1、1939 MOLECULAR,CELLULAR,AND DEVELOPMENTAL BIOLOGY 2009 Poultry Science 88:19391947doi:10.3382/ps.2009-00032 Key words:reduced folate carrier,folate supplementation,molecular cloning and tissue distribution,messenger ribonucleic acid expression,laying hen ABSTRACT The reduced folate carrier(RFC)has b
2、een postulated to be a major entity for folate trans-port activity in humans and other mammals.However,there are limited reports of the importance of RFC in an avian system.In the current study,therefore,the molecular cloning and tissue distribution of RFC,as well as the effect of dietary folate sup
3、plementation on the expression of this transporter,were investigated in the chicken.Shaver White laying hens(n=8 per diet)received 3 wheat-based diets containing the following:1)no supplemental folate,2)folic acid(10.00 mg/kg),or 3)5-methyltetrahydrofolate(11.30 mg/kg)for 21 d.The mRNA expression le
4、vels were analyzed by quantita-tive real-time PCR.The results showed that the cloned partial RFC cDNA containing the full coding region from duodenum was 99%homologous to the reference gene available in GenBank.A broad expression profile of RFC transcripts was observed,with RFC mRNA detected in the
5、brain,liver,kidney,spleen,lung,intes-tine,ovary,and testis,as well as other tissues.Real-time PCR analysis revealed that no significant differ-ences(P 0.05)due to diet were found in the mRNA levels of RFC in the duodenum and cecum.However,compared with the basal diet,jejunal mRNA levels of RFC were
6、decreased(P 0.05)in hens fed with the 5-methyltetrahydrofolate diet,but the reduction did not reach significance(P=0.077)in the hens fed the folic acid diet.Overall,the current study demonstrated that the RFC cDNA containing the full coding region was successfully cloned from the duodenum of laying
7、hens.The wide tissue distribution of RFC transcripts is suggestive of an important role of RFC in the process of folate transport in the chicken.Moreover,dietary folate supplementation could downregulate the jejunal mRNA expression of RFC.Such findings will lay the foundation of future work involvin
8、g the RFC in avian systems,including laying hens.Molecular cloning and tissue distribution of reduced folate carrier and effect of dietary folate supplementation on the expression of reduced folate carrier in laying hens M.Jing,*G.B.Tactacan,*J.C.Rodriguez-Lecompte,*A.Kroeker,*and J.D.House*1 *Depar
9、tment of Animal Science,and Department of Human Nutritional Sciences,University of Manitoba,Winnipeg,Manitoba,R3T 2N2,Canada INTRODUCTION The compounds of the folate family,consisting of folic acid,5-methyltetrahydrofolate(5-MTHF)and other derivatives,are vitamins that act as coenzymes in reac-tions
10、 of 1-carbon metabolism.They are required for pu-rine and pyrimidine biosynthesis and for the conversion of homocysteine to methionine,among other reactions(Stover,2004).A deficiency of folate can lead to a va-riety of abnormalities including megaloblastic anemia,growth retardation,cardiovascular di
11、sease,and neural tube defects(Refsum et al.,1998;Scott,1999;Titus and Moran,2000).Because folates are essential vita-mins that cannot be synthesized by animals,efficient intestinal absorption processes are required.After in-testinal absorption,folates are delivered via the hepatic portal system to t
12、he liver.Here they are taken up by specialized transporters and stored as polyglutamate derivatives.Upon the release of the folate monogluta-mate(almost exclusively 5-MTHF,a bioactive form of folate)from the liver,folates are then transported to peripheral tissues and taken up by specific transporte
13、rs(Steinberg,1984;Mason et al.,1990).Folates are highly hydrophilic bivalent anions that can only minimally traverse biological membranes by simple diffusion(Lucock,2000).Therefore,transporter systems are needed to provide folates to cells.In mam-mals,3 folate transport systems have been identified:
14、the reduced folate carrier(RFC;also called SLC19A1;Dixon et al.,1994;Moscow et al.,1995),the folate receptor(McMartin et al.,1992),and the newly de-Received January 19,2009.Accepted April 22,2009.1 Corresponding author:J_Houseumanitoba.ca 2009 Poultry Science Association Inc.scribed proton-coupled f
15、olate transporter(also called SLC46A1;Qiu et al.,2006).The RFC,the best charac-terized transporter,has been suggested to be a major route for the transport of folates in mammalian cells(Matherly and Goldman,2003;Balamurugan and Said,2006).Reduced folate carrier is a bidirectional trans-porter,with f
16、olate movement reliant on the outward flow of organic phosphates from cells(Matherly and Goldman,2003).In addition to its generalized role as a folate trans-porter,the RFC may have other functions,including the facilitation of transport across renal proximal tu-bule basolateral membranes and the blo
17、od-brain bar-rier(Dudeja et al.,2001;Wang et al.,2001).Previous studies in humans and mammals showed that the level of expression of the RFC system paralleled changes in intestinal folate uptake observed under a variety of con-ditions,including folate deficiency(Said et al.,2000)and folate oversuffi
18、ciency(Ashokkumar et al.,2007),developmental maturation during early stages of life(Balamurugan and Said,2003),and cell differentiation along the crypt-villus axis(Said et al.,1996).Although considerable studies on the RFC have been performed in mammals,those in nonmammals,like the chicken,are less
19、prevalent.Caldwell et al.(2004)described the RFC mRNA in Leghorn chickens;however,studies re-lated to the factors regulating the expression of this gene are not available for poultry.Consequently,the objective of this study was to clone the RFC cDNA and assess tissue distribution of its transcripts,
20、and to examine the effect of dietary folate supplementation on the expression of this transporter in laying hens,which will provide the basis for future studies involving the RFC in an avian system.MATERIALS AND METHODSBirds and DietsShaver White laying hens,22 wk old at peak levels of production,we
21、re used in this study.Hens were kept in confinement housing under semicontrolled environ-mental conditions and exposed to a 16-h photoperiod.Birds were housed individually in battery cages.Feed and water were available ad libitum.Animal care ap-proval was received from the Animal Care Protocol Re-vi
22、ew Committee of the University of Manitoba,and the birds were managed in accordance with recommenda-tions established by the Canadian Council on Animal Care(CCAC,1984).The basal diet was a wheat-based ration,formulated to meet the requirements of laying hens consuming 100 g of feed/d(NRC,1994).The c
23、omposition of the basal diet is presented in Table 1.The basal diet included no crystalline folic acid or commercially produced 5-MTHF,a practice consistent with industry standards(BASF,2000).After a 14-d adaptation period,24 hens were ran-domly divided to receive 3 dietary treatments:1)basal diet(n
24、=8)with no supplemental folate,2)basal diet+10.00 mg/kg of crystalline folic acid(n=8),or 3)basal diet+11.30 mg/kg of 5-MTHF(n=8;adjusted to provide an equimolar concentration with 10 mg/kg of folic acid).At the end of the 21-d feeding period,birds were killed by cervical dislocation and intestinal
25、tissues,including duodenum,jejunum,and cecum,were freshly harvested and immediately frozen in liquid ni-trogen and then stored at 80C until analysis.RNA IsolationDuodenal,jejunal,and cecal samples were removed from 80C storage and put on ice until completely thawed.A total of 70 to 80 mg of tissue w
26、as added to 1 mL of ice-cold Trizol reagent(Invitrogen Canada Inc.,Burlington,Ontario,Canada)and homogenized with a homogenizer at full speed for approximately 1 min.The homogenate was transferred to a 1.5-mL tube and then 200 L of chloroform was added to the Trizol mixture.After centrifugation at 1
27、3,400 g for 10 min at 4C,the aqueous phase was carefully transferred to a new 1.5-mL tube,and the RNA was precipitated with 500 L of isopropanol and washed with 75%ethanol.The RNA pellet was resuspended in 50 to 100 L of DNase-RNase-free water,and total concentration was measured at 260 nm on a DU80
28、0 Spectrophotometer(Beckman Coulter Canada Inc.,Mississauga,Ontario,Canada).The absorbance ratio at wavelength 260:280 nm was within 1.7 to 2.1.The prepared RNA samples were then treated to eliminate the possibility of ge-Table 1.Composition of the basal wheat-based laying hen ra-tion Item%Ingredien
29、t Wheat(13.5%)54.50 Soybean meal(45.8%)25.70 Limestone(38%)10.00 Vegetable oil16.50 Biophos2(21/18)1.60 Vitamin premix31.00 Mineral premix40.50 dl-Methionine0.18 Antioxidant0.02Calculated nutrient composition CP,%19.00 ME,kcal/kg2,950.00 Calcium,%4.20 Phosphorus(available),%0.451Vegetable oil:8,800
30、kcal of ME/kg.2Biophos(21/18)(Feed-Rite Co.,Winnipeg,Manitoba,Canada)pro-vided available phosphorous(21%)and calcium(18%)in the diet.3Provided(per kg of diet):11,000 IU of vitamin A;3,000 IU of vi-tamin D3;20 IU of vitamin E;3 mg of vitamin K3(menadione with Na-bisulfite);0.02 mg of vitamin B12;6.5
31、mg of riboflavin,10 mg of pantothenic acid,40.1 mg of niacin,and 1,000 mg of choline;0.2 mg of biotin;10 mg of niacin;2.2 mg of thiamine(B1;mononitrate);4.5 mg of pyridoxine(B6;HCl);and 125 mg of ethoxyquin(antioxidant).4Provided(per kg of diet):66 mg of MnO;70 mg of ZnO;80 mg of FeSO4;10 mg of CuSO
32、4;0.3 mg of NaSeO3;and 0.4 mg of Ca(IO3)2 pre-mix contains:2.0 g of Ca(IO3)2 and 43 g of iodized salt;and 0.67 mg of iodized salt.JING ET AL.1940nomic DNA contamination(Turbo DNA-free Kit,Ap-plied Biosystems Inc.,Foster City,CA;Kitlinska and Wojcierowski,1995;Sutton et al.,1997).Four birds from each
33、 dietary treatment were selected for individu-al RNA extraction.Reverse Transcription-PCR AnalysisThe cDNA was cloned from duodenal total RNA by reverse transcription(RT)and subsequent PCR,using a 2720 Thermal Cycler(Applied Biosystems Inc.).In brief,a RT reaction was carried out using 2 g of total
34、RNA,and the procedure was performed according to the SuperScript First-Strand Synthesis System for RT-PCR Kit using the Oligo(dT)1218 primer(Invitrogen Canada Inc.;Chomczynski,1993;Takagi et al.,1997).From the obtained cDNA mixture,the full-length cod-ing sequence(CDS)of RFC was amplified by PCR usi
35、ng the PCR Master Mix Kit(Promega,Fisher Scien-tific,Nepean,Ontario,Canada;Saiki et al.,1988).The resulting products were electrophoresed on 1.0%aga-rose gel,stained with ethidium bromide,and the im-ages were captured with the use of a FluorChem Imag-ing System(Alpha Innotech Corp.,San Leandro,CA).T
36、he RFC primers were designed according to the refer-ence gene available in GenBank(NM_001006513)using Primer Premier 5 software(Premier Biosoft,Palo Alto,CA).The sense primer was:5-CCGAGTAGATGACG-GTGCC-3,and the antisense primer was:5-TCAG-GCTTGCACAGTAGCTGC-3.The size of the RFC amplicon,containing
37、the full CDS,was 1,499 bp.The PCR program started with a 2-min denaturation phase at 94C,during which time the hot start Taq DNA polymerase was fully activated.This was followed by 40 cycles of 1 min of denaturation at 94C and 45 s of an-nealing at 58C,and then 2 min of elongation at 72C.Next,an add
38、itional 10 min of incubation at 72C was performed,and the reaction was terminated by placing samples at 4C.Ligation,Transformation,and Sequence AnalysisThe PCR-amplified RFC cDNA from duodenum was purified from 1.0%agarose gel using the QIAquick Gel Extraction Kit(Qiagen Inc.,2008)and ligated into t
39、he pGEM-T Easy vector(Promega,Fisher Scientif-ic)according to the instructions of the manufacturer.Chemically competent Escherichia coli cells(JM109;Promega,Fisher Scientific)were transformed with the ligation products.Transformed cells were plated on se-lective media Luria-Bertani agar plates;in th
40、e pres-ence of 100 g/mL of ampicillin,100 L of 100 mM isopropyl thiogalactoside,and 50 L of 20 mg/mL of X-gal and incubated at 37C overnight.Six positive colonies were then transferred to liquid Luria-Bertani media containing ampicillin and incubated at 37C overnight.The plasmid DNA was purified fro
41、m cells containing the plasmid with the PCR insert of RFC using the Wizard Plus SV Minipreps DNA Purification System(Promega,Fisher Scientific).Then,the purified plasmid DNA from 4 bacterial colonies were confirmed by DNA sequencing using ABI 3730 xl DNA Analyzer(Applied Biosystems Inc.).The bidirec
42、tional sequenc-ing approach was used in this work.The primers T7 and SP6 were used to sequence the RFC gene.Compar-ative analysis with the GenBank nucleic acid database was conducted by BLASTN.Tissue Distribution of RFC TranscriptsTotal RNA,treated to remove genomic DNA con-tamination,from a variety
43、 of chicken(Ross Arbor Acre)tissues,including brain,liver,kidney,spleen,and intestine,was purchased from a commercial source(Zyagen,San Diego,CA)and used to investigate the tissue distribution of RFC transcripts.As described above,first-strand cDNA was reverse-transcribed from 2 g of total RNA for e
44、ach tissue sample,and there-after PCR was performed.Polymerase chain reaction of-actin was conducted on all samples as a control,and a no-template control was also included in each experiment by omitting the cDNA template from the PCR reaction.The sequences of the-actin primers are listed in the fol
45、lowing section.Real-Time PCR AnalysisOne microgram of total RNA from the duodenum,jejunum,and ceca of Shaver White laying hens was reverse-transcribed into cDNA with the SuperScript First-Strand Synthesis System for RT-PCR Kit(In-vitrogen Canada Inc.),as indicated above.The cDNA obtained were used a
46、s a template for PCR expression analysis.The primer sequences used in the real-time PCR were as follws:RFC(amplicon length:103 bp)sense primer,5-CGGGGCTGCTGCTATTCAT-3;RFC antisense primer,5-ATAGCGATGGGAACCA-GAAACT-3;-actin(amplicon length:205 bp)sense primer,5-CAACACAGTGCTGTCTGGTGGTA-3;and-actin ant
47、isense primer,5-ATCGTACTCCT-GCTTGCTGATCC-3.The specificity of the primers was tested by performing a BLASTN search against the genomic National Center for Biotechnology Informa-tion database.-Actin was used as a reference control to normalize quantification of the RFC mRNA.A sample without the cDNA
48、template was used to verify that the master mix was free from contaminants.The real-time PCR reactions were performed with a Step One Real-Time PCR System using SYBR Green I dye chemistry(Ap-plied Biosystems Inc.).The cycling conditions were as follows:10 min at 95C,40 cycles of denaturation at 95C
49、for 3 s,and annealing-extension at 60C for 30 s,then followed by one 3-segment cycle of product melt-FOLATE TRANSPORTER IN LAYING HENS1941ing(95C/15 s,60C/1 min,and 95C/15 s).Dissocia-tion curves confirmed the specific amplification of the RFC and-actin cDNA and the absence of nonspecific products.W
50、ells contained 20 L of PCR mixture(Fast SYBR Green Master Mix,Applied Biosystems Inc.),including 2 L of cDNA at a dilution of 1:10.All sam-ples were amplified in triplicate,and the mean was used for further analysis.Quantitative real-time PCR for RFC and-actin genes was carried out from the same RT
