CA2270417A1 - Transgenic reduction of the sinapine content in brassica napus seeds - Google Patents

Abstract

A method is disclosed for the reduction of sinapine content in Brassica e.g.
canola seeds, using anti-sense methodology.

Description

Attachment to the PSIR by G. Selvaraj, R. Nair, R. Joy IV, W. A. Keller and R.
Datla.
Transgenic reduction of the sinapine content of Brassica napes seeds Introduction:
Canola (Brassica napes) is an important oil seed crop. The portion of the seeds after the extraction of oil is referred as the canola meal. Canola meal, which contains about 36-44 percent crude protein is primarily used as a protein supplement in animal feeds. However, the use of canola meal in animal diet is limited due to presence of several anti-nutritional factors. Among them, sinapine, a major phenolic ester present in the seeds imparts bitter taste to the meal and makes it less palatable to ruminants (Bell, 1993). The presence of sinapine in the meal also limits its use as a feed supplement in the poultry diet as it results in eggs with fishy taint (Bell, 1993).
Sinapine accumulates in canola seeds supposedly as a reserve seed material and constitutes 1-4% of dried canola meal. During seed germination, sinapine is hydrolysed (Strack, 1981 ). However, it is not sure whether sinapine is essential for seed germination and seedling development. Recent identification of an Arabidopsis mutant that fail to accumulate sinapine in seeds but did not affect seed germination suggest that sinapine is dispensable for seed germination and seedling growth (Chapple et al., 1992). Efforts to develop 8. napes breeding lines with low sinapine content have not been successful. We are interested in reducing the sinapine content in 8, napus by genetic modification of the phenylpropanoid pathway that leads to sinapine synthesis.
Sinapine is a sinapic acid ester derived from the general phenylpropanoid pathway (Fig.1 ). Molecular and biochemical characterization has led to the isolation of several genes that are involved in the phenylpropanoid pathway (Whetten and Sederoff, 1995). Among the various enzymes that have been characterized, ferulic acid hydroxylase (FAH), a cytochrome P-450-linked monoxygenase catalyzes the conversion of ferulic acid to 5-hydroxyferulic acid (Fig.1 ). The 5-hydroxyferulic acid being an intermediate in the formation of sinapine and syringyl lignin, down regulation of FAH gene expression may reduce the sinapine content and alter the lignin composition. An Arabidopsis thaliana mutant deficient in FAH has been characterized (Chapple et al., 1992) and the gene encoding the enzyme has been isolated from Arabidopsis (Meyer et al., 1996). We have isolated 3 fah cDNA clones from Brassica napus. One of the cDNA clone was used for reducing sinapine content in canola seeds by the down regulation of fah gene expression using antisense technique.
Materials and Methods Materials Brassica napus plants were grown in a growth chamber maintained at 25°C day and 20°C night temperature under a 16h light and 8h dark photoperiod provided by banks of fluorescent tubes and incandescent bulbs. All plant materials _Z_ needed for the various experiments were collected and immediately frozen in liquid nitrogen and stored at -80°C until needed. Enzymes used in various experiments were purchased from GIBCO BRL. Oligonucleotides synthesis and DNA sequencing were conducted at the DNA technologies unit, National Research Council Canada, Saskatoon.
Identification of cDNA clones A Brassica napes stem cDNA library constructed in Lambda Zap vector (Stratagene) was screened with a 32P-labeled Arabidopsis fah cDNA to isolate 8.
napes fah cDNA clones. Positive plaques were purified and the phagemids excised in vivo (Stratagene). cDNA clones with insertions that were more than 1.5 kb were sequenced in both 5' and 3' directions (Applied Biosystems) using the plasmid-based and internal fah primers designed from DNA sequences as they became available. Lasergene biocomputing software (DNASTAR) was used to analyze the DNA and deduced amino acid sequences.
DNA and RNA analysis 8. napes genomic DNA was isolated from leaves using the Nucleon Phytopure plant DNA extraction Kit (Amersham Life Science). Southern blot analysis was conducted using DNA digested with restriction endonucleases that was separated by 1 % TAE agarose gel electrophoresis and transferred on to a GeneScreen Plus membrane (NEN life Science products). 32P-labeled fah cDNA
probe was synthesized using rediprime II (Amersham) random primer labeling kit. Hybridization with the 32P-labeled fah probe was conducted according to the standard protocols (Sambrook et al., 1989). After hybridization, membrane was rinsed once in 0.5x SSC, 0.1 % SDS, washed twice for 15 min at 50°C in 0.2x SSC, 0.1 % SDS and once for 30 min at 65°C in 0.1 x SSC, 0.1 %
SDS. The washed membrane was exposed to X-ray film for 1-5 days in a Quanta III
(DUPONT) intensifying screen.
Total RNA from various tissues was isolated using TRlzol reagent (Life Technologies) following the manufacturer's protocol. Northern blot analysis was conducted using 15 ~g of total RNA that was electrophoretically separated and transferred to a GeneScreen Plus membrane according to the standard protocols (Sambrook et al., 1989). Hybridization and washing of the membrane was carried out as described previously for genomic DNA analysis.
Isolation of FAH protein and production of antisera A partial fah cDNA lacking the N-terminal 170 amino acids was ligated in frame into a pRSET protein expression vector (Invitrogen) containing N-terminal Histidine tags. The resultant plasmid (PRAMS) was transformed into BL21 DE3 E. coli strain. As a control, pRSET vector alone transformed BL21 DE3 E.coli strain was used. An overnight cultures of the BL21 DE3 E. coli carrying the recombinant plasmid grown at 30°C was inoculated at 1:25 into fresh 50-ml LB
medium. The bacterial culture was then treated with 1 mM IPTG at OD = 0.7 at 600 nm and incubated for another 3h. Bacterial cells were harvested, lysed in guanidine-HCL and the protein purified using Ni-NTA columns (Qiagen) using varying levels (20-200 mM) of imidazole concentration. Protein fractions were analyzed on 10% SDS-PAGE. The protein band that correspond to expected FAH was cut and used for raising antisera in rabbits according to the standard protocols (Harlow and Lane, 1988).
Western blotting Protein concentrations were determined using a protein assay re-agent (Bio-Rad). Proteins (15 fig) extracted from B. napus stem tissues was separated under denaturing condition in a 10% SDS-PAGE and electroblotted onto nitrocellulose membrane (Hybond). Protein-antibody complexes were detected using the HRP conjugated donkey (anti-rabbit Ig) antibodies (Amersham) and the ECL Plus western blotting detection system (Amersham). To check equal loading of proteins, the nitrocellulose membrane after chemiluminescence detection was stained with amido black stain (BIORAD) or duplicate gels were run and stained with coomasive brilliant blue (BIORAD).
-s-Plant transformation constructs Chimeric DNA construct (pJOY42) needed for the agrobacterium transformation was created by inserting an 1.8 kb EcoR I fragment of pJOY9 fah cDNA lacking the polyadenylation signal between a double cauliflower mosaic virus 35S (CaMV35S) and a nopaline synthase terminator (nos) in antisense orientation. The clone was selected for the proper orientation (antisense) using Hind III restriction endonuclease digestion that cleaves the pJOY9 fah cDNA
once. The resultant plasmids were introduced into Agrobacterium and used for the transformation of 8. napes hypocotyls (Moloney et al., 1989).
HPLC analysis of phenolics 8. napes seeds or leaves (100mg) were ground in 500 ~I of 80%
methanol and extracted at 4°C for 2h. The extract was centrifuged (10000 g for 15 min) and the supernatant collected. The pellet was once again extracted with 500 ~I of 80% methanol overnight at 4°C and frozen at -80°C for 1 h. The samples were centrifuged (10000 g for 15 min) and the supernatant pooled with the previous extract. 20 ul of the pooled extract was run on a Nucleosil C18 AB
HPLC column (Alltech) using an acetonitrile/phosphoric acid (1.5%) gradient of 10% to 30% in 35 min. The compounds eluted were detected by UV light at 330 nm absorbance. Authentic sinapine standard extracted from 8. napes and commercially available phenolic ester standards (Aldrich or Sigma) were used to identify eluted compounds. At least 3 independent samples from each transgenic line and controls were extracted and analysed by HPLC for determining the sinapine content.
Results and discussion Isolation of fah cDNAs from Brassica napes A Brassica napes stem cDNA library was screened with an Arabidopsis fah cDNA clone to isolate 8. napes fah cDNA clones. Five positive cDNA clones were isolated, preliminary sequence analysis suggested two of the cDNA clones (pJOY9 and pJOY11 )'represented putative full length fah cDNAs while three of the cDNA clones (pJOY1, pJOY3 and pJOY10) represented partial fah cDNAs.
Restriction map and sequence analysis revealed that the cDNA clones pJOY1 and pJOY10 contained truncated forms of pJOY9 cDNA and were not characterized further. The remaining three cDNA clones pJOY3, pJOY9 and pJOY11 represented unique fah cDNA clones since they exhibited unique restriction sites (Fig. 2). These cDNA clones were sequenced completely in both 5' and 3' directions.
Among the two putative full length cDNA clones, pJOY-9 cDNA clone (1880 bp; Fig. 3) consisted of a 38 by 5'- untranslated region, a 1560 by open reading frame, and a 282 by 3'-untranslated region while pJOY11 cDNA clone (Fig. 4) which is 1884 by long consisted of a 51 by 5'-untranslated region, a 1560 by open reading frame and a 273 by 3'-untranslated region. The pJOY3 cDNA clone (1835 bp; Fig. 5) which appeared to be truncated, lacked the 5'-untranslated regions and 14 nucleotides 3'- of the putative ATG initiation codon present in pJOY9 and pJOY11. We assume that the translation of the FAH
protein initiates at the first ATG codon present in pJOY9 and pJOYl1 open reading frame since it follows the Kozak consensus sequence for eukaryotic translation initiation sites with an A at the -3 position and a G at the +4 position (Kozak et al., 1987). Thus the open reading frame of 1560 base pairs (bp) present in pJOY9 and pJOY11 could encode a 520 amino acid polypeptide (Fig.
6-7) with a predicted molecular weight of 58.5 kilo dalton.
The nucleotide_and the deduced amino acid sequence comparison of the isolated cDNA clones with that of the Arabidopsis fah suggest that the isolated cDNA clones represent putative fah cDNAs. The isolated cDNA clones shared 81-83% sequence identity at the nucleotide level and 93% sequence identity at the deduced amino acid level to that of the Arabidopsis fah. The predicted molecular weight of 8. napes FAH polypeptide (58.5 kDa) also closely agreed to that of the deduced Arabidopsis FAH polypeptide (58.7 kDa).
Isolation of FAH protein and generation of FAH antibodies Recombinant FAH was isolated using an E. coii based heterologous expression system. A truncated fah cDNA that lacks the first 170 amino acids of the FAH coding region was fused in frame with a Histidine tag and expressed in E. coli to produce a FAH-His tag fusion protein. The FAH-His tag fusion protein was purified to apparent homogeneity using Ni-NTA columns. The band corresponding to FAH was cut and used for raising polyclonal antibodies in rabbit. Antibodies raised in rabbit was analysed using Western blots and was _g_ found to recognize a protein of approximately 60 kDa, which was the expected size of FAH in plants.
Analysis of transgenic plants Hypocotyls of 8. napus cv. Westar were transformed with agrobacterium containing plasmid pJOY42 (Fig. 9). A total of 22 putative Kanamycin resistant transgenic plants were isolated using pJOY42 transformation construct. We were interested only in transgenic plants that had single copy inserts. This is to avoid variation in sinapine content due to segregation of multiple copy inserts at a later generation. Southern blot analysis of the putative transgenic plants identified five transgenic plants with single copy inserts for pJOY42 (Fig. 10).
Seeds from the primary transgenic plants that had single copy inserts were analyzed for sinapine content using HPLC. The primary transgenic (T°) plants containing single copy inserts of pJOY42 T-DNA showed 40-60 percent reduction in the sinapine content as compared to that of the controls (Fig. 11 ).
The primary transgenic plants that showed lower levels of sinapine were grown to homozygosity and the sinapine content in the fully mature seeds of the homozygous plants was measured. All of the homozygous transgenic plants with pJOY-42 DNA construct showed varying levels of sinapine reduction with two of the transgenic plants showing up to 40% reduction in sinapine content (Fig.
12).
Northern blot analysis was conducted to determine whether the reduction in sinapine content in the 35S transgenic plants was associated with a reduction in fah transcript. Total RNA isolated from stem tissues of transgenic plants since fah gene expression was highest in stem. Total RNA isolated from the bottom two internodes of transgenic plants and controls were used for analysis and the intensity of the fah transcripts after hybridization was quantified. As compared to that of vector alone transformed plants, only transgenic plant 2758 showed any reduction in fah transcript (Fig. 13). Transgenic plant 2758 showed 45 reduction in fah transcripts as compared to vector transformed plants.
To examine the FAH protein levels in the transgenic plants, crude protein was extracted from the bottom two internodes of the stem sections and used on immunoblots with antibodies raised against 8. napes FAH protein. Except for the transgenic plant 2430, all of the 35 S transgenic plants showed lower levels of FAH protein than the control plants (Fig. 14).
-lo-Figure legends Fig.1. Schematic representation of lignin biosynthesis in plants.
PAL : Phenylalanine ammonia lyase; C4H : Cinnamate 4-hydroxylase;
C3H : Coumarate 3-hydroxylase; 4CL : 4-Coumarate-CoA-ligase; OMT
O-methyltransferase; FAH : Ferulic acid hydroxylase; CCoAOMT
Caffeoyl-CoA O-methyltransferase; CAD : cinnamyl alcohol dehydrogenase; CCR : cinnamyl CoA reductase; SGT : sinapate: UDP-glucose sinapoyltransferase: SCT : sinapate: choline sinapoyltransferase Fig.2. Predicted restriction sites that are unique to each of the three fah cDNA
clones.
Fig.3. Nucleotide sequence of pJOY9 fah cDNA clone. The coding region is represented in capital letters while the 5' and 3'- non-coding region is represented in small letters. The stop codon is marked by shaded box while the putative polyadenylation signal is indicated by an open box.
Fig.4. Nucleotide sequence of pJOY11 fah cDNA clone. The coding region is represented in capital letters while the 5' and 3'- non-coding region is represented in small letters. The stop codon is marked by shaded box while the putative polyadenylation signal is indicated by an open box Fig.S. Nucleotide sequence of pJOY3 fah cDNA clone. The coding region is represented in capital letters while the 3'- untranslated region is represented in small letters. The stop codon is marked by shaded box while the putative polyadenylation signal is indicated by an open box.
Fig.6. Predicted amino acid sequence of a FAH polypeptide encoded by pJOY9 fah cDNA clone.
Fig.7. Predicted amino acid sequence of a FAH polypeptide encoded by pJOY11 fah cDNA clone.
Fig.B. Predicted amino acid sequence of a FAH polypeptide encoded by pJOY3 fah cDNA clone.
Fig.9. Schematic representation of pJOY42 plasmid DNA construct used for the transformation of Brassica napus.
Fig.10. Southern blot analysis of homozygous transgenic plants of Brassica napus originally transformed by pJOY42. Hind III digested genomic DNA
from transgenic plants was probed with a fah cDNA probe (bases 1452 to 1718 of pJOY9). The 1.9 kb DNA fragment present in the transgenic plants corresponds to a Hind III fragment of the T-DNA as shown in fig.9.

Fig. 11. Relative sinapine content in the seeds of primary Brassica napus originally transformed by pJOY42 Fig. 12. Relative sinapine content in the seeds of homozygous Brassica napus originally transformed by pJOY42.
Fig. 13.Northern blot analysis of total RNA from stem tissues of homozygous Brassica napus originally transformed by pJOY42. Northern blot was probed with bases 236 to 662 of pJOY9 fah cDNA.
Fig. 14.Immunoblot analysis of crude protein extracts isolated from stem tissues of homozygous transgenic plants originally transformed by pJOY42. Rabbit antisera against a FAH polypeptide produced in recombinant Escherchia coli was used.
Fig. 15. Is a different version of Fig.1. In this figure the position of the right and left border coincide with the right and left side.

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