九州大学学術情報リポジトリ
Kyushu University Institutional Repository
タバコにおける小胞体局在型ω-3脂肪酸不飽和化酵 素についての研究
濱田, 達朗
九州大学理学研究科生物学専攻
https://doi.org/10.11501/3134903
出版情報：Kyushu University, 1997, 博士（理学）, 課程博士 バージョン：
権利関係：
Study on Microsomal ro-3 Fatty Acid Desaturase in Tobacco Plants
Tatsurou Hamada
Department of Biology Faculity of Science Kyushu University Fukuouka 812-81 Japan
CONTENTS
1. GENERAL INTRODUCTION
2. Part I
Cloning of a eDNA Encoding Tobacco Microsomal OJ-3 Fatty Acid Desaturase
3. Part II
Modification of Fatty Acid Composition by Over and Antisense Expression of a Microsomal OJ-3 Fatty Acid Desaturase Gene in Transgenic Tobacco
4. Part III
1
I l
26
53 Characterization of Biophysical Properties of Plasma Membranes from Transgenic Tobacco with an Increased a-Linolenic Acid Level
5. ACKNOWLEDGMENTS 86
GENERAL INTRODUCTION
A characteristic feature of the cellular membranes in plants is their very high len� I or fatty acid unsaturation c01npared with 1nembrane in other eukaryot s. Wh reas
glycerolipids from animal tissue typically contain an average of 2.0 to 2.5 double bondshnolecule in the hydrocarbon chains (Harwood et al., 1986; Marsh, 1990). the nurnber of double bonds/molecule in glycerolipids from higher plants is 3 to 3A in non photosynthetic tissues and 4.3 to 5.0 in chloroplasts (Harwood, 1980). Thi:-. i mpl ic:-.
that a high level of fatty acid unsaturation 1nay be associated with functions peculiar to plant cells. As a basic step in the understanding of the biological function of
polyunsaturated fatty acids, the n1etabolism of lipids and fatty acids has long been under investigation.
In plant cells, lipid metabolism i initiated y the ynthe is of 16:0-ACP and I > :0- ACP through the reactions of a type II fatty acid synthase (Ohlrogge and Browse. 1995 ).
The 18:0-ACP i, quickly and efficiently de aturated to 18: 1-ACP by a soluble cle:-.aturase in the chloroplast stroma (Shanklin and Somerville, 1991 ). There are two pathways rnr the incorporation of fatty acids into membrane glycerolipid : one in the chloroplast inner envelope (the prokaryotic pathway) and the other in the endoplasmic reticulum (the eukaryotic pathway) (Roughan and Slack, 1982; Browse and Somerville, 1991 ). The prokaryotic pathway uses 18: 1-ACP and 16:0-ACP for the sequential acylation or glycerol-3-phosphate to form phosphatidic acid, which in turn can be hydrolyzed to diacylglycerol. These two intennediates then act as substrates for reactions that produce the rnajor glycerolipid compounds (PG, MGD, DGD, and SL) of the chloroplast
men1branes. The eukaryotic pathway begin with the export of 16:0 and 18: l from the chloroplast as CoA thioesters and their incorporation (again, via phosphatidic acid and diacylglycerol) into PC and other phospholipids that are the principal structural lipid-, nf all nonchloroplast membranes. The diacylglycerol moiety of PC is also returned to the chloroplast nvelope, where it contributes to the production of thylakoid lipids ( Bn)\\'\C
and Sornerville. 199 1 ). The eukaryotic pathway is the predon1inant route of glyccrolipid :-.ynthesis in all non-photosynthetic tissues such as root and oil s eds.
An irnportant feature of the e pathways is that the synthesized glycerolipicb. \Vhich are incorporated into n1ernbrane , contain only 16:0 and 18: L the introduction or rurthcr double bonds into the acyl chains involves a series of additional desaturases. Among the enzymes perforn1ing desaturation of fatty acids, only the 18:0-ACP desatura�e i� -.,oluhlc.
all the others being membrane-bound either within the chloroplast or within the endoplastnic reticulum (Browse and Somerville, 199 1 ). Characterization of these desatura. es by traditional biochetnical approaches is hindered by the problems of solubilizing and purifying membrane-bound proteins. In most cases, procedure�
required to solubilize integral rnembrane proteins have led to the quick loss or de�atura�c activity. Instead, a wealth of understanding regarding the mechanisn1 and regulation of these desaturases ha been obtained through biochemical and genetic analyses or �e,·en classes of Arabidopsis tlwliana mutants, each of which i deficient in the activity of a specific desaturation step. To date, five chloroplast desaturase-deficient mutants (j(u/--1 .
.f([(/5.fad6,fad7 andfad8) and two endoplasmic reticulum desaturase-deficient mutanh
(fod2 and fad3) have been isolated. Two of the chloroplast desaturases are high I y substrate specific. Thefad4 mutant is deficient in a .6.3-de ·aturase that inserts a tran�
double bond into 16:0 esterified to the sn-2 position of PG (Browse et a!.. 1985 ). The
fod5 rnutant is deficient in a ,6. 7 -desaturase responsible for the ·ynthesis of 16: I on :V1 G 0
and possibly DGD (Kunst et al., 1989). In contrast, the other two chloroplast
desaturases act on acyl chains with no apparent specificity for the length of the fatty acid chain ( 16-or 18-carbon), its point of attachment to the glycerol backbone (sn-1 or sn-2 ).
or the nature of the lipid head group. The fac/6 mutant is deficient in the plastid cu-6 desaturase, wherea fad7 andfac/8 mutant are deficient in the plastid cu-3 desaturasc (Brows et al.. 1986, 19 9; McConn et al., 1994 ). The fad2 andfod3 rnutants arc d fie ient in th 1nicrosomal cu-6 and CD-3 desaturases, respectively. and these dc�atura-.,c
tlwliww
has shown that at least five of the seven cla, 'eSoffod
n1utants(f"cul:l. j(td3. f(td6.
fod7, andfod8)
represent the structural loci (Arondel et al., 1992: lba et al.. 1993: C1ih.,on et al.. 1994: Hitz et al., 1994: Okuley et al., 1994 ).Analyses
offad
and other mutants involved in lipid metabolism have shown that unsaturated fatty acids have at least two important physiological roles in plant cell">. Fir"'t.18:3 is a precursor for fatty acid-derived signaling molecules, such as jasn1onic acid (Fanner, 1994 ).
Thefod3fad7 fad8
triple mutant contained negligible levels or I X:3. and showed that conversion of 18:3 to jasmonic acid is essential for pollen development and insect defense (McConn and Browse, 1996, McConn et al., 1997). Second. unsaturated fatty acids have been considered to play an important role in low ten1perature tolerance.Several A.
tlw/iww
mutants in which the content of saturated fatty acids andmonounsaturated fatty acids increased showed altered plant growth. most of \vhich appeared at low temperatures. The
j(n/5
andfad6 mutants showed leaf chlor :-.i:-.. rc !un�d growth rate and impaired chloroplast development at soc (Hugly and Smnerville. 199� ). The A.tlwlianafabl
nmtant in which the 16:0 content increased in leaf polar I ipid� a:-. a result of the reduced activity of 3-ketoacyl-ACP syntha ·e II showed severely inhibited growth after prolonged exposure at 2°C (Wu et al., 1997).Thefod2
n1utant showed reduced stem elongation at l2°C and died at 6°C (Miguel et al., 1993). Analyse.· or transgenic plants in which the level of unsaturated fatty acids had been 1nanipulatcd abo reveal d the modulated chilling sensitivity. When the A.t/wl iano
plant was transformed with the E. coli
glycerol-3-phosphate acyltransferase gene(plsB),
the resultant plant:-.contained n1ore saturated PG and showed increased chilling sensitivity (Wolter et a! ..
1992). On the other hand, the transgenic tobacco plant containing a reduced level or
�aturated PG was produced by an overexpre sion of the A.
tlwliww
glycerol-3-pho-.,phatc acyl transferase gene and showed an increased chilling-tolerant trait (Murata et al . . I l)t)2 ). Transgenic tobacco, in which a cis-double bond was introduc d at the 69 po�ition b) the cyanobacterial 69-desaturase gene, howed a highly reduced 16:0 level and acorresponding accutnulation of 16: lc9 in rno 't membrane lipids. This transgenic tobacco plant e hibited a significant increa e in growth rate at l0°C- l5°C and enhanced chilling
re�istance at 1 °C (I ·hizaki-Nishizawaet al., 1996; Fujii et al., 1997). Taken together.
these results with the mutant and tran genic plants indicate that a high level of fatty acid unsaturation observed in the plant cell membrane contribute to the adaptation to low temperatures.
Liquid-to-gel crystalline phase transition and a reduction in fluidity within the m n1brane lipid bilayer at low te1nperatures has long been assUined to be the trigger or an acute loss of n1embrane integrity, leading to injury and death in higher plant� (Lynn-.,.
1973; WolfeJ 978). However. Hugly and Somerville (1992) denied the application or this hypothesis to the mechanism cau ing chilling injury infad5 andfod6 mutant�
because 1neasurement of the fluidity of thylakoid membranes and differential �canning calorin1etry of thylakoid lipids from tbe wild-type,jad5 andfad6 plants did not \how any significant differences between these lines, within the temperature range of ooc to 55 C.
Miguel et al. ( 1993) also con ·ide red that the chilling- ensitive phenotype of th ./(td:l mutant is not due to the phase transition in the lipid bilayer since the temperature ( 12 -c l at which a reduction in stem elongation was ob erved was higher than the liquid-to-gel crystalline phase transition temperatures for 18:1118:1 and 16:0/l8:1 phosphaticlylcholinc (-l7°C and -3 °C, re pectively), both of which are 1najor constitutents of the micrn-.,omc tnen1branes offod2 plant . Although there are still several research group� which consider that the primary cause of low-temperature-induced injuries is due to change-., in the physical properties of the membrane , 1nea urement of the fluidity of the membrane-..
and detennination of the phase tran ition temperature of 1nembrane lipids were not exanlined in other n1utants or transgenic plants which exhibited altered chilling sensitivities.
The level of 18:3 in both chloropia ·t and extrachloroplast membrane lipid\ ha-., been found to increase in many plant species when plants are expo ·ed to low temperature and thi.· increase is associated with a decrease in 18:2 level (Smolenska and Kuirer.
1977; Clarkson et al., 1980; Kodama et al., 1995). In contrast the level of other ratty
degrees of chilling and freezing tolerance. This adaptive developn1ent of lO'v\·-te mpcrat ure tolerance is referred to as cold acclimation. Low temperatures induce not only an increa�e in 18:3 content but also rnany alterations to other biochemical 111 tabolisms: change-.. in lipid composition. synthesis of low-temperature-induced proteins and cryoprotectanh.
etc. (Guy. 1990). Therefore, the precise role of an increase in 18:3 content in membrane lipids could not have been estimated by a compari on of acclin1ated plants with
nonacclitnated plants (Steponkus, 1984 ). Analyses of the tnutants and transgenic plant\
1nentioned above did not provide any useful information about the significance )ran increase in 18:3 during cold acclimation. Analyses of the
.fctd2, .fctd5
andf
od
o mut<.llll\showed that the fatty acid composition een in the wild-type plants is a pr requisite for healthy growth at low temperatures. Tran genic plants with glycerol-3-phosphate
acyl transferase genes suggest that a high-temperature-melting species of PG i\ in,·oln�d in the deternlination of chilling sensitivity between plant ,pecies. Transgenic plant-.. into which the L19-desaturase gene is overexpressed showed that artificial reduction in the
16:0 level, which would not occur naturally, is effective toward enhancen1ent or chilling
tolerance for higher plant .
Molecular biological techniques with the CD-3 fatty acid desaturase genes enabled U\
to estimate the role played by increases in 18:3 content in membrane lipids on plant cell function. In the transgenic tobacco plants transformed with the A. rlwliono pla\tid Ct)-3 fatty acid desaturase gene (FAD7), the level of trienoic fatty acids ( 16:3 and 18:3) in chloroplast lipids increased (Kodama et al, 1994 ). The re 'Ultant plant exhibited an enhanced chilling resi tance and its degree of chilling tolerance was con1parablc with that of th low-temperature-acclirnated plants (Kodama et al. , 1995). This result �uggc�h that an increase in trienoic fatty acids (16:3 and 18:3) in chloroplast lipids is one or the factor-..
conferring acclin1ation-related chilling tolerance. On the other han l. there ha\'t:- been no reports investigating the physiological function of increased 18:3 content in
extrachloropla 't rnembrane lipids. In this the is, I address this question by U\ing transgenic tobacco plant, in which the 18:3 content of extrachloroplast lipid\ ha� been alter d by n1odulation of the expression of the microsomal CD-3 fatty acid de\atura-..e gene.
In part I. I describe the cloning of the tobacco micros01nal CD-3 fatty acid de�atura�e gene
( VtFAD3. Harnada et al.. 1994 ). The deduced mnino-acid sequence of the VrF AD3 h 719'(' identical to that of th A. tlzaliano microson1al CD-3 fatty acid lesatur<be gene
(FAD3). In part II. I report the production of transgenic tobacco plants in \vhich the transcripts of the NtFAD3 gene were expres ·ed in sense and antisense orientation� under the control of the cauliflower mosaic virus (CaMV) 35S prornoter (Harnada et a!. I 096 ).
I den1onstrate how the up- and down-regulation of the mRN A level in the micro\omal (t)-
3 ratty acid desaturase gene was useful for modifying the 18:3 content in the vegetal i \ e
tissues of higher plants. In part III, I examine the effect- of increased 18:3 content In extrachlorop1ast lipids on the function of higher plant cells. Transg nic tobacco plant-..
containing a high 18:3 content in PC and PE were produced and the 18:3 le\·el in phospholipids was alrnost the san1e as those of low-temperature-acclin1ated fr ezing
tolerant plants. such as wheat and rye, etc.
Abbreviations: 16:0, palmitic acid; 16:1, hexadecenoic acid; 16:3, hexadecatrienoic acid:
18:0. stearic acid; 18:1, oleic acid; 18:2, linoleic acid; 18:3, ex-linolenic acid: ACP_ ac� I
carrier protein; CoA, coenzyrne A; DGD,digalactosyldiacylglyceroL MGD.
monogalacto ·yldiacylglycerol; PC, phosphatidylcholine; PE, phosphatidylethanolaminc:
PG. phosphatidy !glyceroL SL, sulfoquinovo-sy ldiacy lgl ycerol.
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Part I
Cloning of a eDNA Encoding Tobacco Microsomal co-3 Fatty Acid Desaturase
ABSTRACT
A eDNA wa · isolated from a tobacco (Nicotiano tctbocwn cv. SR l) leaf c 0:\
library using, as a hybridization probe, a cON A fragment from th gene (FAD 7) en cod in�
Arohidopsis thaliww chloroplast co-3 fatty acid desaturase. The deduced 379-amino-acid
s quence has 67-71 o/o identity to those deduced frmn the previously described gene-..
encoding the co-3 fatty acid desatura es of A. tlzaliana. The absence of the -terminal extension transit peptide in the deduced amino acid sequence of the cON A clone and the accun1ulation pattern of the mRNA corresponding to this eDNA in leaf and root ti\\ue-..
indicate that the isolated eDNA encodes a tobacco microsomal co-3 fatty acid desatur�be (NtFAD3). The steady-state level of the NtFAD3 mRNA remained approximat ly
constant at temperatures l5°C to 40°C. Southern blot analysis suggested the presence of the additional genes that share the imilarity with NtFAD3 gene.
Abbreviations: 16:2. hexadecadienoic acid� 16:3, hexadecatrienoic: 18:2, linoleic acid:
18:3: linolenic acid: Mr, molecular mass.
INTRODUCTION
One of the remarkable chm·acteristics of the plant cell rnembran s is that they ha' c unusually high contents of lipids containing trienoic fatty acids such as linolenic (I �:.3 l
and hexadecatrienoic (16:3 ) acids (Somerville and Browse, 1991 ). This high I eve I or fatty acid unsaturation is assmned to be important for the low te1np rature fitne�� or plant-..
(Kodmna et al., 1994; 1995). The co-3 fatty acid desaturases catalyze the con\'er�ion of dienoic ( 16:2 and 18:2) fatty acids to trienoic (16:3 and 18:3) fatty acids in lipids. In plant cells. there are two types of w-3 fatty acid desaturase. one located in the micro�omc-.. and
the other located in the plastid membranes. Three genes, FAD3 encoding the micro omal desaturase. FAD7 and FAD8 encoding the plastidial desaturases. were isolated from Arobidopsis tlwliww (Iba et al., 1993; Yadav et al., 1993 ; Gibson et al.. 1994). hnlation of such desatura e genes enables us to modify the fatty acid cornposition in memhranc lipids. Here I isolated a rnicrosomal w-3 fatty acid desaturase gene from a tobacco plant which is one of the model plant species suitable for genetic engineering of the plant cell function.
MATERIALS AND METHODS
Purification of RNA and Construction of eDNA Library
Seeds of Nicotiana tabacum cv. SR 1 were germinated on soil under conti n uou-., light (2.000 lux) at 25°C. The 40-day-old seedlings were transferred to l5°C under the san1e light condition. After 24 h, leaves were harvested. frozen in I iquid nitrogen and stored at -80°C. Total RNA was purified from leaf tissues by the method of Schmidt ct
al. ( 1981 ). Poly( A)-rich RNA was prepared fro1n total RNA by the chron1atography on oligo(dT)-cellulose (Pharmacia). A A.gt l l eDNA library was constructed from the poly(A)-rich RNA by using the eDNA cloning system A.gt 11 (Arn rsham Japan).
Adapters used in this eDNA library had EcoR I, BamH I, Nco I and Kpn I sites. and cDNAs were isolated from the phage DNA by digestion with these restriction
endonucleases.
Screening of Tobacco eDNA Library
A ·ense oligonucleotide 5'-TCTTTGAGTTATGTCGTC-3', cornplementary to the nucleotide sequence +349 through +367 of the A. thaliana FAD7 genomic region ( lba ct al.. 1993). was annealed to the single- tranded A. tlwliana FAD7 eDNA template and extended in the presence of [cx-32p]dCTP (ICN Biomedicals) by the Klenow fragment (Nippon Gene). The product was used as a hybridization probe to screen a total or ahnut 60.000 plaques of the tobacco eDNA library transferred to the Biodyne A membrane"
(Pall Ultrafine Filtration Corporation). The hybridization was carried out at 50°
overnight in the solution described by Singh and Jone ( 1984 ). The membranes were then washed twice for 30 min at 50°C in 2xSET, 0.2o/o (w/v) SDS, and then again in
I x ET, 0.2o/c (w/v) SDS for 30 min at 50°C ( l xSET; 25mM NaCL 1.5rnM Tris-HCI pH 7.5, 0.1 mM EDTA).
The eDNA insert of a positive phage clone was cut out by BwnH I and \Ubcluncd into the plasmid vector, pBluescript II KS+ (Stratagene), to produce a pla. mid clc\l�nc�tcd pF l. Nucleotide sequence was detennined by the dideoxy-chain-tennination method (Sanger et al.. 1977) with double-stranded DNA templates.
Northern Blot Analysis
The steady-state level of the NtFAD3 mRNA in leaf and root tissues were examined in the 9-week-old tobacco plants grown at 26°C. For detennination of mRNA len�! in response to the temperature, the 5-week-old tobacco plants grown at 26°C were incubated at indicated ten1peratures under illumination. Leaf tissues were harvested 2-+ h and-+� h after temperature shift. Total RNA was purified by the method of Sch1nidt et al. ( 1 <)g I l or
the method of Puisc ant and Houdebine ( 1990) and denatured with 6.3o/a ( w/v)
fonnaldehyde and SOo/o (v/v) formamide and then fractionated on 1 o/c (w/\') agaro-..c �cl that contained 2o/o (w/v) fonnaldehyde, by a tnodified version of the procedure or Lchrach et al. (1977). The eDNA insert cut out from the pF l was labeled with [a-32p]c!A TP (ICN Bion1edicals) by random oligonucleotide-pritned synthesis method (Feinberg and Vogelstein, 1983), and used as a hybridization probe. RNAs were transferred to a nylon membrane and hybridized with the probe. Pre-hybridization. hybridization and po t
hybridization steps were perforn1ed essentially a de 'Cribed in the instructions for the nylon 1ne1nbrane (Biodyne B, Pall Ultrafine Filtration Corporation). The cOl'\
fragments that hybridized to the corre ponding RNAs were visualized by
autoradiography. The 32P-labeled rice tRNAGly (GCC) gene (Reddy and Padayatty.
1988) was hybridized to the corresponding tobacco RNA, and used as a rererence in estimation of the amount of loaded RNA.
Southern Blot Analysis
Total genomic DNAs were isolated frmn leaf tissues by the procedure of Murray
·md Thompson ( 1980). and digested with EcoR I or Hind III. DNA fragmenh were separated b lectrophoresis in a 0.8Cfl (w/v) agarose gel, transferred to a nylon
membrane (Biodyne B, Pall Ultrafine Filtration). The blot was hybridized with labeled.
the 0.5-kb Hind III fragment of the NtFAD3 eDNA (from nucleotide 850 to 1381. \CC
also Figure 1) and washed by the method described in the instruction for th nylo n
membrane (Pall Ultrafine Filtration). The eDNA frag1nents that hybridized to the corresponding DNAs were visualized by autoradiography.
RESULTS AND DISCUSSION
Isolation of a eDNA Clone
A Agt11 eDNA library was constructed fro1n poly(A)-rich RNA purified from tobacco (N. tolxtcum cv. SR1) leaves. The A. tlwliona FAD7 eDNA (lba ct al.. llJl)J l ''a"
used as a heterologous hybridization probe to isolate cDNAs related to the co-3 !'att) acid clesaturase genes. One of the cross-hybridizing clones was purified and subjected to nucleotide sequencing. The isolated eDNA, NtFAD3, had a length of 1381 bp that contained an open reading frame of 1137 bp corresponding to 379-amino-acid r �iJuc�
(Mr 44148. Fig. l ). Except for the N-tenninal region, the region corresponding to
arnino-acid sequence. 22-379, of the NtFAD3 showed a continuous stretch or high identity with the A. tlwliana FAD7 gene (67.0o/a) and the A. tlw/iww FAD3 gene ( 71.5r ( l
(Fig. 2). The deduced amino acid sequence of the NtFAD3 eDNA lacked the N-terminal extension transit peptide, a· was the case in the A. tlzaliww FAD3 gene.
Expression of the NtF AD3 Gene
Northern blot analysis revealed that the size of the NtFAD3 mRNA wa� about 1.5 kb. which was si1nilar to the size of the cloned eDNA (Fig. 3). It suggests that thi"
eDNA clone contains a full-length eDNA for the NtFAD3 mRNA. The mRNA corresponding to the NtFAD3 gene was detected both in root and leaf tissues. The A.
tlwliano FAD3 gene was expressed constitutively in root and leaf tissues. while the ,\.
tlwliww FAD7 gene was expressed at a high level in leaf tissues and at a trace lc\·cl in root tissues (Yadav et al., 1993). Simirality with both mnino acid sequ nc and gene
expression of A. tlza/iana FAD3 indicate that the NtFAD3 gene encodes a micro�omal Cr)-
3 fatty acid desaturase.
To d tern1ine whether transcriptional control of the NtFAD3 gene occur� in response to temperatur , the 26°C-grown tobacco seedling· were transferred to ,·ariou"
t mp rature,· (Fig. 4). The steady-state level of the NtFAD3 rnRNA remained approxin1at ly constant for 2 days in leaf tissue· exposed to l5°C. 30°C or -+0 C.
.... L _ _______________________________________________ _
A.rlwliww FAD8 gene is low-temperature inducible; its mRNA 1 vel increa�ed
�ignificantly at temperatures below 20°C, which has been considered to play a po�iti n�
role in acclitnation of the A. tlwliana plants to low temperatures (Gibson et al.. ILJ0-+ l. In tobacco plants. the transcriptional regulation of the NtFAD3 gene had a very restricted role in low tetnperature adaptation.
Southern Blot Analysis of the NtF AD3 Gene
The copy number of the tobacco micros01nal co-3 fatty acid desaturase gene� was
clucidat d by Southern blot analysis (Fig. 5). When 0.5-kb 3' region of the NrFAD3 eDNA was used as a probe, several hybridization bands were detected. sugge�ting the presence of additional genes that are likely to encode the microson1al co-3 fatty acid desaturase.
REFERENCES
Feinberg AP, Vogelstein B ( 1983) A technique for radiolabel ing DNA re�triction endonuclease frag1nents to high specific activity. Anal Bioch m 132: 6-13 GibsonS, Arondel V, lba, K, Somerville C (1994) Cloning of a temperature
regulated gene encoding chloropia t (t}-3 desaturase from Arobidopsis tlwliww.Plant Physiol103: 1615-1621
lba K, Gibson S, Nishiuchi T, Fuse T, Nishimura M, Arondel V, Hugly S, Somerville C (1993) A gene encoding a chloroplast CD-3 fatty acid de-..atura�e c01nplen1ents alterations in fatty acid desaturation and chloroplast copy number or thefad7 n1utant of Arabidopsis tlzaliana. J Biol Chern 268: 24099-24-105
Kodama H, Hamada T, Horiguchi G, Nishimura M, Iba K ( 1994) Genetic enhancement of cold tolerance by expre sion of a gene for chloroplast CD-3 fatty acid desaturase in tran genic tobacco. Plant Physiol 105: 601-605
Kodama H, Horiguchi G, Nishiuchi T, Nishimura M, Iba K ( 1995) Fatt \' acid desaturation during chilling acclimation is one of the factors involved in con krrt ng low-ten1perature tolerance to young tobacco leaves. Plant Physiol 107: 1177-1 I �5 Lehrach H, Diamond D, Wozney JM, Boedtker H ( 1977) RNA molecular
weight determinations by gel electrophoresis under denaturing con lition�. a crittcal reexamination. Biochemistry 16: 4743--4751
l\llurray MG, Thompson WF ( 1980) Rapid isolation of high-1nolecular-weight plant DNA. Nucl Acids Res 8: 4321-4325
Puissant C, Houdebine L-M ( 1990) An improvement of the single-step methnd or RNA i olation by acid guanidinium thiocyanate-phenol-chloroform extraction.
BioTechniques 8: 148-149
Reddy PS, Padayatty JD ( 1988) Effects of 5' flanking sequences and change-; in the 5' internal control region on the transcription of rice tRNAGly (GCC) gene. Plant Mol Bioi 11: 575-583
Sanger F, Nicklen S, Couson AR (1977) DNA sequencing with chain-tcrmin<.lttn�
inhibitiors. Proc Natl Acad Sci USA 74: 5463-5467
Schmidt GW, Bartlett SG, Grossman AR, Chashmore AR, Chua N-H ( 1981) Biosynthetic pathway of two polypeptide subunits of the light-harve�ting chlorophyll alb protein complex. J Cell Biol 91: 468-478
Singh L, Jones KW (1984) The u e of heparin as a simple cost-effective mean" or controlling background in nucleic acid hybridization procedure. N uc I c id� Rc..., 12: 5627-5638
Somerville C, Browse J ( 1991) Plant lipids: metabolisn1, mutants. and meinbranc\.
Science 252: 80-87
Yadav NS, Wierzbicki A, Aegerter M, Caster CS, Perez-Grau L, Kinney AJ, Hitz WD, Booth JR Jr, Schweiger B, Stecca KL, Allen S.\L Blackwell M, Reiter RS, Carlson TJ, Russell SH, Feldmann KA.
Pierce J, Browse J (1993) Cloning of higher plant co-3 fatty acid de�atura...,c Plant Physiol. 103: 467-476
1 GCAGTGTAGAGGAATACGGAGAGCTGTCAAAAGGTTCAGGGTTGAAGACTCTGGK G�C�
61 ATCAATGGGGTCTCTGGGAATAAGTGAGATTTATGATAAGAACAGCTTTAACG�G�TGG�
M G S L G I S E I Y D K N S F E 1 �
121 GTTTGAATTCGACCCGAGTGCCCCTCCTCCGTTTAGGCTGGCTGAGATACGAAATGTC��
F E F D P S A P P P F R L A E I R N
181 CCCTAAGCATTGCTGGGTTAAAGATCCACTTAGGTCCTTGAGCTATGTTGTGAGGGACG�
P K H C W V K D P L R S L S Y V V R 0
241 AATATTTGTTGCTACCTTGATTGGCATAGCAATTCACTTGGATAGTTGGTTATTTTACC
I F V A T L I G I A I H L D S W L F Y ?
3 0 1 ACTTTACTGGGCTATCCAAGGCACCATGTTTTGGGCAA TCTTTGTTCTTGGACA TG.:.. "'r:-r:
L Y W A I Q G T M F W A I F V L G H D C
361 TGGCCATGGCAGCTTTTCAGACAGCCAGTTGCTAAATAATGTGGTTGGTCACATACTTC�
G H G S F S D S Q L L N N V V G H I n
421 TTCTGCCATTCTGGTACCCTACCATGGCTGGAGAATCAGCCATAAAACTCACCATCAGA�
S A I L V P Y H G W R I S H K T H H Q
481 CCATGGAAATGTGGAGACTGATGAGTCTTGGGTGCCGATGCCTGAAAAGCTATACAKC�A
H G N V E T D E S W V P M P E K L Y N K
541 AGTGGGCTATTCAACCAAGTTCCTAAGATACAAGATCCCTTTTCCCTTGTTAGCATATCC
V G Y S T K F L R Y K I P F P L L A Y P
601 AATGTACCTGATGAAGAGAAGTCCAGGAAAATCTGGTTCTCATTTCAACCCATATKG�G�
M Y L M K R S P G K S G S H F N P Y S
661 TTTGTTCCAACCCCATGAGAGGAAGTATGTGGTAACATCAACTCTGTGCTGGACTG�C��
L F Q P H E R K Y V V T S T L C W T �
721 GGCAGCTCTCCTCCTCTATCTCTGCACTGCCTTTGGTTCTCTCCAAATGTTTAAGATT':"�
A A L L L Y L C T A F G S L Q M F K I Y
781 TGGCGCTCCTTACTTGATTTTTGTAATGTGGCTGGATTTTGTTACCTACTTGCATCACCA G A P Y L I F V M W L D F V T Y L H H H
841 TGGTTATGAGAAGAAGCTTCCCTGGTACCGCGGCAAGGAATGGAGTTATCTTAGAGGAGG G Y E K K L P W Y R G K E W S Y L R G G
901 ACTAACCACAGTTGATAGAGACTACGGATTGTTTAACAACATACATCATGACAT�GG��
L T T V D R D Y G L F N N I H H D I G
961 ACATGTTATCCACCATCTATTTCCTCAGATACCACATTATCACTTGAGAGAAGCGACC;�
H V I H H L F P Q I P H Y H L R E A T
1021 AGCAGCAAAGCCAGTACTTGGCAAGTATTACAGGGAACCCAAGAAATCAGGGCCAA TCC
A A K P V L G K Y Y R E P K K S G P I P
1081 TTTTCACTTGGTTAAGGATTTAACAAGGAGCATGAAGCAGGATCATTATGTTAGCGATTC
F H L V K D L T R S M K Q D H Y V S D
1141 CGGGGAGATTGTTTTCTATCAGACCGATCCACACATCTTCCGATCTGCTCCAAAGGKTG�
G E I V F Y Q T D P H I F R S A P K D �
12 0 1 ATGAGTGAGAAACAGAAA TCAAAAGP..A TCTCATT AACTAGTTTCAGAAACTTGGT ;:,.. 'T __ :._r:
1261 CTATAGTTTTATAATTACAACCTATTGAAAAAATAGGTTTGAGAGCTTTTTGTTAGTCG�
1321 TAAGATTGTGTTCCCATTATTTCTCTCTTGAAAACAAAACTACAAG���AA�
13 1 A
Figure 1. Nucleotid and deduced amino acid equences from the isolated cO ' of the tFAD3 gene. (The DDBJ/EMBL/ GeneBank acce sion No. for NtFAD3 i� 026509).
1
7 MA LVLSECGIRPLPRIYTTPRSNFLSNNNKFRPSLSSSSYKTSSSPLSFGLNSRDGFTR
7 mvALNVSTPLTTPIFEESPLEEDNKQRFDPGAPPPFNLADIRAAIPKHCWVKNPWKSLSY 3 MWAMDQR • NVNGDPGAGDRKKEE • • • • S • Q • • • KIG • • • • • • • • • • • • • S • LR • tvi • • 3Nt MGS•GISEIYDKNSFNEMEFE•••S•••••R••E••NV••••••••D•LR••••
7 VVRDVAIVFALAAGAAYLNNWIVWPLYWLAQGTMFWALFVLGHDCGHGSFSNDPKL S � 3 • • • •IIA•A• • •IA•V•VDS•FL• • • • •A• • • •L• • •I• • • • • • • • • • • • •DI•L• • • • • 3Nt • • • • •IF•AT•IGI•IH•DS•LFY• • • •AI• • • • • • •I• • • • • • • • • • • • •DSQL• ._�. •
7 GHLLHSSILVPYHGWRISHRTHHQNHGHVENDESWHPMSEKIYNTLDKPTRFFRFTLPLV 3 • • I • • • F • • • • • • • • • • • • • • • • • • • • • • • • • • • • V • LP • RV • KK • PHS • • ML • Y • V • • P 3Nt • • I • • • A • • • • • • • • • • • • K • • • • • • • N • • T • • • • V • • P • • L • • KVGYS • K • L • YKI • FP
7 MLAYPFYLWARSPGKKGSHYHPDSDLFLPKERKDVLTSTACWTAMAALLVCLNFTIGPIQ 3 • • • • • L • • CY • • • • • E • • • FN • Y • S • • A • S • • • LIA • • • T • • S I • FVS • IA • S • VF • • LA 3Nt L • • • • M • • MK • • • • • S • • • FN • Y • • • • Q • H • • • Y • V • • • L • • • V • • • • • LY • CTAF • SL •
7 MLKLYGIPYWINVMWLDFVTYLHHHGHEDKLPWYRGKEWSYLRGGLTTLDRDYGLIN IH 3 V• •V• •V• •I•F• • • • •A• • • • • • • • •DE• • • • • • • • • • • • • • • • • • •I• • • • •IF• • • • 3 Nt • F • I • • A • • L • F • • • • • • • • • • • • • • Y • K • • • • • • • • • • • • • • • • • • • V • • • • • • F • • • •
7 HDIGTHVIHHLFPQIPHYHLVEATEAAKPVLGKYYREPDKSGPLPLHLLEILAKSIKEDH 3 • • • • • • • • • • • • • • • • • • • • •D• •K• • •H• • •R• • • • •KT• •AI•I• •V•S•VA• • •K• • 3Nt ••••••••••••••••••••R•••K•••••••••••••K••••I•F••VKD•TR•M•Q••
7 YVSDEGEVVYYKADPNLYGEVKVRAD 446 3 ••••T•DI•F•ET••D••VYASDKSKIN 386 3Nt • • • • S • • I • F • QT • • HIFRSAPKDE 3 7 9
6
:7- 17 38
36
4:2
Figure 2. Alignn1ent of the amino acid sequences derived from the NtFAD3 gene (3Nt) and the A. tlwliona FAD7 (7) and FAD3 (3) genes. Identical amino acid residues are indicated by a clot.
NtFAD3
t RNAGiy
...
0 0 a:
Figure 3. Expression of the NtFAD3 mRNA in leaf and root tissues. Total RNA
(19.4
!lg) extracted from each tissue was probed with the 32P-labeled NtFAD3 eDNA and then reprobed with a rice tRNAGly gene as a loading control.
NtFAD3
C") a
i1
1�
0 <i5>
� <(
z a:
E
0.5
� Q)
(j) :>,
"'0 cu Q)
(j) 0
() 0 () 0 () 0 () 0 () 0 () 0 () 0 () 0
l() <0 0 0 l() <0 0 0
T""" N Ct') � T""" N Ct') �
t t t t t t t t
() 0 () 0 () 0 () 0 () 0 () 0 () 0 () 0
<0 <0 <0 <0 <0 <0 <0 <0
N N N N N N N N
24h 48h
Time after temperature shift
Figure 4. Expression of the NtFAD3 mRNA in leaf tissues exposed to various temperature conditions. Twenty micrograms of total RNA per lane were subjected to Northern analysis. The blot was visualized and quantitated with BAS 1000 II imaging system (Fuji Film, Japan). The amount of the NtFAD3 mRNA in plants after
temperature hift was normalized to that of the 26oC-grown plants (second lane from the left).
,....
-
a: - ,
0 c:
aH ... :X:
(kb)
23.1
--9.4
--6.6
--4.4
- 2.3 -- 2.0
-- 0.6
Figure 5. Southern blot analysis of total DNAs. Total DNAs (10 mg) were digested with EcoR I and Hind III and hybridized with 0.5-kb Hind III fragment of pFl. The fragment izes of the A, DNA digested with Hind III are indicated on the right.
Part II
Modification of Fatty Acid Composition by Over and Antisense Expression of a Microsomal co-3 Fatty Acid Desaturase Gene
in Transgenic Tobacco
ABSTRACT
CD-3 Fatty acid desaturase catalyzes the conversion of linoleic acid ( 18:2) to 1 inolenic acid (18:3) in lipids. In Nicotiana tabacum cv.SRl, two cDNAs for the CD-3 fatty acid desaturases have been isolated: one encodes the micros01nal desaturase (N! FA D3) and the another encodes the plastidial desaturase (NtFAD7). In root tissues. the main enzyme for the production of 18:3 is considered to be the microsomal CD-3 fatty acid desaturase. I
produced transgenic tobacco plant that express the transcripts of the NtFAD3 gene in antisense and sense orientation under the control of the cauliflower 1nosaic virus 35S promoter. The antisense construct has the 0.5-kbp fragment of the NtF AD3 cDN A that contains a 3'-flanking region and a part of the coding region in antisense orientation.
Several antisense-transformant lines showed decreases of the steady-state le\·eb or the NtFAD3 and NtFAD7 mRNAs to 30o/o and 70o/o of the control plants. respectively. In these lines, the 18:3 content decreased to about 80o/o in root tissues and to about 60-70 r in leaf tissue· when compared with the control plants. The sense construct has the I.-+
kbp full-length eDNA of NtFAD3. In one of the sense-transfonnant lines. the rFAD3 mRN A level increased 8 times when compared with that of the control plan h. Th i. line had increased 18:3 content by about 1.5-fold in root tissues and by about I. !-Cold in lear tissues when compared with the control plants. These results indicate that the up- and down-regulation of the transcript level in the micros01nal CD-3 fatty acid desaturase gene is useful to modify the 18:3 content in the vegetative tissues of higher plants.
INTRODUCTION
The following two reasons may be presented for the broad inter st in controlling the fatty acid compositions of higher plants. Fir t, vegetative oils are of economic imrortance as human and animal foods and in many industrial applications. The fatty acid
compositions of Inost vegetative oils are not optimal for these uses (reviewed by Ohlrogge, 1994 ). Second, the changes in unsaturation of the fatty acids have been considered to be one of the factors in metabolic adaptation of higher plants to
environn1ental stresses, e pecially to temperature (reviewed by Son1erville and Brow�c.
1991 ). The isolation of genes involved in lipid metabolis1n and the development or
techniques for producing tran genic plants in which target genes are overexpres�ed or suppressed will allow us to produce more valuable plant oils and also to enhance the tolerance of plant to temperature stress by 1nodifying plant lipid metabolism. For
example, antisense expression of the stearoyl-acyl carrier protein desatura�e gene re�ulted in a decrea ·e of oleic acid ( 18:1) and an increa e of stearic acid ( 18:0) in seed oil. or t\VO Brossica specie, (Knutzon et al., 1992). An alteration of chilling sensitivity wa\ reported in tran 'genic tobacco (Murata et al., 1992) and Arubidopsis tlzalimw (Wolter ct al.. 19l)2) in which the glycerol-3-pho phate acyltransferase gene was overexpressed. Th
transgenic tobacco plants harboring a cyanobacterial L19-desaturase gene exhibit d enhanced resi tant traits for low temperatures (lshizaki-Nishizawa et al. 1996 ).
The co-3 fatty acid desaturases are membrane-bound enzymes found in micro�omc�
and in plastid envelopes (reviewed by Mazliak, 1994) and catalyze the conv r�ion of linoleic acid (18:2) to linolenic acid (18:3) in lipids. The plastid-localized type cataly;c"
also th conversion of hexadecadienoic acid (16:2) to hexadecatrienoic acid ( 16:3). The ./c1d mutants of A. tlwliana were isolated a being defective in fatty acid de\aturation. The .fcl(/3 (Lemieux et a!., 1990) andfac/7 (Browse et al., 1986) were characterized by a
18:3 I g:2
wu� i ·alated by T-DNA tagging fro1nA. tlzaliana (Yadav et aL 1993). The FtiD7 gene.
too. was i ·alated from A. tlwliana by using of a technique of chron1o�omal \\'a! king ( Iha
et al.. 1993).
These genes for the co-3 fatty acid desaturases have been introduced to �e\·eral plant cells. The A. rlwliww hairy root in which the B. nopus FAD3 gene was O\'erexpre�"ed under the control of the cauliflower mo aic virus (CaMV) 35S pro mot r had a
significantly increased amount of 18:3 (Arondel et al., 1992). The A. rlwliww FtiD3 eDNA was introduced into A. thaliana in the sense orientation behind a seed-�peciric pron1oter. The R2 seeds from the transgenic plants ·bowed about 2-f lcl increa�c nr IS:�
level when compared with those of the wild-type plants (Yadav et a!.. 1993 ). The overexpression of the A. tlwliona FAD3 gene by using the CaMV 35S promoter Gtu..,ed the conversion of mo ·t 18:2 to 18:3 in the transgenic carrot hairy roots (Yadav et al..
1993 ). In soybean plants transformed with a chimeric gene consisting of an anti�en. e
soybean FAD3 eDNA under the control of a seed-specific prmnoter. the seed I �:3 le\·el decreased fron1 9% to les than 2o/o (Hitz et al., 1995). However. there has been no report concerning the effects of overexpres ion of the FAD3 gene on the fatty acid compositions of vegetative tissues including leaf and normal root tissues. n the other
hand. the A. tlwliana FAD7 eDNA was overexpressed in the A. tlwliww j({(/7 mu tant. and the transformants showed a complementary phenotype (Iba et al., 1993 ). Tran�genic tobacco plants that contain increased trienoic fatty acid ( 16:3 and 18:3) and
correspondingly decreased levels of their precursors. 16:2 and 18:2. in lear ti�"uc:-. \\ere engineered by the introduction of the A. tlwliana FAD7 eDNA in sense orientation he hind the CaMV 35S promoter. The resultant transgenic plants show d enhanced chilling tolerance (Kodama et al., 1994; 1995). These results suggest that application or molecular genetic technique· to control the expression of the co-3 fatty acid cksatura\c gen s can cause effective modifications of fatty acid cmnposition. I describe here the :uccessful production of transgenic tobacco plants that express the transcript� 0 r tobacco micro-on1al co-3 fatty acid desaturase gene (NtFAD3, Hamada et a!.. 1994) in en-..e and anti: nse orientations under the control of the CaMV 35S promoter. The relation�hip
between the level of the expression of the NtFAD3 gene and the change in the fatty acid compositions in leaf and root tissues is discussed.
MATERIALS AND METHODS
Plasmid Construction
The 1.4-kbp full-length eDNA of NtFAD3 (Hamada et al.. 1994) was �ubcloncd into the unique BanzH I site of pUC18. The Xba !-Sac I fragment of the resulting recOinbinant plas1nid was then cloned into the binary plasmid pBI 121 (Jefferson ct al..
1987) so as to replace the
�
-glucuronidase gene. The resulting plasmid, pTF IS. contain�a full-length NtFAD3 gene in sense orientation relative to the CaMV 35S promoter (Fig.
I ) .
The 1.3-kbp EcoR I fragment of the NtFAD3 eDNA was cloned into the unique
£coR I site of pBluescript II KS+. Thi plasmid was digested with Sac II and �c If
ligated. The EcoR V-Sac I fragrnent (about 500 bp) of the resulting plasn1id cloned into the Snw 1-Sac I site of pBI 121. The resulting plasmid, pTF 1 AS500. contains the NtFAD3 cDN A fragn1ent from nucleotide 867 to 1381 (see also the NtF AD3 c Of\: A sequence in part I, the DDBJ/EMBL/GeneBank accession No. 026509) in anti�en�c orientation relative to the CaMV 35S promoter (Fig. 1 ).
Plant Transformation
Nicotiana tabacum cv. SR 1 was transformed by the leaf-disk method ( Hor. c h ct
al.. 1985) using Agrobacteriwn tumefaciens LBA4404 containing the pla�mid (pTF IS.
pTF 1 AS500 or pBI 121 ). Individual kanamycin-re ·istant regenerated shoot� were
selected, and the plants were rooted in MS medimn with no growth regulator� ( M ura-..hige and Skoog, 1962) and transferred to soil. Primary transforn1£.lDt · RO were sel fed by bagging their inflore ·cences. The R1 seeds resulting fron1 self-pollination were aseptically genninated in continuous light (3,000 lux) at 26°C on MS rnedium
supplemented with 100 mg/L kanamycin. The kanamycin-resistant R I seedling� \\'L�rc subjected to further analy ·es of lipids .
Fatty Acid Analysis
Fatty acid compositions of whole tissues were determined by gas chromatography (GC-14B: Shirnadzu. Kyoto, Japan) as previously described (Kodama et al.. 190-+l.
Northern Blot Analysis
Leaf tissues fr01n 30-day-old tobacco plants grown at 26°C were harve�tcd and frozen in liquid nitrogen. Total RNA was purified from leaf tissues as de�cribed
(Puissant and Houdebine, 1990). Twenty microgrmns of total RNA were denatured and fractionated as described previously (Kodmna et al., 1991 ). The full-length sequence or the NtFAD3 eDNA (Hamada et al., 1994) and a 1.4 kbp-BamH I fragment of the
tFAD7 eDNA (Hamada et al., 1996) were used as probes. The NtFAD7 cDN
encodes a tobacco plastidial ffi-3 fatty acid desaturase. RNAs were transferred to nylon membrane, and hybridized with labeled probes. The 32P-labeled rice tRNA-Giy(GCCl gene (Reddy and Padayatty, 1988) was used as a reference probe to norn1alize the transcript levels of the above genes. Pre-hybridization, hybridization and post
hybridization steps were perfonned e entially as described in the instruction for nylon membrane (Pall Ultrafine Filtration). Northern blot was visualized and quantitated with a BAS-1 000 II irnaging system (Fuji Photo Film).
Southern Blot Analysis
Total genomic DNAs were isolated from leaf tissues by the procedure of Murray and Thompson ( 1980), and digested with EcoR I. DNA fragments were s parated hy electrophore 'is in a 0.8o/o (w/v) agarose gel, and transferred to nylon tnetnbrane ( 8 i odyne B. Pall Ultrafine Filtration). Southern blot was hybridized with labeled, full-length eDNA fragment of the NtFAD3 gene and washed by the method described in the instruction for nylon n1embrane (Pall Ultrafine Filtration). The hybridized D wa: vi ·ualized with a BAS-1000 II irnaging system (Fuji Photo Film).
fragment
RESULTS
Fatty Acid Compositions in the Leaves and Roots of Transgenic Tobacco Plants Expressing the Antisense NtF AD3 Construct
A O.S-kbp fragment of the NtFAD3 eDNA containing a 3'-flanking region ( 163 hp l and a part of the coding region (349 bp) was linked in antisense orientation to the Ca\1V 35S promoter (Fig. 1). This construct pTF 1AS500, wa introduced into tobacco plant�
by the Agrohocterium-1nediated method. Two transformed lines with an empty vector.
pB I 1 2 L were prepared as control plants and designated C 1 and C2. Twenty-six independent transformants with pTFlASSOO were generated, and the fatty acid
compositions of the leaf and root tissues of each kanmnycin-resistant R 1 seedling were determined. Of the 26 transformants, 3 line (designated ASS. AS9 and A 18 l \hO\H:d significantly reduced contents in 18:3 of root tissues when compared with tho�e or control plants (Fig. 2A). The 18:3 content of the remaining 23 lines slightly decreased. or were aim st the same as those of the control plants. Figure 2A shows the 18:1 content� or J lines, AS 10, AS 13 and AS23, which were selected a representative I ines for the
remaining 23lines. In leaf tissues of the Cl, C2, ASlO. AS13 and AS23 plant�. the 1<1:3 contents were almo t imilar (Fig. 2B). The 18:3 contents of leaf tissu s or the A '�.
AS9 and AS 18 plants were lower by about 10-20o/o (Fig. 2B ). Therefore, I cla��i !'ied the tranc forn1ants with pTF 1 AS500 into two groups; antisense plants (AS 10. AS 13 and AS23) that apparently showed insignificant differences in fatty acid composition� from the control plants (type I) and those (ASS, AS9 and AS 18) showing considerable
reduction of the 18:3 contents in root and leaf tissues (type II). In the type-ll plant\
(ASS ). the 18:3 content· decreased by about 4o/o in root tissues and the 16:3 and I <1 :3 level· in leaf tissue· decreased by about 6o/o and 14o/o, respectiv ly. which were a.·sociated with increa ed mnount of 16:2 and 18:2 (Table 1 ).
The kanmnycin-resistance segregation test of R 1 seedlings revealed that the
transfonned lines, C L AS 10. AS 13 and AS23, contained a single functional in�enion of T-D :the C2 line contained two unlinked insertions ofT-DNA: th AS9 and ·I c1
lines contained three insertions of T -DNA; and the ASS line contained probably rnur in�ertions of T-DNA (Table 2).
Fatty Acid Compositions in the Leaves and Roots of Transgenic Tobacco Plants Expressing the Sense NtFAD3 construct
A construct pTF lS, containing a full-length NtFAD3 eDNA behind the CaYIV .35 promoter (Fig. l ). was introduced into tobacco plants. Of the 7 ind pendent
rransforn1ants with pTF l S. the 18:3 levels of root tissues of Rl seedlings of-+ line-., (designated s L S7, s 10. and s 14) increased substantially when COI11pared \Vith lhn e or the control plants (Fig. 3A). Among these 4 line , S 10 transforn1ants showed the highe'->t amount of 18:3 in root ti sues. In leaf tissues, the 18:3 levels of the S I. S 7 and S 1-t I i nes were aln1ost sin1ilar to those of the control plants, and those of the S I 0 line increa\cd slightly (Fig. 3 B). In fact the 18:3 content of the S 10 line increased by about I (Y r and the 18:2 contents correspondingly decreased in root ti ·sues (Table I). The 18:3 lcn�lnf leaf tissues of S 10 increased by about 6o/o, and the 16:3 level apparent! y dec! in eel. 'nl i kc the case with the antisense tran formant (the ASS line), the amount of 16:2 �tayecl at a lo,,·
level (Table 1 ). These 4 transgenic lines were segregated as single functional T -0 ·A insertion plants (Table 2).
Levels of the NtFAD3 Transcript in the Transformants with the Sense and Antisense Constructs
The transgenic tobacco plants with the sense and antisense NtFAD3 coiv-.truct. "ere subjected to Northern blot analysis to determine whether the level of the NtF A D3
transcript was correlated with the altered profile of the fatty acid composition. The
. t ady-state level of NtFAD3 mRNA was measured in total RNAs isolat d from Jca,·e-., or
each s edling grown at 26°C for 30 day . The RNAs were hybridiz d with a labeled
