pB19hpc psy 4m daffodil+ seq 4 bac phytotene desaturase(crt1) 4m erwinia uredonora under cntrl of plant endosperm sp glutalin promoter(Gt1) and constitutive CaMV 35S promoter. pB19hpc- direct formation of lycopene in endosperm plastids. Immature rice embryos were inoculated with agrobacterium LBA4404/pB19phc.pB19hpc digested with I Sce1 – released 10Kb insertion of aph4, psy, crt1. 1 copy number
pZPsC carries psy and crt I bt lack aph4. pZLcyH PROVIDE LYCOPENE B-CYCLASE FROM NARICISSUS PSEUDONARICISSUS CONTROLLED BY RICE GLUTELIN PROMOTER AND APH4 GENE UNDER CAMV 35S PROMOTER. IMMATURE RICE EMBROYES INOCULTED WITH AGROBACTERIUM LBA4404/pZPsC and LBA4404/pZLcyH 6.6kb for excised psy and crt1 expression cassete 4m pzpsc and 9.5kb 4 lcy and aph4 4m pzcych. All showed normal phenotype and were fertile.
Introduction Vitamins are the very basic building blocks required over a long period of time (lifelong) to build a strong, healthy, disease free body. Vitamin A essential for vision (also known as Retinol), cell growth, reproductive functions and maintaining immune system. Dependance on rice as predominant food source-VAD(vit A deficiency).because in rice – no b carotene(vitamin A). VAD- 250,000-500,000 chilldren blind every year. Biofortified crop like Golden rice – offer sustainable solution to VAD.
Golden rice prototype (1999) – accumulated around 1.6ug/g β carotene in the grain. New line GR2 (by tissue specific promoter) – produced 31ug/g β carotene . Vitamin A - first synthesized in 1947 by two Dutch chemists, David Adriaan van Dorp and Jozef Ferdinand Arens.
Increase severeness of common childhood infection Impaired Epithelial integrity Impaired Impaired skeletal vision growth VAD Reduced Impaired Immune haemopoises response
Improving the nutritional value of Golden ricethrough increased pro-vitamin A contentJacqueline A Paine1, Catherine A Shipton1, Sunandha Chaggar1,Rhian M Howells1, Mike J Kennedy1, Gareth Vernon1, Susan YWright1, Edward Hinchliffe2, Jessica L Adams3, Aron L Silverstone3& Rachel Drake1
Golden Rice Variety of rice engineered to produce β-carotene (pro-vitamin A) to combat vitamin A deficiency. Carotenoids- plant pigment- precursor of vitamin A so known as pro-vitamin A Produce Carotenoids in the endosperm of grain so, giving characteristic “ yellow colour ”. Phytotene synthase – limiting regulatory steps for Carotenoid biosynthesis.
In case of Canola seed, crtB (gene encoding bac phytotene synthase) expression alone – increased carotenoids production. In wild type Rice endosperm, first barrier to Carotenoid biosynthesis – phytotene synthase and carotene desaturase – provided by daffodil psy and crt transgenes.
Expression of a psy transgene increases thecarotenoid content of maize callus Gene cassettes in the two plasmid used to cotransform maize callus. Both contain the maize polyubiquitin1 promoter (Ubi1) and the nos terminator (nos). (i) The seven similar plasmids constructed with the phytoene synthase-coding region (psy) from each of the species listed below. (ii) The phosphino N-acetyl transferase (pat) selectablemarker and beta- glucuronidase (gus) gene cassettes. Paine et al.Nature biotechnology(2005)
Photograph showing individual maize calli cotransformed with theplasmid containing the maize psy (right, Zm psy) and an empty vector(EV) control (left). Paine et al.Nature biotechnology(2005)
Histogram showing the total colored carotenoid content of maize calli transformed with agiven psy gene (from Arabidopsis thaliana (At), Daucus carota (Dc), Narcissuspseudonarcissus (Np), Zea mays (Zm), Capsicum annuum (Ca), Oryza sativa (Os) orLycopersicon esculentum (Le)). Data shown represents the 75th percentile for eachpopulation of transgenic calli expressed as a percentage of the median empty vector (EV)control value. The second y-axis (diamonds) shows the percentage of calli from eachpopulation with a carotenoid content more than fivefold that of the EV median. Paine et al.Nature biotechnology(2005)
High level of carotenoid accumulate by the mechanism of carotenoid sequestration including crystallization, oil deposition, membrane proliferation or protein lipid sequestration. Starchy food matrix of the rice grain + fat content → facilitate intestinal β carotene uptake.
Carotenoid enhancement of the rice endosperm by transformation with psy orthologues and crtI.T-DNAs used to generate transgenic rice plants. The T-DNA comprisedthe rice glutelin promoter (Glu) and the first intron of the catalase genefrom castor bean (I), E. uredovora crtI functionally fused to the peaRUBISCO chloroplast transit peptide (SSUcrtI) and a phytoene synthasefrom each of five plant species (psy), with a nos terminator, as well as aselectable marker cassette comprising the maize polyubiquitin (Ubi1)promoter with intron, hygromycin resistance (hpt) and nos terminator. Paine et al 2005 ,Nature biotechnology
Photograph of polished wild-type and transgenic rice grainscontaining the T-DNA with the daffodil psy (Np) or maize psy(Zm) showing altered color due to carotenoid accumulation. Paine et al.Nature biotechnology(2005)
Histogram showing the total carotenoid content of T1 rice seedcontaining a T-DNA (as above) with the psy gene from either rice,maize, pepper, tomato or daffodil from the five events with thehighest carotenoid content for each T-DNA. Paine et al.Nature biotechnology(2005)
Analysis of T2 seed showed -carotenogenic ability was stable and heritable for all psy cDNAs, and high levels of carotenoids were again observed in seed from homozygous progeny containing the maize psy/crtI transgenes (over 16 mg/g).
Why daffodil psy show low expression? daffodil psy – itself barrier to even higher level of carotenoid accumulation in Golden rice. Although there was no shortage of precursor geranyl geranyl diphosphate and no problem with product sequestration. Overcome by providing psy of other species. Reason for differing efficacy of psy1 – difference in transgene transcription under the control of same promotor.
The increase in total carotenoid content brought about by the more highly effective psy genes is largely due to a preferential increase in b-carotene rather than a proportional increase in all carotenoids. increases in the amount of b-carotene in transgenic tomato were associated with a reduced total carotenoid content possibly because of feedback inhibition at the level of phytoene synthase activity.
Explanation for high b-carotene level:- Downstream processing of carotene to xanthophylls does not keep pace with the rate of flux through the pathway when an efficacious PSY is expressed. so, b-carotene accumulate.e. Pathway endpoint may be influenced by sequestration, rendering b-carotene inaccessible to downstream hydroxylases.
Golden rice 2Schematic diagram of the T-DNA in pSYN12424 used to create Golden Rice 2. The T-DNA components with a selectable marker cassette comprising the maize polyubiquitin(Ubi1) promoter with intron, phosphomannose isomerase gene (pmi) and nosterminator. The use of an intron was abandoned because it was shown to have no effecton carotenoid accumulation.The golden rice reported here has up to 37 µg/g carotenoid of which 31 µg/g is b-carotene (23 fold increase).
Engineering the b-carotene biosynthetic pathway into rice endosperm Heldt-Plant biochemistry
Engineering the provitamin A biosynthetic pathway into rice endospermXudong Ye,1*† Salim Al-Babili,2* Andreas Klo¨ ti,1‡ Jing Zhang,1Paola Lucca,1 Peter Beyer,2§ Ingo Potrykus1§ Immature rice endosperm – synthesize geranylgeranyl diphosphate – uncoloured carotene. β carotene synthesis require – Phytotene synthase (psy) Phytotene desaturase (crt) ζ carotene desaturase Introduce 2 double bond Lycopene β cyclase Agrobacterium mediated transformation – to introduce the entire β carotene biosynthetic pathway in rice endosperm.
Structure of T-DNA region and representative Southern blots of pB19hpc used in single transformation LB, left border; RB, right border; “!”, polyadenylation signals; p, promoters; psy, phytoene synthase; crtI, bacterial phytoene desaturase; tp, transit peptide.
Structure of T-DNA region and representative Southern blots of pZPsC and pZLcyH used in co-transformation