Transformation of HBsAg (Hepatitis B Surface Antigen) Gene into Tomato Mediated by Agrobacterium tumefaciens

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1 Trnsformtion of HBsAg (Heptitis B Surfe Antigen) Gene into Tomto Medited y Agroterium tumefiens Tin Li, Jing Kun Sun, Zho Hu Lu nd Qing Liu Shndong Provinil Key Lortory of Eo-Environmentl Siene for Yellow River Delt, Binzhou University, Binzhou, Chin Astrt: The plnt expression vetor pbrsag ws onstruted s suitle for trnsformtion vi Agroteriummedited pproh. It ontins ll elements for plnt expression, suh s CMV 35S promoter, oth left nd right order sequene for trnsferred DNA (T-DNA) in Agroterium, plnt reporter gene gus, nd plnt seletion mrker gene hpt. The reominnt inry vetor pbrsag ws trnsformed into Agroterium tumefiens strins y using the freeze-thw method. Tomto otyledon explnts were trnsformed y A. tumefiens nd plnts were regenerted on seletion medium. GUS stining, PCR nd PCR-Southern nlysis of the plnts were positive. It ws for the first time shown tht the HBsAg (heptitis B surfe ntigen) gene ws introdued into tomto plnts. The expression of trnsgene is under investigtion. Keywords: Agroterium; HBsAg gene; tomto; trnsformtion With the development of gene engineering tehnology, the study of using gene modified plnts to produe vines nd meditions hs eome hot spot. Plnt-sed vines hve ttrted the ttention of worldwide sholrs for its sfety nd eonomy, most of whih hve een expressed through geneti trnsformtions nd hve shown stisftory effets nd hve een put into ommeril prodution, suh s the vines of Heptitis B (Mson et l. 1992), pulmonry tuerulosis (Rigno et l. 6), ries (Modelsk et l. 1998), mesles virus (Bouhe et l. 3) et. Heptitis B virus (HBV) is glol disese. It n e trnsmitted y lood nd ody fluid nd its pthogenesis is omplex. With the inresing infetion of HBV, it hevily thretens people s helth. Epidemiologil dt revel tht there re out 36 million rriers of heptitis B virus throughout the gloe nd 78% of the world popultions hil from Asi (Behl et l. 8). Nowdys, n injetion of HBV vine is the only eonomi nd effetive wy to ontrol this disese. The ommerilly ville HBV vine is yest-derived nd the high ost hs led to the reserh for heper nd sfer methods to produe effetive heptitis B vines (Higshihshi et l. 1991; Shodel et l. 1994). Although vst mount of informtion is ville out the gene trnsformtion of heptitis B surfe ntigen (HBsAg), there re still mny truths to seek. Suessful tretment of HBV rriers with HBsAg plnt vine from plnts must hve enefiil effet on regions without fvourle environmentl snittion. Geneti engineering n e used to produe desirle gronomi trits quikly nd effiiently, nd lso to introdue genes enoding high-vlue reominnt proteins (Arokirj et l. 2). In omprison with the trditionl expression systems, plnt-derived heptitis B vine hs ttrted the ttention of the phrmeutil industry for resons of sfety nd eonomy (Wlmsley & 69

2 Arntzen ). In the experiment with mie nd humn eings, prenterl immuniztion of plntderived heptitis B surfe ntigen stimulted B nd T ell immune responses similr to those found with ommeril yest-derived heptitis B vine (Thnvl et l. 1995). So, it is fesile to produe heptitis B vine vi trnsgeni plnts. The ojetive of presented study ws to trnsform tomto s ndidte rop for edile vine prodution with HBsAg gene vi Agroteriummedited pproh. MATERIALS AND METHODS Plnt mteril The seeds of tomto Yugung 41 were purhsed from the Ademy of Agriulturl Sienes in Fujin Provine. The seeds were surfe sterilized with 75% lohol for 4 6 s nd wshed 3 4 times in sterilized distilled wter. Then the seeds were sterilized with 2% NClO for min nd wshed gin. Finlly, the seeds were sown nd germinted on hlf-strength Murshige nd Skoog (1962) (MS) medium. The otyledons of germinted seedlings were isolted, their pil prts were removed nd otyledons were setioned trnsversely into two segments. Plsmids psprok, pcambia-131, nd pbsag were preserved in our lortory, of whih the plsmid psprok ontins CMV35s promoter nd nos termintor, plsmid pcambia-131 ontins hyg-resistne gene, kn-resistne gene nd gus gene, nd plsmid pbsag ontins the 681 p DNA frgment (nmed HBsAg). The frgment ontining P35s-Tnos ws isolted y gel extrtion from plsmid psprok fter EoR I nd Hind III restritive digestion nd inserted into plsmid pcambia-131, whih hd een digested y the sme restrition endonulese to yield the reonstruted plsmid pcbrok. A 681 p DNA frgment nmed HBsAg ws otined y gel extrtion from plsmid pbsag fter X I nd Kpn I restritive digestion nd then suloned into plsmid pcbrok, whih hd een digested y the sme restrition endonulese to yield the reonstruted plnt inry expression plsmid pbrsag (Figure 1). All plsmids were isolted y the lkline lysis method nd purified y phenol nd hloroform extrtion (Smrook et l. 1989). Agroterium strin The A. tumefiens strins LBA444, EHA5 nd C58 were trnsformed with the reominnt inry vetor pbrsag vi the freeze-thw method (Hofgen & Willmitzer 1988). Trnsformnts were seleted on LB gr (sein enzymi hydrolyste mg/l, yest extrt 5 mg/l, sodium hloride 5 mg/l) plte ontining knmyin 5 mg/l (Sigm, St. Louis, USA), rifmpiin 5 mg/l nd streptomyin mg/l (Shndong Xinhu Phrmeutil Compny, Zio, Chin)nd the resistnt olonies were verified y restrition enzyme digestion nd used in trnsformtion experiments. Agroterium medited trnsformtion nd multiplition of trnsgeni plnts Tomto otyledon explnts were pre-ultivted on MS medium with 6-BA 2. mg/l nd IAA.2 mg/l (Shnghi Chemil Regent Compny, Shnghi, Chin) for 2 dys, then they were trnsformed y A. tumefiens with pbrsag. The otyledon explnts fter o-ultivtion for 2 dys on MS with 6-BA Constrution of plnt expression vetors Figure 1. Struture of plsmid pbrsag 7

3 2. mg/l nd IAA.2 mg/l were trnsferred to MS seletion medium with 6-BA 2. mg/l, IAA.2 mg/l, timentin mg/l (SmithKline Beehm Phrmeutils, Worthing, UK) nd hygromyin 7 mg/l (Sigm, St. louis, USA). Shoots were generted from the trnsformed llus on seletion medium fter 4 5 weeks. The shoots whih grew into 2 3 m were trnsferred for rooting on MS with IAA.5 mg/l, timentin mg/l nd hygromyin 7 mg/l. The rooting plnts growing into plntlets were trnsplnted into soil. GUS stining Both trnsformed nd untrnsformed of tomto plnts were immersed into GUS retion uffer for 12 to 24 h t 37 C nd then lehed with solute lohol for 3 5 h. Moleulr nlysis PCR nlysis. The modified method of CTAB (Stewrt & Vi 1993) ws used to extrt the genomi DNA of trnsgeni tomto plnts nd untrnsformed tomto plnts. PCR method ws used to onfirm the trnsgene integrtion into the GUS positive puttive trnsformnts. The primer pirs were: forwrd: 5 -CGCAGTCCCAAATCTCC-3, nd reverse: 5 -TGGTAACAGCGGCATAAA-3. ml of PCR mix ontined 25 ng of genomi DNA s templte, mm of eh primer,.3 ml of Tq DNA polymerse (5 U/ml) (Tkr, Dlin, Chin), 1.8 ml of eh dntp (2.5mM), 2. ml of PCR uffer. Cyling prmeters were t 94 C for 6 min, followed y 35 yles t 94 C for 1 min, 52 C for 1 min, nd t 72 C for 1 min, nd finl extension t 72 C for min, 4 C preservtion. Southern nlysis of PCR produts. PCR-Southern lotting ws rried out to further verify the integrtion of the trget gene (HBsAg) into the genome of tomto plnts. The lotting nd susequent hyridiztion were rried out ording to Smrook et l. (1989). Sttistil nlysis All dt on the trnsformtion frequeny represent the men vlues of three independent experiments with minimum of 5 explnts per tretment. RESULTS AND DISCUSSION Effets of mjor ftors on tomto trnsformtion The geneti trnsformtion medited y Agroterium is omplited proess whih is influened y mny prmeters suh s Agroterium strin (Crne et l. 6), explnt genotype (Chen et l. 8), o-ultivtion durtion (Brik et l. 5) nd signl moleules (Sthel et l. 1985). High-frequeny trnsformtion relies on the highly effiient T-DNA delivery from Agroterium into plnt ells (Ko et l. 3). Different strins of A. tumefiens vry in their trnsforming ilities. A omprtive study ws performed on the intertion of three different A. tumefiens strins (LBA444, EHA5 nd C58). Aording to the results of the GUS expression frequeny, A. tumefiens strin LBA444 ws found to e the most suitle, s GUS expression ws the highest (21%) in these explnts when ompred to EHA5 nd C58 (Figure 2A). So A. tumefiens strin LBA444 ws used in ll susequent experiments. The results were in line with those of Bhtthrjee et l. (). In their study, three different Agroterium strins (LBA444, EHA5 nd AGL1) were used nd the highest numer of lue-stined tissue tivity ws otined from the puttive trnsgeni tissue infeted y LBA444 strin. Figure 2B shows omprison of the trnsformtion frequeny fter different infetion time. The trnsformtion frequeny ws inresed with the infetion time inresing from to min. Results showed tht the -min infetion time in the presene of Agroterium produed the highest perentge of GUS positive explnts (19.6%). However, teri grew on the seletion medium signifintly t the time eyond min nd the trnsformtion frequeny ws ritilly redued. The results indited tht too short infetion time ws not fvourle for trnsformtion. However, too long infetion time resulted in the overgrowth of Agroterium nd therefore it ws hrmful to explnts (Shivni et l. ). Bteril onentrtion plys n importnt role in the prodution of trnsformnts. More nerosis of explnts ws seen t the highest onentrtions of teri for ll ultivrs. There ws signifint liner reltionship etween the perentge of neroti explnts nd teril onentrtion, 71

4 nd there ws signifint intertion of ultivr with teril onentrtion (Dvis et l. 1991). Our results showed tht the trnsformtion frequeny ws inresed signifintly s the teril onentrtion rose from.1 to.4 (OD 6 ), while the trnsformtion frequeny ws redued s the teril onentrtion rose from.6 to 1. (OD 6 ) (Figure 2C). The results were similr to those of Sini nd Jiwl (7), who reported tht higher Agroterium onentrtions ould (A) GUS frequeny (%) 4 3 (B) d LBA444 EHA5 C Bteril strin Infetion time (min) (C) GUS frequeny (%) e d d (D) Bteril onentrtion (OD 6 ) Co-ultivtion time (dys) (E) 4 GUS frequeny (%) 3 d 3 As onentrtion (mg/l) Figure 2. Effet of vrious ftors on the trnsformtion effiieny (GUS positive expression); (A) effet of A. tumefiens strins on trnsformtion frequeny, (B) effet of infetion time on trnsformtion frequeny, (C) effet of A. tumefiens ell density on trnsformtion frequeny, (D) effet of o-ultivtion time on trnsformtion frequeny, (E) effet of AS onentrtion on trnsformtion frequeny; mens of trnsformtion frequenies were ompred using Fisher s LSD test (P <.5) nd the sme letters re not signifintly different 72

5 led to hypersensitive responses of explnts. Thus, reltively lower inoulum density improved the trnsformtion effiieny nd redued the Agroterium overgrowth in this study. Co-ultivtion is very importnt in the trnsformtion proess. Bteri tthment, T-DNA trnsfer nd integrtion were rried out during this stge, whih ould e elerted y supplementing some ingredients to the o-ultivtion medium or prolonging o-ultivtion so tht it ould e terminted suessfully (Brik et l. 5). The influene of o-ultivtion on Agroterium-medited trnsformtion hs een reported in numer of plnt speies (Bnerjee et l. 2; Mohn & Krishnmurthy 3). In our study, the highest GUS expression frequeny ws oserved with 2-dy o-ultivtion nd it ws remrkly different from the other o-ulture durtion (Figure 2D). Two dys were lso onfirmed s the optimum o-ultivtion durtion wheres 3-dy o-ultivtion might use the overgrowth of A. tumefiens leding to dmge of the plnt ells nd onsequently resulting in low trnsformtion frequeny. On the other hnd, shorter o-ultivtion time might disrupt A. tumefiens ell prolifertion, therey reduing its virulene nd leding to low trnsformtion frequeny. Some relitrnt plnt speies n e trnsformed y induing the vir genes of the teri y signl moleules or in vitro y o-ultivtion of Agroterium with wounded tissue or in medi tht ontin signl moleules (Sthel et l.1985). Aetosyringone (As) or relted ompounds funtioning s signl moleules hve een reported to improve the Agroterium-medited trnsformtion in severl plnt speies (Šváová & Grig 8; Wng et l. 9). In our study, mg/l nd mg/l As signifintly improved tomto trnsformtion effiieny, while 3 mg/l As signifintly redued tomto trnsformtion effiieny, the mximum frequeny of trnsformtion ws oserved t mg/l As (Figure 2E). Our experiments showed tht tomto trnsformtion effiieny ws enhned under low As onentrtion, ut it ws redued under high As onentrtion. The present results were in line with those of Priy & Shivendr (9). Trnsformed plnts were seleted on the sis of their resistne to hygromyin. The integrtion of trnsgene in different lines ws onfirmed y GUS stining. Tken together, tomto shoot prodution under the optiml onditions determined ove ws onduted: strin LBA444,.4 teril onentrtion (OD 6 ), -minute infetion, o-ultivtion for 2 dys nd mg/l etosyringone. Regenertion nd GUS stining of trnsgeni plnts Trnsformed tomto otyledon explnts showed the development of shoots on the hygromyin seletion medium. The shoots were exised nd ultured on rooting medium with 7 mg/l hygromyin. The trnsformed shoots ould develop roots nd grow into omplete plntlets on the seletion medium of this onentrtion wheres untrnsformed shoots hrdly developed ny roots nd hrdly grew into plntlets. The shoots were trnsferred to new seletion medium every two weeks. Most untrnsformed shoots were eliminted during out 4 6 weeks of seletion nd rooting ulture. Twenty-one independent hyg-resistnt plntlets were otined fter out 2 months of seletion (Figure 3). The trnsformtion events of puttive hygromyin-resistnt trnsgeni lines were onfirmed y GUS histohemil nlysis. After tomto lef tissues were stined with GUS retion solution nd lehed with solute lohol, the whole of the trnsformed tissues ppered nerly deep lue olour while untrnsformed tissues ppered olourless. GUS histohemil ssy is onvenient nd fst tehnique to detet the integrtion nd expression of trget gene whih ws widely used in mny plnt speies (Šváová et l. 5; Sri Shilp et l. ). The GUS histohemil ssy of the of T plnts showed lue olourtion, onfirming the presene of the trnsgene (Figure 3F). In this study, we used stringent hygromyin seletion, so GUS expression ws oserved in most of tomto ultivrs. This result lso indited the reliility of the seletion regime for otining trnsformed tomto plnts. Moleulr nlysis of trnsgeni tomto plnts PCR ws used to onfirm the trnsgene integrtion into the GUS positive puttive trnsformnts. The prtil gene-speifi HBsAg frgment (495 p) of the expeted size ws oserved in 73

6 Czeh J. Genet. Plnt Breed., 47, 11 (2): Figure 3. Trnsgeni tomto plnts otined fter Agroterium-medited trnsformtion; (A) llus nd shoots egn to pper t the wounded sites of the otyledon explnts fter 2 weeks of seletion, (B) shoots regenerted from llus 4 weeks lter, (C) shoots were trnsferred onto MS medium for elongtion, (D) shoots growing well on the rooting medium, (E) mture plnts trnsplnted in plsti pots, (F) GUS stining of the trnsgeni T plnts: I II trnsgeni tomto plnts; III non-trnsgeni tomto plnts (negtive ontrol) the trnsgeni plnts while it ws sent in the untrnsformed wild-type tomto plnts (negtive ontrol). The length (495 p) of the PCR mplifi74 tion frgment ws identil with tht mplified from pcambia-131 plsmid (positive ontrol). The results primrily verified tht the trget gene

7 Figure 4. PCR nlysis of trnsgeni tomto plnts; 1 DNA mrker II (DL p); 2,4 negtive ontrol; 3 positive ontrol; 5 8 trnsgeni tomto plnts Figure 5. PCR-Southern lot nlysis of trnsgeni tomto plnts; 1 6 trnsgeni tomto plnts; 7 non-trnsgeni tomto plnt (negtive ontrol); 8 enzyme shering of pbrsag with X I/Kpn I (positive ontrol) ws integrted into the genomi DNA of trnsformed tomto plnts (Figure 4). The PCR produts fter 1% gel seprtion were lotted onto nylon memrnes. The plsmid DNA ontining the HBsAg gene ws lelled with DIG nd used for the hyridiztion with the mplified PCR produts to onfirm the suess of trnsformtion. The trnsformed plnts gve the sme hyridiztion spots s did the positive ontrol, wheres the untrnsformed plnts showed no detetle hyridiztion signl (Figure 5). The results of PCR-Southern lot nlysis lerly demonstrted the presene of the trget gene in the tomto plnts. CONCLUSION Plnts n e used s the most effetive systems for the prodution of therpeuti proteins, inluding reominnt vines. In our study, tomto otyledon explnts were trnsformed with the plnt expression vetor pbrsag hrouring HBsAg gene vi Agroterium-medited pproh. The trnsgeni nture of the plnts ws onfirmed y GUS stining, PCR nd PCR-Southern nlysis. A numer of ftors whih re importnt for the onsistent prodution of trnsgeni tomto plnts inluding Agroterium strins, infetion time, o-ultivtion time, Agroterium onentrtions nd etosyringone onentrtions were evluted. The present results demonstrted the fesiility nd effetiveness of A. tumefiens strin LBA444 hrouring plsmids with HBsAg gene under the optimized onditions for tomto trnsformtion. This study would ly the foundtion for the development of new type of plnt-derived heptitis B vine or other edile vines nd promote their prtil pplition. However, dditionl study would e required to e rried out. The trnsformnts should e further onfirmed y Southern lot nd Northern lot, whih re superior to PCR nd PCR-Southern. The trget gene/gene of interest (GOI) expression should e deteted y Western lot while the mount of HBsAg protein should e deteted y ELISA. In ddition, more experiments re needed to onfirm the inheritne of trnsgenes in suessive genertions (T 1, T 2 ) nd the mode of GOI heritility. Aknowledgements. This study ws supported y the Eleventh Five-yer Pln for Supporting Siene nd Tehnology of Chin (No. 6BAC1A13) nd y the Ntionl Nturl Siene Foundtion of Chin (No ). Referenes Arokirj P., Rueker F., Oermyr E., Shmsul Bhri A.R., Hfsh J., Crer D.C., Yeng H.Y. (2): Expression of humn serum lumin in trnsgeni Heve rsiliensis. Journl of Ruer Reserh, 5: Bnerjee A.K., Agrwl D.C., Nlwde S.M., Krishnmurthy K.V. (2): Trnsient expression of β-gluuronidse in emryo xes of otton y Agroterium nd prtile omrdment methods. Biologi Plntrum, 45: Brik D.P., Mohptr U., Chnd P.K. (5): Trnsgeni grsspe (Lthyrus stivus L.): ftors influening Agroterium-medited trnsformtion nd regenertion. Plnt Cell Reports, 24:

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9 Thnvl Y., Yng Y.F., Lyons P., Mson H.S, Arntzen C. (1995): Immunogeniity of trnsgeni plntderived heptitis B surfe ntigen. Proeedings of the Ntionl Ademy of Sienes of the United Sttes of Ameri, 92: Wlmsley A.M., Arntzen C.J. (): Plnts for delivery of edile vines. Current Opinion in Biotehnology, 11: Wng B., Liu L.J., Wng X.X., Yng J.Y., Sun Z. X., Zhng N., Go S.M., Xing X.L., Peng D.X. (9): Trnsgeni rmie [Boehmeri nive (L.) Gud.]: ftors ffeting the effiieny of Agroterium tumefiensmedited trnsformtion nd regenertion. Plnt Cell Reports, 28: Reeived for pulition Jnury 21, 11 Aepted fter orretions My 9, 11 Corresponding uthor: Prof. Zhohu Lu, Binzhou University, Shndong Provinil Key Lortory of Eo-Environmentl Siene for Yellow River Delt, Binzhou 25663, P.R. Chin e-mil: lu-zhh@263.net 77