Apr 27, 2000 - Centre for Plant Molecular Biology and Department of Plant Molecular Biology, Uni versity of Delhi, South Campus,. Benito Juarez Road, New ...
Indi an Jou rnal of Biochemistry & Biophysics Vol. 37, December 2000, pp. 447-452
Analysis of the activity of promoters from two photosynthesisrre psaF -_ .. ated gene ..and petH of spinach in a monocot plant, rice
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. Amitabh 'M ohanly,~o nendr, Gr; ver, l.,Ashok, haudhury( Quazi,'Rizwan-ul-H aq, Arun
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, Salish 9 Maheshwari and Akhilesh K,fTyagi
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Cent re for Pl ant Molecular Biology and Department of Plant Molecular Biology, Uni versity of Delhi , South Campus, Benito Juarez Road, New Delhi 110~ ndi a Received 27 April 2000; accepted 22 May 2000
The subunil III of ~l o tosys le m .l and ferredoxin-NADP+-oxidoreductase are encoded by nuclear genes, namely ]J.wF and petH. J he ac ti vity of their pro moters from sp in ach has been eva luated in transge ni c tobacco earlier. Evaluati on of the acti vi ty of these Dicotyledoll eae-spec ilic promoters has bee n carried out in a monocot sys tem (i.c. ri ce) by transient ge ne expression sys tem, based on elec troporati on-medi ated gene delivery into protoplasts from leaves and roo ts. It has been found th at vari ous promoter deletions show hi gher ac ti vity in leaf protoplasts and elements for quantitative respo nse are widely di stributed. Transgenic rice has also been produced with a petH promoter and g lls reporter gene construct. Although petH promoter is a weak promoter in compari so n Lo the 3 ~ r moter, it eXPfesses well in green ti ssues and could be useful for plant ge neti c engi neerin g. J J l .~ ..-
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In the last few years , iso lati on of genes for prote in s invo lved in ph otosy nthes is and c haracte ri zati on of the ir promoters from hi g her pl a nts has led to a better understandin g of the mo lecular bas is of l photosynthes is -., . In mos t cases, act ivity of promoters fro m dicotyledonous as well as mon ocotyl ed o no us plants has bee n eva lu ated in to bacco, a dicot mode l 4 system o r a ho mo logous syste m .5. It has been sugges ted th at the mechanism of regulati o n of monocotyledonous a nd di cotyledono us ge nes or the genes from C3 and C4 plants may va ry and the regul atory e lements may no t be commo n o r ex pressed 9 correc tl y across the divide 6- . It is, the refo re, imperative to in vestigate these obse rvat io ns furth e r. In recent yea rs, because o f its sma ll geno me size, ava il ab ility of large numbe r of mo lec ul ar marke rs, expressed seq ue nced tags (ESTs) and cDNAs, together with a relatively we ll established syste m for tran sformati o n, rice has e me rged as a mode l c rop plant for in vest igatin g regulation of gene e xpress ion ' vestl.gate t Ile as we II as f un ct .to na I ge no ml.cs 10- 1S' . T 0 III regul ati o n by Dicotyledon eae-s pecific ge ne promoters
*AUlhor for correspondence Tel: + 11-46732 16, - 4675095 Fax: + I 1-6885270, -6886427 E-mail: aklpmb@holmai l. com
in a monocoty ledonou s plant, we have e mpl oyed . . 16 d . tran s ie nt gene ex press io n an tran sgenIc tec hn o logy' 7 of rice pl a nts w ith ph o tosy nthesisrelate d gene promoters 0 f psaF 18 and petH 19 f ro m s pin ac h driving gus reporter gene.
Materials and Methods Constructs for transient gene exp ression For trans ie nt ge ne exp ress ion studi es promote r fragments o f two photosynthetic genes, psaF and petH , a lo ng with glls gene, as desc ribed in F li egel' et al. l s and Oelmliller et al. 19 , res pec tive ly, were used . The followin g de letions of psaF gene promoter were used: -1074 to + 163, -687 to + 163, -220 to + I 63, ~ I 78 to +163 and -124 to +16 3 bp. Similarly, for petH two deletion constru cts (-753 to +23 I bp, -I 18 to +23 I bp) we re used . In both cases, the co nstructs in c lu de o nl y part o f the first exo n upstream to ATG codon.
COllstructsfor rice tral/stor/,/lation To stud y regulati o n of hetero logous gene pro mo te rs in ri ce, largest fragments from pro moters of psaF and petH of s pinach were cloned as SalIlBamHI fra g me nts in th e c lo nin g site of the promo te r c lo nin g vector pCAMBIA I 39 1Z , kindly provided by Dr. R. A. Je fferson , CAMBIA, Australia.
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INDIAN J BIOCHEM. BIOPHYS. , VOL. 37, DECEMB ER 2000
The recombinant plasmids are named as pCAMF and pCAMH, respectively. The gene construct pCAMH was used for stable genetic tran sformation. Transient gene expression
Seed material and plant growth Dehusked seeds of O. sativa var. Basmati 370 were surface-sterilized in 2.5 % (w/v) sodium hypochlorite for 30 min and washed several times with steri le di stilled water before inoculation on nutrient MS medium 20 . Seedlin gs were germinated in cu lture tubes at 26±2°C and -2411mol m·2s· 1 light intensity . Protoplast isolation Protoplast isolati on from leaves and roots was performed as desc ribed by Chaudhury et al. 16 Briefly , leaves from 10-day old light-grown seedlings were shredded longitudinally, chopped into small pi eces and incubated in an enzyme solution ( I g mr l) comprising of CPW salts (0.2 mM KH 2 P04 , I mM KNO.1, 10 mM CaCh, 1 mM MgS0 4 , I 11M KI and I 11M CUS04), I % Cellu lase R-IO and 0.5 % Macerozyme R-IO (Yakult Pharmaceuti ca ls , Japan), 0.6 M mannitol and 0.1 % MES (p H 5.8) at 28°C for 15 hr without shaking. Protoplasts from root ex plants were isolated from 5 to 6-day old dark-grown seedlin gs. Roots were shredded into small pieces and incubated in an enzyme so lution ( I g mrl ) comprising of CPW sa lts, 2% cellu lase RS (Yakult Pharmaceuti cals, Japan ), 3% Macerozyme R-IO, 0.1 % Pectolyase Y-23 (Seishin Pharmaceuti ca ls, Japan) , 0.5 M mannitol and 0.1% MES (PH 5.8) at 30°C for 15 hr on a shaker (20 rpm).
Gene delivery by electroporatio/l. The plasmid DNA was iso lated on large scale using CsCI density gradient and was qu antified spectrophotometerically by taking absorbance at 260 nm using a UV 3000 spectrophotometer (Shimadzu, Japan)1 6. Its quality was checked by ge l electrophoresis after digestion with unique restriction enzymes. Aliquots of solution containing 100 Il g DNA were removed in autoclaved 1.5 ml tubes and DNA was precipitated by the addition of 0.1 vol of 3 M sodium acetate (PH 5.2) and 2 vo l of ethan ol followed by incubation at -20°C for 15 min. The pe llet was obtained by centrifugation at 10,000 rpm and 4°C for 15 min in a microcentrifu ge tu be and washed in 70% ethanol, air-dried in a laminar fl ow and disso lved with 100 III of steri Ie disti lled water. For gene delivery experiments, protoplasts were resuspended in 1.0 ml of electroporation buffer (0.6 M mannitol, 100 mM MgC I2, 0. 1% MES , pH 5.8), float ed on 20% sucrose and centrifu ged at 100 g for 5 min . Protoplasts form ing a rin g at the interface were collected. An electroporat or compri sing a bank of capacitors was empl oyed to generate pul ses 16. The electroporation chamber co nsi stin g of a plex iglass cuvette with stainless stee l electrodes (0.5 cm apart) was sterilized in 70% ethan ol and dri ed. The ( 1-2x 106 mr l) in protoplasts suspensi on electroporation buffer was incubated on ice for 5 min and then mixed wit h sterili zed plasmid DNA (50 Ilg mrl). Protoplasts were given a single electri cal pulse ( 100 IlF, 300 V), incubated on ice for 10 min , pelleted at 100 g for three minutes as above and subsequently cultured in R2 medium 21 with 0.6 M glucose added at 25±2°C in diffuse li ght. Agro!Jacfer;/l1I/ -mediated t ransforma tion
PurifIcation of pro top lasts The protoplast suspension was passed through a sterili zed stee l mes h (45 11M) to remove undigested ti ssue and the protoplasts in the filtrate were pelleted in a centrifu ge (Clay Adams Co., Inc., USA) by spin at 100 g for fi ve min . Pelleted protoplasts were was hed three times with washing so lution (C PW salts, 0.6 M mannitol, pH 5.8). Protoplasts were purified by fl oatin g on 20% sucrose and centrifu gation. Those forming a rin g at the interface of 20% sucrose and protoplast suspension medium were fi nall y co llected and used for cou nting th e de nsity by a hemocytometer and furth er ex perimentati on.
Preparation ofAgrobacferilllll for co-clilli va fion For mobilizati on of pCAMH into AgrobacferiLIIIl fum efaciens LBA4404, triparental mating was performed22 with minor modifi cati ons. Before plant transformation , a singl e co lony was inoc ul ated into 30 ml liquid AB medium with kanamyc in (50 mg 1'1 ) and rifampicin ( 100 mg 1'1 ) and grow n for two days at 28°C. One ml of this bacterial culture was in ocul ated in fres h medium and grown for an other 36-48 hr. Bacteria were pelleted in SS-34 tubes at 4,000 rpm and 4°C for 10 min . The pell et was sus pended in I ml of AAM liquid medium and an aliqu ot was diluted to I ml to measure absorbance at 600 nm . Bacterial
MO HANTY
el al.:
ANALY SIS OF THE ACTIVITY OF PROMOTERS
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density was calcul ated and adj usted to 3-5x 10 mr l 17 with AA M medium .
Cal/lis induction Oryza sativa var Pusa Bas mati I seeds, after surface-s terilizat ion, were soaked in sterile water overni ght and nex t day they were blotted dry o n a steril e ti ssue paper and inocul ated on 3/2 MS medium l7 at 26±2°C in dark for 2 1 days. After thi s period, the pro li ferat ing calli deri ved fro m scutell a were dissected out an d sub-cultured on fresh 312 l7 MSCA medium fo r another 4 days. Fast grow ing, nodul ar, healthy loo king, embryogeni c calli were used for transformati o n. Co-cultivation, regeneration and Southern analysis These proced ures were performed as desc ribed 17. After co-culti vati o n for fo ur days, calli were washed with sterile water co ntaining cefotax ime (250 mg I- I), blotted on a steril e ti ssue paper to remove excess l7 moisture and in cubated on 3/2 MS C HCA medium at 26±2°C fo r three weeks. Th e hygro myc in resistant calli obtained after first round o f selecti o n were transferred to plates co ntaining fresh 3/2 MSCHCA medium fo r two to three weeks. Fresh and healthy look ing hygromyc in res istant ca lli were transferred to regenerati on medium co ntal I1lng BAP (0.5-1.0 mg r l)1 7 and in cubated at 26±2°C under 16 hr photoperiod. T he li ~ht intens ity was 100-1 25 )..lmo l m·2 S- I. Shoots of abo ut 3-4 cm length were transfen-ed to MS basal med ium contai ning hygro myc in (50 mg r l) fo r root fo rmati on. If required , frag il e loo king pl ants were cond iti oned in liquid ri ce growth medium for a week before transfer to soil. Rooted pl antl ets were transferred to pots containing a mi xture of soilrite (Kel Perli te, Bangalo re, Indi a) and so il (l : I) and grown to maturity in Gro wth C hamber (Co nviron, Control Environments Lim ited, Winnipeg, Canada) operating at 24-28°C, 16 hr li ght at 100-125 )..lmo l m~S-I and 70-75 % re lative humidity . The plants were supplied with ri ce growth medium 23 . So uthern analysis was performed as described by Mohanty et al 17 . Analysis of GUS activity ~-Glu curo nid ase (G US) acti vity was assayed as · 16.17.24 f rom electropo rated protopl asts e d earl ler d esc n. b and different parts of the pl ants. As a control, transgeni c ri ce containin g gus dri ven by 35S 17 promoter was also employed .
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All the ex peri ments were carri ed out at least twi ce w ith essenti all y similar results and data from re presentati ve ex periments are given.
Results and Discussion Promoter analysis using transient gene expression Investi gati ons with heterologous gene ex press ion sys tems have show n that Dicotyledoneae-s pecific gene pro moters are not efficientl y utili zed in monocotyledo no us plants5 .7-9. In order to study regul ation of some of the Dicotyledoneae-s pecific gene promoters and the regul atory systems in ri ce, promoters of two photosy nthes is-related genes psaF and petH I S• 19 fro m spinach were empl oyed in the present study. ]n these pro moters, there is o nl y limi ted ho mo logy at nucleotide sequ ence level but various elements know n to be present in the pro moters of photosy nthetic genes are present 3 . The data fro m ex periments of transient gene ex press io n in leaf protopl asts in terms of G US spec ific acti vity sho w that fo r psaF promoter, - 1074 to +163 bp construct has max imal acti vity (Fig. l a) . Except fo r fragment - 687 to + 163 bp that showed lower acti vity, the pattern of ex press ion was si milar in ri ce leaf protoplasts and transgeni c tobacco leaves IS. The promoter fragment (-220 to +1 63 bp) resulted in very signifi cant increase in G US acti vity as compared to the shorter fragments. It contains extra 42 bp which have been shown to co nfer si mil ar regul ation in transgeni c tobacco plants IS. However, lower g us express io n dri ven by -687 to + 163 bp pro moter frag ment in rice may refl ect di ffe rences in regul ati on in a mo nocot and a di cot and reveals th at seq uences present between +220 to +687 bp may act as negat ive regul atory elements. Further, regul atory elements present between - 1074 to -687 bp can nullify repress ion.
It was reported by OelmUller e l al. 19 that regul atory elements o f petH promoter are present in the fi rst 75 3 bp . Hence thi s frag ment (-75 3 to +23 1 bp), together w ith small er promoter fragment (-11 8 to +23 1 bp), whi ch was re ported to be sufficient to confer li ght regul ati on, were chosen fo r transient gene ex pression in ri ce. Analysis o f thi s promoter in rice protoplasts revealed a pattern of ex press ion similar to that o f transgeni c tobacco pl ants. The larger promoter fragment (-75 3 to +23 1 bp) showed hi gher expression (Fig. Ib)
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