 73, pp 2067-2077 94 Jefferson R A., Burgess S M., Hirsh D (1986), -Glucuronidase from Escherichia coli as a gene-fusion marker, Proc Natl Acad Sci USA,83, pp 84478451 95 Jefferson R A., Kavanagh T A., Bevan M W (1987), GUS fusions: betaglucuronidase as a sensitive and versatile gene fusion marker in higher plants, Embo., 6, pp 39013907 96 Jones M L., Mathewson C S., Adkins V K., Ayllon T (2003), Use of behavioral contingencies to promote prevention of recurrent pressure ulcers, Arch Phys Med Rehabil., 84 (6), pp 796-802 97 Karimi M., Inzộ D., Depicker A (2002), Gateway vectors for Agrobacterium - mediated plant transformation, Trends Plant Sci., 7, pp 193 -195 98 Kasai M., Kanazawa A (2012), RNA silencing as a tool to uncover gene function and engineer novel traits in soybean, Breed Sci., 61, pp 468479 99 Kasschau K D., Cronin S., Carrington J C (1997), Genome amplification and long - distance movement functions associated with the central domain of tobacco etch potyvirus helper component-proteinase, Virology, 228, pp 251-262 100 Keller K E., Johansen I E., Martin R R., and Hampton R O (1998), Potyvirus genome-linked protein (VPg) determines peaseed-borne mosaic virus pathotype-specific virulence in Pisum sativum, Molec Plant Micr Inter 11: pp 124-130 101 Khalafalla M M., Rahman S M., El S H A., Nakamoto Y., Wakasa K., Ishimoto M (2005), Optimization of particle bombard-ment conditions by monitoring of transient sGFP(S65T) expression in transformed soybean, Breed Sci., 55, pp 257263 102 Kim Y H., Kim O S., Lee B C., Roh J H., Kim M K., Im D J., Hur I B., Lee S C (1999), Detection of soybean mosaic virus using RT-PCR, Korean J Crop Sci., 44, pp 253-255 S húa bi Trung tõm Hc liu http://www.lrc-tnu.edu.vn/ 119 103 Kirichenko A N (2013), The characterization of bean yellow mosaic virus isolated from soybean, Mikrobiol Z., 75 (3), pp 68-73 104 Kirthi N., Maiya, S P., Murthy M R., Savithri H S.(2002), Evidence for recombination among the tomato leaf curl virus strains/species from Bangalore, India Arch Virol., 147 (2), pp 255 272 105 Kirthi N., Priyadarshini C G., Sharma P., Maiya S P., Hemalatha V., Sivaraman P., Dhawan P., Rishi N., Savithri H S (2004), Genetic variability of begomoviruses associated with cotton leaf curl disease originating from India, Arch Virol.,149, (10), pp 20 47 2057 106 Kita Y., Nakamoto Y., Takahashi M., Kitamura K., Wakasa K., Ishimoto M (2010), Manipulation of amino acid composition in soybean seeds by the combination of deregulated tryptophan bio-synthesis and storage protein deficiency, Plant Cell Rep., 29, pp 8795 107 Kita Y., Hanafy M S., Deguchi M., Hasegawa H., Terakawa T., Kitamura K., Ishimoto M (2009), Generation and characterization of herbicide - resistant soybean plants expressing novel phosphino-thricin N-acetyltransferase genes, Breed Sci., 59, pp 245251 108 Koning, G., TeKrony, D M., Ghabrial, S A and Pfeiffer, T W (2002) Soybean mosaic virus (SMV) and the SMV resistance gene (Rsv1): influence on homopsis Spp seed infection in an aphid-free environment Crop Sci., 42, pp.178-185 109 Krause S R., Legall O., Fakhfakh H., Peypelut M., Marrakchi M., Varveri C., Pavan M A., Souche S., Lot H., Zerbini M., Candresse, T (2002), Molecular and biological characterization of Lettuce mosaic virus (LMV) isolates reveals a distinct and widespread type of resistancebreaking isolate: LMV-Most, Phytopathology, 92, pp 563-572 110 Kumar D., Yusuf M A., Singh P., Sardar M., Sarin N B (2013), Modulation of antioxidant machinery in -tocopherol-enriched transgenic Brassica juncea plants tolerant to abiotic stress conditions, Protoplasma, DOI 10.1007/s00709-013-0484-0 111 Li R., Yu K., Hatanaka T., Hildebrand D F (2010), Vernonia DGATs increase accumulation of epoxy fatty acids in oil, Plant Biotechnol., 8, pp 184195 S húa bi Trung tõm Hc liu http://www.lrc-tnu.edu.vn/ 120 112 Li Z., Meyer S., Essig J S., Liu Y., Schapaugh M A., Muthukrishnan S., Hainline B E., Trick H N (2005), High-level expression of maize -zein protein in transgenic soybean (Glycine max), Mol Breed., 16, pp 1120 113 Lim H S., Ko T S , Hobbs H A., Lambert K N., Yu J M., McCoppin N K., Korban S S., Hartman G L., Domier L L (2007), Soybean mosaic virus Helper Component-Protease Alters Leaf Morphology and Reduces Seed Production in Transgenic Soybean Plants, Phytopathology, 97(3), pp 366-372 114 Lopez S J., Kumar R R., Pius P K., Muraleedharan N (2004), Agrobacterium tumefaciens-mediated genetic transformation in Tea (Camellia sinensis [L.] O Kuntze), Plant molecular biology reporter, 22, pp 201-201 115 Maruthi M N., Seal S., Colvin J., Briddon R W., Bull, S E.(2004), East African cassava mosaic Zanzibar virus a recombinant begomovirus species with a mild phenotype, Arch Virol., 149, pp 23652377 116 Masuta C., Nishimura M., Morishita H., Hataya T (1999), A single amino acid change in viral genome- associated protein of potato virus Ycorrelates with resistance breaking inVirgin A Mutant tobacco, Phytopathology, 89, pp 118 -123 117 McCabe D E., Swain W F., Martinell B J., Christou P (1988), Stable transformation of soybean (Glycine max) by particle acceleration, Nat Biotechnol., 6, pp 923926 118 McKern N M., Barnett O W., Whittaker L A., Mishra A., Strike P M., Xiao X W., Ward C W., Shukla D D (1993), Sequence relationship among the coat proteins of strains of peamosaic, white lupin mosaic and bean yellow moasaic potyviruses, Phytopathology, 83, pp 255-361 119 Nicolas O., Dunnington S W., Gotow L., Pirone T P., Hellmann G M (1997), Variation in the VPg protein allow a potyvirus to overcome va gene resistance in tobacco, Virology, 237, pp 452-459 120 Nishizawa K., Kita Y., Kitayama M., Ishimoto M (2006), A red fluorescent protein, DsRed2, as a visual reporter for transient expression and stable transformation in soybean, Plant Cell Rep., 25, pp 13551361 S húa bi Trung tõm Hc liu http://www.lrc-tnu.edu.vn/ 121 121 Nishizawa K., Takagi K., Teraishi M., Kita A., Ishimoto M (2010), Application of somatic embryos to rapid and reliable analysis of soybean seed components by RNA interference-mediated gene silencing, Plant Biotechnol., 27, pp 409420 122 Olhoft P M., Donovan C M., Somers, D A (2006), Soybean (Glycine max) transformation using mature cotyledonary node explants Methods in molecular biology: Agrobacterium protocols, 2nd ed Humana Press Inc, Totowa, N, pp 385-396 123 Owens L D., Cress, D E (1985), Genotypic variability of soybean response to Agrobacterium strains harboring the Ti or Ri plasmids, Plant Physiol., 77, pp 8794 124 Padgette S R., Kolacz K H., Delannay X., Re D., LaVallee B J., Tinius C N., Rhodes K., Otero Y I., Barry G F., Eichholtz D A (1995), Development, identification, and characterization of a glyphosate-tolerant soybean line, Crop Sci., 35, pp 14511461 125 Parrott W A., Williams E G., Hildebrand D F., Collins G B (1989), Effect of genotype on somatic embryogenesis from immature cotyledons of soybean, Plant Cell Tissue Organ Cult., 16, pp 1521 126 Paz M M., Shou H., Guo Z., Zhang Z., Banerjee A K., Wang K (2004), Assessment of conditions affecting Agrobacterium - mediated soybean transformation using the cotyledonary node explant, Euphytica, 136, pp 167179 127 Paz M M., Martinez J C., Kalvig A B., Fonger T M., Wang K (2006), Improved cotyledonary node method using an alternative explant derived from mature seed for efficient Agrobacterium - mediated soybean transformation, Plant Cell Rep., 25, pp 206213 128 Pfosser M F., Baumann H (2002), Phylogeny and geographical differentiation of Zucchini yellow mosaic virus isolates (Potyviridae) based on molecular analysis of the coat protein and part of the cytoplamsic inclusion protein genes, Arch Virol., 147, pp 1599-1610 129 Pradeep K., Satya V K., Selvapriya M., Vijayasamundeeswari A., Ladhalakshmi D., Paranidharan V., Rabindran R., Samiyappan R., Balasubramanian P., Velazhahan R (2012), Engineering resistance against Tobacco streak virus (TSV) in sunflower and tobacco using RNA interference, Biologia Plantarum, 56 (4), pp 735-741 S húa bi Trung tõm Hc liu http://www.lrc-tnu.edu.vn/ 122 130 Qi Q., Huang J., Crowley J., Ruschke L., Goldman B S., Wen L., Rapp, W D (2011), Metabolically engineered soybean seed with en-hanced threonine levels: biochemical characterization and seed-specific expression of lysineinsensitive variants of aspartate kinases from the enteric bacterium Xenorhabdus bovienii, Plant Biotechnol., 9, pp 193204 131 Qiong H., Yanbing N., Kai Z., Yong L., Xueping Z (2011), Virus-derived transgenes expressing hairpin RNA give immunity to Tobacco mosaic virus and Cucumber mosaic virus, Virology Journal, : 41 132 Qusus S (1997), Molecular studies on soybean mosaic virus-Soybean interactions Ph.D Dissertation, Virginia Polytechnic Institute and State University, pp.163 133 Rao S S., Hildebrand D (2009), Changes in oil content of transgen-ic soybeans expressing the yeast SLC1 gene, Lipids, 44, pp 945951 134 Reinhart B J., Weinstein E G., Rhoades M W., Bartel B., Bartel D P (2002), MicroRNAs in plants, Genes Dev., 16, pp 16161626 135 Restrepo H M., Carrington J C (1994), The tobacco etch potyvirus 6kilodalton protein is membrane associated and involved in viral replication, J Virol., 68, pp 2388-2397 136 Rhoades M W., Reinhart B J., Lim L P., Burge C B., Bartel B., Bartel D P (2002), Prediction of Plant MicroRNA Targets, Cell, 110, pp 513-520 137 Roberts C A., Dietzgen R G., Heelan L A., Maclean D J (2000), Real time RT-PCR fluorescnet detection of tomato spotted wilt virus, J Virol Meth., 88, pp.1-8 138 Saghai M M A., Soliman K M., Jorgensen R A., Allard R W (1984), Ribosomal DNA spacer - length polymorphisms in barley: Mendelian inheritance, chromosomal location and population dymnamics, Proc Natl Acad Sci USA, 81, pp 8014-8018 139 Sambrook J., Fritsch E F., Maniatis T (2001), Molecular Cloning: A Laboratory Manual New York, Cold Spring Harbor Laboratory Press 140 Schaad M C., Haldeman C R., Cronin S., Carrington J C (1996), Analysis of the VPg-proteinase (NIa) encoded by tobacco etch potyvirus: Effects of mutations on the subcellular transport, proteolytic processing, and genome amplification, J Virol., 70, pp 7039-7048 S húa bi Trung tõm Hc liu http://www.lrc-tnu.edu.vn/ 123 141 Schwarz D S., Hutvagner G., Haley B., Zamore P D (2002), Evidenc that siRNAs funtion as guides, nt primers in the Drosophila and human RNAi pathways, Mol Cell, 10, pp 537-548 142 Selvaraj T., Nisha M C., Rajeshkumar S (2009), Effect of indigenous arbuscular mycorrhizal fungi on some growth parameters and phytochemical constituents of Pogostemon patchouli Pellet, Maejo international journal of science and technology, (1), pp 222-234 143 Setchell K D (1998), Phytoestrogens: the biochemistry, physiology, and implications for human health of soy isoflavones, J Clin Nutr., 68, pp 13331346 144 Shukla D D., Ward C W (1988), Amino acid sequence homology of coat proteins as a basis for identification and classification of the potyvirus group, J Gen Virol., 69, pp 2703-2710 145 Shukla D D., Ward C W., Brunt, A A (1994), The Potyviridae, CAB International, University Press, Cambridge, UK 146 Smith N A., Singh S P., Wang M B., Stoutjesdijk P A., Green A G., Waterhouse P.M (2000), Total silencing by intron-spliced hairpin RNAs, Nature, 407, pp 319320 147 Srivastava V., Vasil V., Vasil I K (1996), Molecular characterization of the face of transgenes in transformed wheat (Triticum aestivum L.), Theor Appl Genet., 92, pp 1031-1037 148 Stewart C N J., Adang M J., All J N., Boerma H R., Cardineau G., Tucker D., Parrott W A (1996), Genetic transformation, recovery, and characterization of fertile soybean transgenic for a synthetic Bacillus thuringiensis cryIAc gene, Plant Physiol., 112, pp 121129 149 Sugano M (2005), Soy in Health and Disease Prevention, CRC Press, New York, USA 150 Sunkar R., Zhu, J.K (2004), Novel and Stress-Regulated MicroRNAs and Other Small RNAs from Arabidopsis, Plant Cell, 16, pp 2001-2019 151 Tabashnik B.E (1994), Evolution of resistance to Bacillus thuringiensis, Annu Rev Entomol., 39, pp 4779 S húa bi Trung tõm Hc liu http://www.lrc-tnu.edu.vn/ 124 152 Takagi K., Nishizawa K., Hirose A., Kita A., Ishimoto M (2011), Manipulation of saponin biosynthesis by RNA interference-mediated silencing of -amyrin synthase gene expression in soybean, Plant Cell Rep., 30, pp 18351846 153 Tavva V S., Kim Y H., Kagan I A., Dinkins R D., Kim K H., Collins G B (2007), Increased -tocopherol content in soybean seed overexpressing the Perilla frutescens -tocopherol methyltransferase gene, Plant Cell Rep., 26: pp 6170 154 Tomlin E S., Branch S R., Chamberlain D., Gabe H., Wright M S., Stewart, C.N (2002), Screening of soybean, Glycine max (L.) Merrill, lines for somatic embryo induction and maturation capability from immature cotyledons, In Vitro Cell Dev Biol Plant, 38, pp 543548 155 Topping J.F (1998), Tobacco transformation, Methods of Mol Biol., 81, pp 365 372 156 Topping J F., Wei W., Lindsey, K (1991), Functional tagging of regulatory elements in plant genome, Development, 112, pp 1009-1019 157 Tougou M., Yamagishi N., Furutani N., Shizukawa Y., Takahata Y., Hidaka S (2007), Soybean dwarf virus -resistant transgenic soybeans with the sense coat protein gene, Plant Cell Rep., 26, pp 19671975 158 United Soybean Board (2000), World Soybean Production 1999, Report, Chesterfield, MO, USA 159 Urcuqui I S., Haenni A L., and Bernardi F (2001), Potyvirus proteins: a wealth of functions Virus Research 74: pp 157-175 160 Valente M A S., Faria J A Q A., Soares-Ramos J R L., Reis P A B., Pinheiro G L., Piovesan N D., Morais A T., Menezes C C., Cano M A O., Fietto L G (2009), The ER luminal binding protein (BiP) mediates an increase in drought tolerance in soybean and delays drought-induced leaf senescence in soybean and tobacco, J Exp Bot., 60, pp 533546 161 Vanderveken J J., Harris K F.,Maramorosch K (1977), Oils and other inhibitors of nonpersistent virus transmission, In Aphids as Virus Vectors Academic Press, London, pp 435-454 S húa bi Trung tõm Hc liu http://www.lrc-tnu.edu.vn/ 125 162 Walker D., Boerma H R., All, J Parrott W (2002), Combining cry1Ac with QTL alleles from PI 229358 to improve soybean resistance to lepidopteran pests, Mol Breed., 9, pp 4351 163 Walsh K., North J., Barker I., Boonham N (2001), Detection of different strains of Potato virus Y and their mixed infections using com petetive RTPCR, J Virol Meth., 91, pp 167-173 164 Wang D., Maule A.J (1995), Inhibition of host gene expression associated with plant virus replication, Science, 267, pp 229-231 165 Waterhouse P.M., Graham M.W., Wang M.B (1998), Virus resistance and gene silencing in plants can be induced by simultaneous expression of sense and antisense RNA, Proc Natl Acad Sci USA, 95, pp 1395913964 166 Wesley S V., Helliwell C A., Smith N A., Wang M B., Rouse D T., Lie Q., Gooding P S., Singh S P., Abbott D., Stoutjesdijk P A., Robinson S P., Gleave A P., Green A G., Waterhouse, P M (2001), Construct design for efficient, effective and high - throughput gene silencing in plants, Plant, 27, pp 581590 167 Wingard S A (1928), Hosts and symptoms of ring spot, a virus disease of plants, J Agric Res., 37, pp 127153 168 Won S L, Yul H K., Kook H K (2003), Complete Genome Sequences of the Genomic RNA of Soybean mosaic virus Strains G7H and G5, Plant Pathol., 19 (3), pp 171-176 169 Xue R G., Xie H F., Zhang B (2006), A multi-needle-assisted transformation of soybean cotyledonary node cells, Biotechnol Lett., 28, pp 15511557 170 Yadav N S (1996), Genetic modific ation of soybean oil quality In : Verma, D P S and R.C.Shoemaker (eds.) Soybean, Genetics, Molecular Biology and Biotechnology, Cab International, USA, pp 165188 171 Yamada T., Takagi K., Ishimoto, M (2012), Recent advances in soybean transformation and their application to molecular breeding and genomic analysis, Breeding Science, 61, pp 480494 172 Yamada Y., Nishizawa K., Yokoo M., Zhao H., Onishi K., Teraishi M., Utsumi S., Ishimoto M., Yoshikawa M (2008), Anti-hypertensive activity of genetically modified soybean seeds accumulating novo kinin, Peptides, 29, pp 331337 S húa bi Trung tõm Hc liu http://www.lrc-tnu.edu.vn/ 3,0kb 126 173 Yan P Q., Bai X Q., Wan X Q., Guo Z K., Li L J., Gong H Y., Chu C C (2007), Expression of TMV coat protein gene RNAi in transgenic tobacco plants confer immunity to tobacco mosaic virus infection, Yi Chuan, 29 (8), pp 1018-22 174 Ye F., Signer E R (1996), RIGS (repeat - induced gene silencing) in Arabidopsis is transcriptional and alters chromatin configuration, Proc Natl Acad Sci USA, 93, pp 10881-10886 175 Yin Z., Malepszy S (2003), The transgenes are expressed with different level in plants, Biotechnologia, (61), pp 236-260 176 Zeng P., Vadnais D A., Zhang Z., Polacco J.C (2004), Refined glu-fosinate selection in Agrobacterium-mediated transformation of soybean [Glycine max (L.) Merrill], Plant Cell Rep., 22, pp 478482 177 Zhang C., Song Y., Jiang F., Li G., Jiang Y., Zhu C., Wen F F (2012), Virus resistance obtained in transgenic tobacco and rice by RNA interference using promoters with distinct activity, Biologia Plantarum, 56 (4), pp 742748 178 Zhang X., Sato S., Ye X., Dorrance A E., Morris T J , Clemente T E., Qu F., (2012), Robust RNAi-based resistance to mixed infection of three viruses in soybean plants expressing separate short hairpins from a single transgene, Phytopathology, ,101 (11), pp 1264-1269 179 Zhang Z., Xing A., Staswick P., Clemente T E (1999), The use of glufosinate as a selective agent in Agrobacterium-mediated trans-formation of soybean, Plant Cell Tissue Organ Cult., 56, pp 3746 180 Zhu S., Walker D R., Boerma H R., All J., Parrott W A (2008), Effects of defoliating insect resistance QTLs and a cry1Ac transgene in soybean nearisogenic lines, Theor Appl Genet., 116, pp 455463 181 Zilberman D., Cao X., Jacobsen S E (2003), Argonaute4 control of locus specific siRNA accumulation and DNA and histone methylation, Science 299 (5607), pp 716-719 182 www.ncbi.nlm.nih.gov/GenBank/nucleotide S húa bi Trung tõm Hc liu http://www.lrc-tnu.edu.vn/ [...]... chung Tạo đƣợc dòng cây chuyển gen kháng virus gây bệnh khảm trên cây đậu tƣơng bằng kỹ thuật RNAi 2.2 Mục tiêu cụ thể (i) Xác định đƣợc trình tự đoạn gen CP từ SMV gây bệnh khảm ở cây đậu tƣơng Việt Nam (ii) Phát triển đƣợc vector chuyển gen mang cấu trúc RNAi chứa CPi SMV và gen (iii) Tạo đƣợc cây thuốc lá chuyển gen mang cấu trúc RNAi có khả năng kháng SMV và BYMV cao hơn cây đối chứng ; (iv) Tạo. .. loài virus gây bệnh ở thực vật Dựa trên nguyên lý bất hoạt gen sau phiên mã, một số tác giả đã ứng dụng thành công kỹ thuật RNAi trong chiến lƣợc tạo cây chuyển gen kháng virus [3], [31], [98] Xuất phát từ những lý do trên, chúng tôi tiến hành đề tài: Phân lập đoạn gen CP từ Soybean mosaic virus và phát triển vector chuyển gen mang cấu trúc RNAi phục vụ tạo cây đậu tương chuyển gen kháng bệnh 2 Mục... thành công vector chuyển gen mang cấu trúc gen RNAi chứa đoạn gen CPi SMV, tạo đƣợc cây thuốc lá chuyển gen có tính kháng đối với SMV BYMV và BYMV cao hơn cây đối chứng đã khẳng định cơ sở khoa học và tính hiệu quả của biện pháp sử dụng các gen có nguồn gốc từ chính loài virus gây bệnh để chuyển vào cây trồng nhằm tạo cây chuyển gen kháng virus theo nguyên lý của kỹ thuật RNAi Năm bài báo đăng tải... kiểu gen đậu tƣơng khác nhau [106] Các quá trình vật lý trong chuyển gen có xu hƣớng dẫn đến sự kết hợp, phân mảnh và tái cấu trúc gen chuyển, điều này đôi khi dẫn đến làm bất hoạt gen chuyển Việc sử dụng các gen thông báo và gen chỉ thị chọn lọc có thể trợ giúp làm giảm sự bất hoạt gen chuyển của hệ thống chuyển gen vật lý và tạo điều kiện cho sự phục hồi cây chuyển gen cũng nhƣ sự thể hiện của gen. .. xác định sự có mặt của gen chuyển trong tế bào cây chủ, kiểm tra sự biểu hiện của gen chuyển thông qua xác định sản phẩm biểu hiện là mRNA, protein và phân tích sự biểu hiện chức năng sinh học của gen chuyển Có thể mô tả khái quát các thí nghiệm chuyển gen và phân tích cây chuyển gen ở sơ đồ hình 1.3 Hình 1.3 Sơ đồ phương pháp phân tích cây chuyển gen Quá trình tạo cây chuyển gen đòi hỏi một phƣơng... Nghiên cứu thu thập thông tin về gen liên quan đến đặc tính, tính trạng quan tâm và phân lập gen; (ii) Thiết kế vector chuyển gen; (iii) Tạo vi khuẩn mang cấu trúc gen chuyển; (iv) Lây nhiễm vào mô tế bào thực vật; (v) Chọn lọc các thể biến nạp và tái sinh cây biến nạp; (vi) Phân tích cây chuyển gen Agrobacterium là loài vi khuẩn đất Gram âm gây bệnh khối u ở thực vật và có khả năng lây nhiễm một cách... 4.1 Đoạn gen CP đƣợc phân lập từ SMV dòng SL1 và S nucleotide, 720 240 amino acid Hai trình tự đoạn gen CP của SMV đã đƣợc đăng ký trên Ngân hàng gen quốc tế với mã số HG965102, HG965103 4.2 Phát triển mang cấu trúc Số hóa bởi Trung tâm Học liệu http://www.lrc-tnu.edu.vn/ 4 19 RNAi DT2008, thu 5 n gen 12 19 2008 5 Ý nghĩa khoa học và thực tiễn 5.1 Về khoa học Việc phát triển thành công vector chuyển. .. đƣợc cây đậu tƣơng chuyển gen mang cấu trúc RNAi Số hóa bởi Trung tâm Học liệu http://www.lrc-tnu.edu.vn/ 3 3 Nội dung nghiên cứu (1) Nghiên cứ (2) Nghiên cứu phát triển vector gen CPi của hai loài SMV và BYMV bằng kỹ thuật Gateway CPi đã thiết kế vào A tumefaciens (3) Nghiên cứu b Phân tích sự có CPi (4) Nghiên cứu thử nghiệm ch u tƣơng thông RNAi qua A tumefaciens CPi trên cây đậu tƣơng chuyển gen. .. mosaic virus – SMV), virus gây bệnh khảm vàng hại đậu đỗ (Bean yellow mosaic virus – BYMV) và một số virus gây bệnh khác Trong đó, SMV và BYMV là những loại virus gây bệnh khảm nhất đến cây đậu tƣơng SMV và BYMV đƣợc lây nhiễm từ cây bệnh sang cây khoẻ do rệp là môi giới virus có thể truyền qua hạt Khi cây đậu tƣơng bị bệnh, lá cây có những phần xanh nhạt, đậm và biến vàng xen kẽ, lá non ở ngọn bị biến dạng,... gợi ý sự cần thiết và kinh nghiệm tổ chức và quản lý chặt chẽ quá trình nghiên cứu chuyển gen ở đậu tƣơng ở các quốc gia trên thế giới, trong đó có Việt Nam 1.2.1.3 Phân tích cây đậu tương chuyển gen Kỹ thuật chuyển gen ở thực vật nói chung và chuyển gen ở cây đậu tƣơng nói riêng đƣợc hiểu đẩy đủ là: (i) gen ngoại lai (gen chuyển) phải đƣợc hợp nhất vào hệ gen tế bào chủ; (ii) gen chuyển phải đƣợc biểu