Fatty Acid Composition and Seed Quality Traits of the Transgenic Rapeseed W-4(Brassica napus L.)with Down-regulated Expression of fad2 Gene

Song CHEN,Qi PENG,Jianqin GAO,Xiaoying ZHOU,Jiefu ZHANG,Huiming PU,Cunkou QI

Institute of Industrial Crops,Jiangsu Academy of Agricultural Sciences/Nanjing Sub-Center (Oilseed Rape),National Oilseed Crops Improvement Center/Key Lab of Cotton and Oilseed Rape of Lower Reaches of Yangtze River,Ministry of Agriculture,Nanjing 210014,China

Responsible editor:Tingting XU Responsible proofreader:Xiaoyan WU

R apeseed is the world’s third largest oil crop second only to palm and soybean.The cultivation area of rapeseed in China is about 7.0 ×106hm2,and across the world,the cultivation area and yield of rapeseed in China all rank first.According to the statistics,the yield of rapeseed oil accounted for 57% of the total vegetable oil yield in China in 2009.Rapeseed oil is China’s largest source of edible vegetable oil[1-2].Compared with other edible vegetable oils,the low-erucic content rapeseed oil has balanced fatty acid composition.In rapeseed oil,the saturated fatty acid content is relatively low(accounting for about 7% of total fatty acid content),but oleic acid content is relatively high(accounting for about 60%of total fatty acid content).Oleic acid is a monoethylenically unsaturated fatty acid,and its chemical properties are more stable compared with those of polyunsaturated fatty acids.High-oleic acid low-linolenic acid vegetable oils have good oxidation resistance,and they are ideal alternatives to hydrogenated vegetable oils[3-4].Oleic acid can reduce LDL cholesterol level in blood,maintain high-density lipoprotein cholesterol level and reduce the accumulation of cholesterol in blood vessels[5].Compared with polyunsaturated fatty acids,oleic acid can more effectively prevent cardiovascular diseases[6].In addition,high oleic acid rapeseed oil is also a high-quality raw material for producing industrial lubricants and biodiesel.Given the nutritional value and versatility of high oleic acid rapeseed oil,the breeding of high oleic acid rapeseed has become another important goal,following the breeding of double-low rapeseed,in rapeseed quality improvement.

The breeding of high oleic acid rapeseed germplasm is mainly achieved through mutagenesis and genetic engineering.Chemical or physical mutagenesis is usually carried out with rapeseed seeds or microspore embryoids.The oleic acid desaturase(fad2)gene mutant line is selected systematically for obtaining high oleic acid rapeseed material[7-8].In addition,the expression of exogenous fad2 gene in rapeseed seeds can be down-regulated by transgenic approaches,such as anti-sense RNA technology[9],co-suppression technology[10]and RNAi technology[11-12],and thus the high oleic acid rapeseed is obtained.Transgenic approaches often specifically down-regulate the expression level of fad2 gene in developing seeds.Therefore,the fatty acid composition only in rapeseed seeds,instead of in other organizations and organs,will be changed[13].Currently,the successful application of transgenic approaches in improving seed oleic acid content has been reported in soybean[14-15],peanut[16],cotton[17]and other crops.

The high oleic acid W-4 (Brassica napus L.) is bred by transforming the expression cassette of inverted repeat of fad2,driven by napin promoter,into Westar with Agrobacterium-mediated transformation system[12].Previous studies have shown that W-4 has only one inserted transgenic copy[12],and the high oleic acid content is a dominant trait in W-4[13].So W-4 has a certain application value and can be used to breed high-quality and highyielding cultivars.In the synthesis and metabolism of fatty acids in rapeseed seeds,oleic acid plays a key role.On one hand,oleic acid can be used to synthesize eicosenoic acid and erucic acid by extending the carbon chain;and on the other hand,oleic acid can be used to synthesize linoleic acid and linolenic acid by desaturation.The effects of down-regulated expression of fad2 on improving oleic acid content and reducing linoleic acid and linolenic acid contents in seeds have been widely reported[9-18].However,there are rare reports on the effects of downregulated expression of fad2 on the contents of other fatty acid components in seeds.In addition,rapeseed seeds also contain other various nutrients and anti-nutrients,such as storage protein,which contains balanced essential amino acid composition and is an excellent potential protein source[19].The anti-nutrients mainly include glucosinolates,cellulose,sinapine and phytic acid.The presence of these anti-nutrients affects the further use of rapeseed oil cakes.It is rarely reported about the effects of down-regulated expression of fad2 on amino acid composition,crude fiber content and glucosinolate content,as well as fatty acids in rapeseed seeds,which are also important parts of biological safety evaluation of transgenic rapeseed.In this study,the main seed quality traits were compared between the transgenic high oleic acid rapeseed W-4 and its non-transgenic receptor (control) Westar so as to clarify the effects of down-regulated expression of fad2 on seed quality,thereby providing references for the application of high oleic acid rapeseed germplasm and the biological safety evaluation of genetically modified rapeseed.

Materials and Methods

Materials

The transgenic rapeseed W-4 and its non-transgenic control Westar were all bred and preserved by the rapeseed research laboratory of Institute of Industrial Crops,Jiangsu Academy of Agricultural Sciences.The seeds of both W-4 and Westar were all directly sowed in a screenhouse in late September.There were 6 rows in each plot,and the row and plant spacing was 15 cm×40 cm.The final thinning was carried out at the three-leaf stage,and total 18-20 plants were remained in each row.The field management was same with that in the general field.In the early flowering stage,the W-4 and Westar plants were all isolated with 60-mesh nylon net from the outside world,and in the final flowering stage,the nylon net was removed.At the maturity stage,the seeds from all of the plots of each treatment were mixed together for quality analysis.

The RNA was extracted with the SV Total RNA Extraction Kit (Z3105,Promega).

For the real-time quantitative analysis,PrimeScript?RT Master Mix Kit(DRR036A,TaKaRa)was used.

The used instruments mainly included PCR instrument (7500,ABI)and gas chromatography (6890N,Agilent).

Methods

Detection of expression level of fad2 in rapeseed seedsFor each of W-4 and Westar,certain amounts(100 mg for each)of seeds collected on day 7,14,21 and 28 since the beginning of the flowering were sampled for RNA extraction.The total concentration of extracted RNA was determined using UV spectrophotometer,and the final concentration of extracted RNA was adjusted to 250 ng/μl with DEPC water.The cDNAsynthesis system was as follows:5× PrimeScript buffer (for Real Time) 2 μl,total RNA 500 ng,RNase free dH2O up to 10 μl.The reaction conditions were as follows:37 ℃15 min,85 ℃5 s,preservation at 4 ℃.

The actin gene in rapeseed was used as the reference gene,and the relative expression levels of fad2 gene in W-4 and Westar were calculated using the ΔΔCT method.The RT-PCR reaction system was as follows:SYBR Premix Ex TaqTM 10 μl,forward primer (10 μmol/L) 0.4 μl,reverse primer (10 μmol/L)0.4 μl,cDNA template 2 μl,sterile water 7.2 μl.The reaction conditions were as follows:95℃,5 min,40 cycles:95 ℃for 5 s,64℃,34 ℃.

The sequences of primers for fad2 gene were as follows:F:5’-AAGTGTTTGTCCCCAAGAAGAAGTC-3’;R:5’-AAGCGAAGCCGCCGTCGTAAG-3’.The sequences of primers for actin gene were as follows:F:5’-CGAGGCTCCTCTTAACCCAAAGG-3’; R:5’-CACCAGAATCCAGCACAATACCG-3’.

Determination of oil content in rapeseed seedsTotal 5 samples of each of W-4 and Westar were used for oil content determination using GB2906-1982.

Fatty acid composition analysisTotal 5 samples of each of W-4 and Westar were used for fatty acid composition analysis using GB/T 17376-2008.

Determination of glucosinolate content in rapeseed seedsTotal 5 samples of each of W-4 and Westar were used for glucosinolate content determination using the glucose oxidase/peroxidase method[20].

Amino acid composition analysisTotal 2 samples of each of W-4 and Westar were used for amino acid composition analysis using GB/T 5009.124-2003,and this was completed by the Physical and Chemical Testing Center in Jiangsu Province.

Determination of crude fiber content in rapeseed seedsTotal 2 samples of each of W-4 and Westar were used for crude fiber content determination using GB/T 5009.10-2003,and this was also completed by the Physical and Chemical Testing Center in Jiangsu Province.

Determination of protein content in rapeseed seedsThe protein content was calculated by the sum of contents of various amino acids determined above.

Results and Analysis

Down-regulated expression of fad2 gene in developing seeds of W-4

The relative expression levels of fad2 gene in seeds of W-4 and Westar on day 7,14,21 and 28 were detected using real-time PCR.The results showed that the expression of fad2 gene could already been detected in seeds of W-4 and Westar on day 7.The expression level of fad2 gene in seeds of Westar was increased with the proceeding of flowering,but there were significant differences in fad2 expression level between W-4 and Westar(Fig.1).On day 21 and 28,the expression levels of fad2 in seeds of W-4 were significantly lower than those of Westar.Especially,on day 28,the expression level of fad2 in seeds of W-4 was only 40% of that in seeds of Westar.

Fatty acid composition in seeds of W-4 and Westar

The fatty acid composition in mature seeds of W-4 and Westar was detected using gas chromatography.Total 7 fatty acids,including palmitic acid,stearic acid,oleic acid,linoleic acid,linolenic acid,erucic acid and eicosenoic acid,were detected (Table1).The results showed that the fatty acids contents differed significantly between W-4 and Westar.The total unsaturated fatty acid content in W-4 was 93.89%,while in Westar was 93.27%.The oleic acid content in W-4 was 86.03%,which was increased by 29.36% compared with that in Westar(P ＜0.01); the linoleic acid content in W-4 was 2.86%,which was 86.03%lower than that in Westar(P＜0.01);the linolenic acid content in W-4 was 3.04,which was 57.60% lower than that in Westar (P＜0.01).The total saturated fatty acid content in W-4 was 5.24%,which was reduced by 10.88% compared with that in Westar.Especially,the palmitic acid content in W-4 was 18.63%lower than that in Westar (P＜0.01).Compared with those in control,the stearic acid content in W-4 was increased slightly (P ＞0.05); the eicosenoic acid content in W-4 was increased by 18.46%(P＜0.01);the erucic acid content in W-4 was increased by 13.15%(P＜0.05).

Amino acids contents in seeds

Rapeseed seeds have a variety of amino acids.Total 18 kinds of amino acids were detected in rapeseed seeds (Table2).Since asparagine is hydrolyzed into aspartic acid and glutamine is hydrolyzed into glutamic acid,the contents of aspartic acid and glutamic acid referred to the sums of asparagine and aspartic acid and glutamine and glutamic acid,respectively.The contents of amino acids in W-4 were almost all lower than those in Westar.The total amino acid content in W-4 accounted for 18.18% of the seed dry weight,while in Westar accounted for 19.0%.The rapeseed seeds of W-4 and Westar all contained the 8 kinds of essential amino acids.In W-4,the content of essential amino acids accounted for 37.05% of the total amino acid content,and in Westar,the content of essential amino acid content accounted for 37.36% (P ＞0.05).

Oil,glucosinolate,protein and crude fiber contents in seeds

Table1 Fatty acid composition in seeds of W-4 and Westar %

Table2 Amino acid composition in seeds of W-4 and Westar

Table3 Contents of oil,proteins,crude fiber and glucosinolates in seeds of W-4 and Westar

The contents of oil,glucosinolates,proteins and crude fiber in seeds of W-4 and Westar were analyzed quantitatively.As shown in Table3,the average oil content in W-4 was 45.40%,which was slightly higher than that in Westar (P＞0.05); the average glucosinolate contents in W-4 and Westar were 18.20 and 18.66 μmol/g cake meal,which were all lower than the national standard (30 μmol/g).Based on the amino acids contents in seeds,the protein contents in seeds of W-4 (18.18% ±0.91% ) and Westar(19.0±0.34%)were deduced(P＞0.05).There was also no significant difference in crude fiber content between W-4 and Westar.

Discussion

The biosynthesis of fatty acids in seeds is a complex process,including mainly carbon chain elongation and desaturation.The synthesis of oleic acid is mainly completed in plastids.With malonyl-CoA as a substrate,the fatty acid is synthesized with fatty acid synthase through successive condensation,reduction,dehydration and reduction reactions,and two carbons is added to the carbon chain each time until the generation of stearic acid,which can be further catalyzed with Δ9 stearoyl-ACP desaturase in plasmids into oleic acid.Oleic acid enters into the endoplasmic reticulum,and it is transformed into oleoyl-phosphatidyl choline through enzymatic reactions.The oleoyl-phosphatidyl choline can be catalyzed into linoleic acid (Δ-12)and linolenic acid (Δ-15)respectively by the desaturases (fad2 and fad3)in endoplasmic reticulum.Studies have shown that the mutation or down-regulated expression of fad2 gene all can hinder the conversion from oleic acid to linoleic acid,reducing the synthesis of linolenic acid,and a large amount of oleic acid will be accumulated in seeds[9-17].

The results of this study showed that compared with those in control,the expression levels of fad2 in developing seeds of W-4 were all reduced on day 21 and 28.It indicates that the inverted repeats of fad2 gene,expressed in seeds of W-4,effectively interfere with the expression of fad2 gene.This interference is started in seeds about 21 d after the beginning of flowering,which is determined by the expression characteristics of napin,the promoter for inverted repeat expression cassette of fad2 gene in W-4.Hoglund et al.reported that the accumulation of Napin protein in rapeseed (Brassica napus L.) seeds was started 20 d after the beginning of flowering,and it reached the peak 30-35 d after the beginning of flowering[21].In this study,the expression level of fad2 gene in seeds of W-4 was also reduced on day 21 since the beginning of flowering.According to the contents of oleic acid,linoleic acid and linolenic acid,the oleic acid desaturation index(ODP) can be speculated.The ODP values of W-4 and Westar are about 6.41% and 27.38%,respectively.The ODP value of W-4 is 76.58% lower than that of Westar,so it can be speculated that the FAD2 enzyme activity in W-4 is reduced by 76.58% compared with that in Westar.However,in this study,the expression level of fad2 gene in W-4 was reduced by 60%compared with that in Westar,which might be caused by that the expression activity of napin promoter had not reached its peak in seeds 28 d after the beginning of flowering.This still needs further study.

This study found that down-regulated expression of fad2 gene also changed the amino acid composition in seeds of W-4.The contents of oleic acid,linoleic acid and linolenic acid were changed most significantly.Since the transformation of plant expression vector ihpRNAi interfered with the expression of fad2 gene in seeds,the expression level of fad2 gene was down-regulated during the developing of W-4 seeds,resulting in reduced FAD2 enzyme activity in seeds.Thus the desaturation of oleic acid would be affected,resulting in reduced linoleic acid content in W-4 seeds.The reduced linoleic acid content in seeds would reduce the substrate for FAD3 enzyme,thereby affecting indirectly the synthesis and accumulation of linolenic acid in seeds.So the linolenic acid content in W-4 seeds was also reduced compared with that in control.The hindered desaturation of oleic acid caused the accumulation of a large amount of oleic acid in seeds.In this study,the oleic acid content in W-4 seeds was even up to 86%.

The down-regulated expression of fad2 gene not only significantly changed the unsaturated fatty acid content in seeds but also affected the accumulation of saturated fatty acid in seeds.The palmitic acid,stearic acid and oleic acid are all synthesized in the plasmid,and their contents are determined by the competition among palmitoyl-ACP elongase,stearoyl-ACP desaturase and acyl-ACP thiolase for the common substrate.The palmitoyl-ACP,stearoyl-ACP and oleoyl-ACP synthesized in plasmid will be converted into palmitic acid,stearic acid and oleic acid respectively under the effects of 2 kinds of acyl-ACP thiolases(FATA and FATB).FATB is mainly included in the thiolysis of palmitoyl-ACP and stearoyl-ACP.After entering the cytoplasm,the generated fatty acid molecules by thiolases will be converted into acyl-coenzyme A molecules,which provide substrates for the synthesis of triglycerides.The fatB gene silencing could reduce the content of saturated fatty acid in seeds[22-23].

It has been reported that the fad2 gene silencing causes reduced palmitic acid content in seeds of transgenic high oleic acid soybean.Kinney et al.increased the oleic acid content in soybean to 75%-85%though downregulating the expression level of fad2 gene in soybean seeds,and with the decreased content of polyunsaturated fatty acids,the palmitic acid content was also decreased by nearly 20%[24].The fatty acid composition in seeds of various oil crops is actually the ultimate reflection of fatty acid composition from glycerol molecule esterification,which may depend on the relative concentrations of various acyl-CoA molecules in the cytoplasmic acyl-CoA pool and the substrates selectivity of acylase.Therefore,it could be hypothesized that the down-regulated expression of fad2 gene caused that the oleoyl-CoA concentration in the acyl-CoA pool was much higher than the palmitoyl-CoA concentration,so oleoyl-CoA was dominant in the esterification reactions with glycerol-3-phosphate,lysophosphatidic acid or 1,2-sn diglycerides,resulting in more oleoyl-CoA molecules binding to the TAG molecules; Or the triglyceride-phosphate acyltransferase,lysophosphatidic acid acyltransferase and 1,2-sndiacylglycerol acyltransferase might showed stronger affinity to oleoyl-CoA than to palmitoyl-CoA,leading to higher oleic acid content but lower palmitic acid content.

The synthesis of long-chain fatty acids,such as eicosenoic acid and erucic acid in Brassica seeds all uses oleic acid as the precursor.Under the catalysis of specific acyl-CoA elongase complex (FAE),two C groups are transferred consecutively from malonyl-CoA to the carboxyl terminus of oleoyl-CoAs,synthesizing eicosenoic acid and erucic acid.The extension of fatty acid carbon chain in the ER is very similar to the de novo synthesis of fatty acids in plastids.The down-regulated expression of fad2 gene results in the accumulation of a large amount of oleic acid in seeds,theoretically providing ample substrates for the synthesis of long-chain fatty acids.Ujjal K.Nath improved the erucic acid content in seeds using the fad2 mutant that was combined with LPPAT and FAE modification[25].This study found that the eicosenoic acid (18.46%)and erucic acid (13.15%) contents in W-4 seeds were all increased compared with those in Westar,indicating that the increased oleic acid level promotes the synthesis of long-chain monoenoic acids.

Using W-4 and its receptor control Westar as materials,this study investigated the effects of down-regulated expression of fad2 on contents of oil,proteins,crude fiber and glucosinolates in rapeseed seeds.The results showed that the contents of oil,proteins,crude fiber and glucosinolates in seeds of W-4 and Westar were all within the reasonable limits,and no significant differences were found between them.It suggested that the down -regulated expression of fad2 gene showed small effects on the quality indexes above.In fact,fad2 gene is a modified gene in the synthesis of fatty acids,and its main function is to desaturate oleic acid.So in theory,the effects of down-regulated expression of fad2 gene on none-fatty acid components in seeds should be very small.This also explained why there were no significant differences in contents of oil,proteins,crude fiber and glucosinolates in seeds between W-4 and Westar.In addition,the down-regulated expression of fad2 gene also showed small effects on amino acids contents in seeds.The results showed that the contents of various amino acids in seeds of W-4 and Westar were all within the reported ranges[21].The contents of non-essential amino acids in seeds of W-4 were lower than those of Westar (P ＜0.05),but there were no significant differences in contents of the other amino acids between W-4 and Westar,the reason for which is still unclear and needs further study.

In conclusion,the down-regulated expression of fad2 gene significantly improves the fatty acid composition in rapeseed seeds.Not only the linolenic acid content is reduced,but the saturated fatty acid content is reduced as well.Thus the oxidation resistance of rapeseed oil is improved,and at the same time,the nutritional value of rapeseed oil is also improved.

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