Cloning of Formate dehydrogenase Gene and Effect on the Waterlogging Tolerance of Brassica napus L

A Formate dehydrogenase (FDH) gene, named BnFDH-1, was cloned from oilseeds (Brassica napus L.) by the Rapid Amplification of cDNA Ends (RACE) method. The Complementary DNA (cDNA) consisted of 1488 bp and had an open reading frame of 1140 bp encoding a polypeptide of 384 amino acids with a molecular weight of 42.3 kD and an isoelectric point of 8.17. BnFDH-1 showed high homology to known FDH genes, especially FDH from Arabidopsis thaliana. Conserved domain search and motif characterization identified BnFDH protein as Rossmann-fold NAD (P) (+)-binding proteins. NetPhos 2.0 search predicted 18 significant phosphorylation sites. To investigate the effect of BnFDH on waterlogging tolerance of B. napus, 12 B. napus cultivars with different waterlogging tolerance were used in the research and waterlogging Tolerance Index (WTI) was calculated by multiplying relative percentage germination and the relative seedling height. BnFDH-1 was cloned from the 12 materials also, but the results indicated all of them had same protein sequence. BnFDH expression level in 12 B. napus cultivates with different waterlogging tolerance after waterlogging treatment was measured. The results indicated expression level of BnFDH in 12 cultivates had significant difference. But correlation analysis proved that BnFDH expression level was uncorrelated with Waterlogging tolerance index (WTI). So it was not the BnFDH that was key gene for the waterlogging tolerance of B. napus.


INTRODUCTION
Oilseed rape (Brassica napus L.), is one of the most important oil crops in the world, especially in China, but it is generally susceptible to waterlogging stress.Water is critical for plants, but excess water is also harmful because it results in lower oxygen concentration in the soil and may block the exchange of CO 2 and O 2 between the soil and the atmosphere.waterlogging may result in necrosis, stunting, defoliation, even plant death (Cornelious et al., 2005), which can cause to yield loss.According to reports, waterlogging leaded to 10-15% yield loss for rapeseeds and wheat and about 11% yield loss for cotton in the world.
Oxygen dissolved in soil water reduced and resulted in hypoxia because of the slow diffusion rate of oxygen and limited solubility in water during waterlogging (Sairam et al., 2008).Two-dimensional electrophoresis found that more than 20 anaerobic proteins over expressed in maize roots under low oxygen conditions and most of them were involved in sugar metabolism and fermentation (Dennis et al., 2000;Sachs et al., 1980).In the limiting oxygen conditions, the starting of fermentation metabolism was regarded as an adaptive phenomenon to maintain the energy of ATP and the growing (Good and Muench, 1993;Kennedy et al., 1992).And a number of enzymes including Formate dehydrogenase (FDH, EC 1.2.1.2.), alcohol dehydrogenase (ADH, EC 1.1.1.1),lactate dehydrogenase (LDH; EC 1.1.1.27)and pyruvate decarboxylase (PDC; EC 4.1.1.1)were induced during waterlogging (Kennedy et al., 1992;Ricard et al., 1994).Waterlogging tolerance of plants was closely related with ATP concentrations and the induced expression levels of ADH and PDC based on some reports (Drew, 1997;Waters et al., 1991), because reduction in oxygen supply leaded to an 18-fold reduction in ATP production and plants had to compensate for the loss by accelerating sugar metabolism and glycolysis (Dolferus et al., 2001).
As waterlogging, arerobic metabolism was transformed into annerobic metabolism.According to Davis-Roberts pH-stat hypothesis, the primary response of plants to O 2 limitation was characterized by a burst of lactate production, which resulted in lower cytoplasm pH and activated the over expression of PDC and ADH and inhibited the expression LDH.Then PDC and ADH were active and ethanol metabolism started (Agarwal and Grover, 2006).
FDH induced by one-carbon metabolites, such as methanol, formaldehyde and formate and FDH transcripts increased quickly and strongly in response to various stresses (Li et al., 2001).In the study, FDHs were cloned from 12 different waterlogging tolerance oilseeds and FDH expression was detected by real time PCR in 12 lines.We want to know if there has any correlation between FDH and WTI.

MATERIALS AND METHODS
Plant materials: Oilseeds with different waterlogging tolerance ability, Zhongshuang 9, H305, Qing 8, 9558, H04, H1020, PH36, Huyou 17, GH01, Zhongshuang 6, 2021 and Zhongshuang 11 selected from more than 4000 lines based on 4 years waterlogging experiment from 2005-2009 in the field, were used as experiment materials.In 2010, the 12 cultivates were grown in experimental field of Chinese Academy of Agricultural Sciences, China.In the flowering stage, plants were treatment with waterlogging in the field.Roots treated with waterlogging and untreated were used as samples and preserved at -80°C.Gene cloning, enzyme activity and gene expression level analyzing were finished in Key Laboratory of Biology and Genetic Improvement of Oil Crops, Ministry of Agriculture of P. R. China.
Extraction of total RNA: Total RNA was isolated with CTAB method from different tissues of the 12 materials (Jaakola et al., 2001) and the contaminated DNA in RNA samples was removed by digestion with RNasefree DNase I (TaKaRa).

RACE and 5' RACE for cloning FDH cDNA ends from B. napus:
Five µg of equally proportioned (w/w) mixture of total RNA from various organs of Zhongshuang 9 was used as template to carry out Rapid Amplification of cDNA Ends (RACE) in terms of the user manual (Gene Racer kit, Invitrogen, USA).Based on multi-alignment of FDH sequences from A. thaliana and other plants, two forward and two reverse primers were designed corresponding to conserved sites of FDH: sense primers FDH-31 (5'-TGTACCA TGA CAGGCTCCAGAT -3') and FDH-32 (5'-GGACCA TCCATGGCGTTACAT -3') for 3' cDNA end(s) amplification of B. napus FDH gene(s), while antisense primers RFDH-51 (5'-GTCATG GTACAA CAAG TTGCA -3') and RFDH-52 (5'-TGCCTT CCAG ATCATACGCTCT -3') for 5' cDNA end(s).FFDH-31 and FFDH-32 were paired with kit primers 3' Primer and 3' Nested Primer respectively for primary and nested amplification of 3' cDNA ends.As for primary and nested amplification of the 5' cDNA end, kit primers 5' Primer and 5' Nested Primer were paired with RFDH-51 and RFDH-52, respectively.Polymerase Chain Reactions (PCRs) were carried out in kitrecommended 50-µL standard amplification system containing 1.5 units of Taq DNA polymerase (TaKaRa) and other ingredients.For primary amplification, 1 µL of total first strand cDNA was used as template.Then 0.1 µL of primary amplification product was used for nested amplification.The 4 reactions were carried out on PTC-200 (BIO-RAD, US) under following conditions: predenaturation at 94°C for 2 min, 30 cycles of amplification of 94°C for 1 min, 50°C for 1 min and 72°C for 1.5 min and followed by a final extension at 72°C for 10 min.
Amplification of full-length cDNA sequence: Based on the obtained 5' and 3' cDNA ends, a pair of primers was designed to amplify the cDNA of BnFDH: FBnFDH (5'-GGTTCTACAAAAATATCGACACGA -3') and RBnFDH (5'-GTCACGAAAT ATCAAC GTTACATAC-3').In the cDNA amplification, 0.5 µL of total cDNA as template and 1.25 units of Pyrobest DNA Polymerase (TaKaRa) were used in a kitrecommended 50-µL PCR reaction.Cycling conditions were as follows: predenaturation at 94°C for 2 min, followed by 35 cycles of amplification (94°C for 1 min, 50°C for 1 min and 72°C for 3 min), succeeded by a final extension at 72°C for 10 min.After the reaction, 1.5 units of Taq DNA polymerase was added into each tube and the tubes incubated at 72°C for 20 min for dAtailing of the PCR product.
PCR products detection, subcloning and sequence analysis: PCR products were analyzed by 1% agarose gel with GoldView/1×TAE electrophoresis and detected by UV light.DNA of target bands was recovered using a Gel Extraction Mini Kit (Watson Biotechnologies, Inc., China) and ligated to the pMD18-T vector.Competent cells of DH5α were prepared by a CaCl 2 method for transformation of recombinant vectors.Transformants were subjected to antibiotic selection and Isopropyl-beta-D-Thiogalactopyranoside (IPTG) /X-gal blue-white screening.White colonies were cultured and identified by PCR.Positive clones were sequenced at Beijing Sunbiotech co., Ltd., China.Sequence alignment, open reading frame translation and parameter calculation of the predicted protein were carried out on Vector NTI Advance 9.0.BLAST analyses of nucleotide and protein sequences were done on NCBI website (http://www.ncbi.nlm.nih.gov/).Protein structure predictions were carried out on websites (http:// www.expasy.org and http://www.softberry.com/berry.phtml).
Evaluation of waterlogging tolerance of 12 materials: One-hundred seeds were submerged in 10 mL of deionized water in tubes for 24 h in an incubator at 20°C (Ueno and Takahashi, 1997).After the treatment, the seeds were removed from the water and rinsed with distilled water.The seeds were placed on a moist filter paper in 9 cm Petri dishes as seeds without flooding treatment served as control.The germination tests were evaluated on the 7 th day according to the ISTA Handbook of Vigour Test Methods.The percentage of germination and radicle length of germinated seeds were recorded.Relative germination rate, radicle length, seedling height and fresh weight were expressed as a percentage relative of flooding treatment to control.The seed flooding tolerance was calculated by multiplying relative percentage germination and the relative seedling height (Zhang et al., 2007).

Sequence cloning of BnFDH:
The results of 3' RACE showed a band about 400 bp and T-vector colonies showed plentiful variation of insert length.Representative colonies were sequenced.The accurate length of the 3' cDNA ends was 339, 369 and 384 bp, respectively (poly (A) not included) and the only difference of them was splicing site.NCBI Nucleotidenucleotide BLAST (Blastn) (Altschul et al., 1997) proved sequences showed high homologies to FDH genes reported.The results of 5' RACE showed a bright band of about 700 bp and an accurate length of 727 bp, which were also in consensus with homology-based length prediction.Its homology to FDHs was proved by Blastn.
Gel detection of the cDNA amplification showed one band about 1400 bp and the accurate length was 1383 bp and the sequence showed identities to 5' and 3' cDNA ends in corresponding regions.BnFDH-1 encoded a polypeptide of 384 amino acids with a calculated molecular weight of 42.3 kD and an isoelectric point of 8.17.NCBI blastp indicated that BnFDH-1 showed wide similarities to FDHs from other plants, 73.2 and 73.2% identity to A. thalina and Populus trichocarpa FDH sequence, respectively.SignalP 3.0 (Bendtsen et al., 2004) predicted that BnFDH-1 had not signal peptide.Both WoLFPSORT and TargetP 1.1 predict BnFDH-1 probably located in Mitochondrion.TMpred prediction revealed 1 transmembrane helice (I 133 -A 157 ) in BnFDH-1.NetPhos 2.0 search (Altschul et al., 1997) predicted 18 significant phosphorylation sites (10 for Ser, 4 for Thr and 4 for Tyr, respectively) (Fig. 1) suggesting that phosphorylation may be a posttranslational modification of BnFDH-1.NCBI Conserved Domain  search (Marchler-Bauer and Bryant, 2004) found it belongs to Rossmann-fold NAD (P) (+) -binding proteins.All proved BnFDH-1 was a representative FDH protein.SOPMA (Geourjon and Deleage, 1995) predicted that the secondary structure of BnFDH-1 was mainly composed of alpha helices (41.93%) and random coils (30.73%), while extended strands (18.75%) and beta turns (8.59%) contribute a little (Fig. 1).Alpha helices mainly distributed in the N terminal and the middle, which was similar with the predicted 3D structure (Fig. 1 and 2).BnFDH-1 was cloned from other 11 materials also, but the results indicated all of them had same protein sequence.
Waterlogging Tolerance Index (WTI) of 12 materials: Waterlogging Tolerance Index (WTI) of 12 cultivates with different waterlogging tolerances was measured and the results were listed in Table 1.The results indicated WTI among 12 lines had significant difference, so the 12 lines could be used as materials for waterlogging research.

Transcription of BnFDH in root of 12 different waterlogging tolerances materials:
The results suggested that transcription level of the BnFDH gene had significant difference in 12 cultivates with different waterlogging tolerances ability.BnFDH expression in Zhongshuagn 11 increased quickly after waterlogging treatment and slowly in some lines, for example, H305 and GH01 (Fig. 3).But all the BnFDH expression in 12 lines was induced by waterlogging and reached their peak at 24 h after treatment.The expression level decreased from 24 to 48 h.

BnFDH expression was uncorrelated with WTI:
Correlation between WTI and BnFDH expression at 24 48 h after waterlogging treatment was analyzed and the results indicated the correlation coefficient was 0.31 and 0.04, respectively (Fig. 4).It indicated there was no significant correlation between them.The results proved that the transcription level of BnFDH gene was not the reason leading to different of waterlogging tolerance of B. napus.

DISCUSSION
The results of the response of plant to hypoxia stress in maize roots by two-dimensional electrophoresis shown that about 20 kinds of proteins were synthesized during hypoxia stress (Sachs et al., 1980), including sucrose synthase, phosphohexose isomerase, fructose-1, 6-diphosphate aldolase, PDC, FDH, LDH and ADH (Chung and Ferl, 1999;Zeng et al., 1999).The difference genotypes in waterlogging tolerance could be distinguished according to the changed patterns of enzymatic activities because POD and CAT activity under waterlogging determine the status of oxidative stress (Zhang et al., 2007).A short anoxic stress (1-2 h) increased the production of superoxide production in soybean roots and longer time of anoxia (3-5 h) show less injury because of increased ability to cope with oxygen free radicals (Vantoai and Bolles, 1991).ADH deficient mutant can produce LDH and result in pH declining, so the mutant was very sensitive to waterlogging (Roberts et al., 1984a;1984b;Sairam et al., 2008), but over-expression of ADH gene could not improve plant waterlogging tolerance.It may be some genes induce by waterlogging and response to hypoxia stress, but not decide the tolerance.In this research, BnFDH also induced by waterlogging, but its expression was not correlated with WTI.It indicated BnFDH was not the key gene for waterlogging tolerance.Our research also indicated BnADH also responded to hypoxia stress and had no correlation with WTI (unpublished).

Fig. 1 :
Fig. 1: Predicted secondary structures and phosphorylation sites of BnFDH-1 h: Helix; e: Extended strand; t: Beta turn; c: Random coil; The predicted significant phosphorylation sites were boxed

Table 1 :
WTI of 12 different waterlogging tolerances materials