At-HPPD
,
VTE2
,
VTE3
,
VTE1
, and
VTE4
(Genbank accession Nos.AY072329,AY089963,AB054257,NM119430andAF104220)wereprepared by using TaKaRa One Step RT-PCR kit (Code No.:DRR024A, TaKaRa, Japan). The amplified cDNA fragments of
At-HPPD
,
VTE2
,
VTE3
, and
VTE1
with an introduced
Hin
dIII site at thestart codon (ATG) and an introduced
Bam
HI site before the stopcodon were digested, and sub-cloned into
Hin
dIII–
Bam
HI site of vector pBSmyc to obtain the expression cassette with recombina-tion sequence of target gene and
myc
. The amplified fragment of
VTE4
with an introduced
Xho
I site at the start codon and anintroduced
Bam
HI site before the stop codon was digested, andsub-cloned into
Xho
I–
Bam
HI site of pBSmyc. The reading frame of pBS-target gene–
myc
was confirmed to be correct by sequencingfrombothstrands.Therecombinantsequencesoftargetgene–
myc
wereintroducedintopET28avectorandover-expressedin
E.coli
inorder to detect biological activity of fusing proteins
in vitro
. Therecombinant sequences of target gene–
myc
were sub-cloned intothe botany expression vector PHB (a gift from Prof. HongquanYang, SIPPE, CAS)[24], located downstream of double 35S CaMVpromoters and upstream of rbcS polyA terminator (Fig. 2A).In order to explore the function of two enzymes in planttocopherol biosynthetic pathway, three of them were chosen fordual expression vector construction. Two co-expression combina-tions were designed:
VTE2
+
VTE4
and
VTE3
+
VTE4
. The targetgeneswerealsorecombinantwith
myc
.Inordertoputtwogenesinonevector,theexpressioncassetteofvectorpBI121(Clontech)wasdigested with
Hin
dIII and
Eco
RI, and then sub-cloned into the
Hin
dIII–
Eco
RI site of pCAMBIA1304 vector to construct the co-expression vector.
VTE4
–
myc
fraction was sub-cloned into
Xba
I–
Sac
Isite,while
VTE2
–
myc
and
VTE3–myc
fragmentwereclonedinto
Bgl
II–
Bst
EII site, respectively (Fig. 2B).[The primers used for cloning were listed inSupplement Table2].
2.3. Transformation and screening of transgenic plants
The binary vector PHB contains hygromycin resistant gene andanti-herbicide gene (BAR) inside the T-DNA for the selection of transformants. The PHB:target gene constructs were transformedinto
Agrobacterium tumefaciens
C58C1 (pGV3101; rifampicinresistance) and the floral-dip method was used for
Arabidopsis
transformation[25]. The plants harboring empty plant expressionvector were used as non-transgenic control (NC). T
0
seeds werescreened on Murashige–Skoog (MS) basal medium supplementedwith 50
m
g mL
À
1
hygromycin. More than fifty independent linesexpressingtransgeneweretransferredtosoilandscreenedwith1%herbicide (glufosinate-ammonium). Screening of T
1
and T
2
seedswere performed with herbicide in order to obtain transgenic lineswhich accorded with Mendel’s Law. Polymerase chain reaction(PCR) and protein gel blot analysis were also performed onindependent transgenic lines (random chosen) of each generation,inordertofurtherconfirmandcheckthehomozygosisandgeneticstability. Homozygous seeds could be obtained in T
4
generation.The screening of co-expression transgenic plants was performedwith PCR and western blot analysis, while herbicide could not beused because anti-herbicide gene was not contained in the binaryvectors.
2.4. Molecular analysis of transgenic lines
Genomic DNA was isolated from the transgenic plants and NCplant with a CTAB method[26]. The presence of the transgene wasdetected by PCR.Proteingelblotanalysiswasperformedasdescribedpreviouslywith minor modifications[27]. Fifty
m
g of total protein, deter-mined by using the DC Protein Assay Kit (Bio-Rad, Hercules, CA),was fractionated on 12% SDS-PAGE mini-gel and blotted onto anitrocellulose membrane (PerkinElmer, 0.45
m
m). The blots wereprobed with the primary antibody c-Myc (9E10) (sc-40, mousemonoclonal IgG1, Santa Cruz Biotechnology) [diluted in PBST(80 mmol L
À
1
Na
2
HPO
4
, 20 mmol L
À
1
NaH
2
PO
4
, 100 mmol L
À
1
NaCl, and 0.1% Tween 20)], washed with PBST three times, reactedwith goat anti-mouse IgG-AP (sc-2047, Santa Cruz Biotechnology),washed, and exposed with alkali phosphatase for 3 min.
2.5. Real-time PCR
Total RNA was isolated and genomic DNA was removed bytreatment with RQ1-RNase free DNase (Promega, Madison, WI).One microgram of total RNA was reverse transcribed to generatecDNAin20
m
LreactionsforeachsampleusingaToyoboReverTra-Plus-Kit according to manufacturer’s recommendations (Toyobo,Osaka, Japan). An aliquot of cDNA corresponding to 10 pg – 10 ngtotal RNA was used in each real-time PCR assay (SYBR
1
ExScriptRT-PCR Kit, TaKaRa, Shiga, Japan). Ubiquitin RNA was used tonormalize RNA concentrations. Standard curves were constructed
Fig. 2.
Schematic representation of the transformation vector construct used in this study. (A) Schematic representation of the single-gene transformation vector. (B)Schematic representation of dual-gene transformation vector. LB, left border; RB, right border; 35S, cauliflower mosaic virus 35S promoter; 35S polyA, cauliflower mosaicvirus 35S polyA terminator; Nos, nopaline synthase gene terminator.
Y. Li et al./Plant Science 178 (2010) 312–320
314
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