Plant transformation
Transformed roots ofM. truncatulacv. Jemalong
were obtained usingAgrobacterium rhizogenesARqua1
according to the protocol of Boisson-Dernier et al.
(2001). About 3–6 weeks later, plants with transformed
roots were put individually into square 12-cm plastic
dishes (Greiner Labortechnik, Kremsmu¨nster, Austria)
on Fahraeus medium containing 1% agar.A. thaliana
plants (ecotype Columbia) were transformed using the
A. tumefaciens-mediated floral dip method. Cultivation
of Arabidopsis plants was performed on half MS
enriched with vitamins, 1% sucrose and 0.4% phytagel.
Microscopy
M. truncatularoots growing on agar were covered
with bioFolie 25 (Sartorius AG, Vivascience Support
Center, Go¨ttingen, Germany) and observed with a
LEICATCS 4D confocal microscope (Leica, Germany)
using a 63water-immersion objective. Four-day-old
A. thalianaseedlings were mounted in liquid half MS
medium containing 1% sucrose using a spacer of one
layer of parafilm between slide and coverslip, and
observed by confocal microscopy. Plants were adapted
to liquid medium overnight to allow application of
drugs.
For the observation of transgenicA. thalianaexpressing Ara6-GFP (Ueda et al., 2001), serial images were
obtained every 1mm using a fluorescence microscope
(Olympus, Japan) equipped with 40oil-immersion
objectives and a confocal unit, CSU10 (Yokogawa
Electric Corporation, Japan). Images and movies were
digitally processed with Image-Pro Plus 4.1 (Media
Cybernetics, L.P.), Adobe Photoshop 4.0 (Adobe Corp.,
Mountain View, CA) and VideoMach 2.7.2.
Drug treatments and FM4-64 staining
Growing root apices were exposed to the following
drugs: 2,3-butanedione monoxime (10 mM), latrunculin
B(1mM forA. thalianaand 10mM forM. truncatula),
jasplakinolide (1mM/5mM), brefeldin A(10mM for
A. thalianaand 100mM forM. truncatula), wortmannin
(10mM), and oryzalin (1mM). The drugs were diluted in
culture medium and directly added to the roots of
transgenic M. truncatulaor A. thaliana. Regarding
FM4-64, stock solution (1 mg/ml) was prepared in
DMSO and used at 17.5mM forM. truncatula. Before
FM4-64 treatment, plants were incubated for 25 min at
61C to slow down endocytosis.
Results
A tandem FYVE construct recognizes plant
endosomes
In both yeast and mammals, the phosphoinositide
PI(3)P accumulates preferentially in endosomal membranes (Gillooly et al., 2000), and binding of FYVEdomain proteins to PI(3)P is sufficient to target
endosomal proteins to these subcellular compartments.
ForA. thalianait is known that the classical FYVE
domain binds specifically to PI(3)P in vitro (Jensen et
al., 2001). Therefore, we were interested to know if the
tandem FYVE domain would be sufficient for targeting
to endocytic compartments in plants. To this end, the
FYVE domain from the mouse Hrs protein was
Plant transformation
Transformed roots ofM. truncatulacv. Jemalong
were obtained usingAgrobacterium rhizogenesARqua1
according to the protocol of Boisson-Dernier et al.
(2001). About 3–6 weeks later, plants with transformed
roots were put individually into square 12-cm plastic
dishes (Greiner Labortechnik, Kremsmu¨nster, Austria)
on Fahraeus medium containing 1% agar.A. thaliana
plants (ecotype Columbia) were transformed using the
A. tumefaciens-mediated floral dip method. Cultivation
of Arabidopsis plants was performed on half MS
enriched with vitamins, 1% sucrose and 0.4% phytagel.
Microscopy
M. truncatularoots growing on agar were covered
with bioFolie 25 (Sartorius AG, Vivascience Support
Center, Go¨ttingen, Germany) and observed with a
LEICATCS 4D confocal microscope (Leica, Germany)
using a 63water-immersion objective. Four-day-old
A. thalianaseedlings were mounted in liquid half MS
medium containing 1% sucrose using a spacer of one
layer of parafilm between slide and coverslip, and
observed by confocal microscopy. Plants were adapted
to liquid medium overnight to allow application of
drugs.
For the observation of transgenicA. thalianaexpressing Ara6-GFP (Ueda et al., 2001), serial images were
obtained every 1mm using a fluorescence microscope
(Olympus, Japan) equipped with 40oil-immersion
objectives and a confocal unit, CSU10 (Yokogawa
Electric Corporation, Japan). Images and movies were
digitally processed with Image-Pro Plus 4.1 (Media
Cybernetics, L.P.), Adobe Photoshop 4.0 (Adobe Corp.,
Mountain View, CA) and VideoMach 2.7.2.
Drug treatments and FM4-64 staining
Growing root apices were exposed to the following
drugs: 2,3-butanedione monoxime (10 mM), latrunculin
B(1mM forA. thalianaand 10mM forM. truncatula),
jasplakinolide (1mM/5mM), brefeldin A(10mM for
A. thalianaand 100mM forM. truncatula), wortmannin
(10mM), and oryzalin (1mM). The drugs were diluted in
culture medium and directly added to the roots of
transgenic M. truncatulaor A. thaliana. Regarding
FM4-64, stock solution (1 mg/ml) was prepared in
DMSO and used at 17.5mM forM. truncatula. Before
FM4-64 treatment, plants were incubated for 25 min at
61C to slow down endocytosis.
Results
A tandem FYVE construct recognizes plant
endosomes
In both yeast and mammals, the phosphoinositide
PI(3)P accumulates preferentially in endosomal membranes (Gillooly et al., 2000), and binding of FYVEdomain proteins to PI(3)P is sufficient to target
endosomal proteins to these subcellular compartments.
ForA. thalianait is known that the classical FYVE
domain binds specifically to PI(3)P in vitro (Jensen et
al., 2001). Therefore, we were interested to know if the
tandem FYVE domain would be sufficient for targeting
to endocytic compartments in plants. To this end, the
FYVE domain from the mouse Hrs protein was
การแปล กรุณารอสักครู่..
Plant transformation
Transformed roots ofM. truncatulacv. Jemalong
were obtained usingAgrobacterium rhizogenesARqua1
according to the protocol of Boisson-Dernier et al.
(2001). About 3–6 weeks later, plants with transformed
roots were put individually into square 12-cm plastic
dishes (Greiner Labortechnik, Kremsmu¨nster, Austria)
on Fahraeus medium containing 1% agar.A. thaliana
plants (ecotype Columbia) were transformed using the
A. tumefaciens-mediated floral dip method. Cultivation
of Arabidopsis plants was performed on half MS
enriched with vitamins, 1% sucrose and 0.4% phytagel.
Microscopy
M. truncatularoots growing on agar were covered
with bioFolie 25 (Sartorius AG, Vivascience Support
Center, Go¨ttingen, Germany) and observed with a
LEICATCS 4D confocal microscope (Leica, Germany)
using a 63water-immersion objective. Four-day-old
A. thalianaseedlings were mounted in liquid half MS
medium containing 1% sucrose using a spacer of one
layer of parafilm between slide and coverslip, and
observed by confocal microscopy. Plants were adapted
to liquid medium overnight to allow application of
drugs.
For the observation of transgenicA. thalianaexpressing Ara6-GFP (Ueda et al., 2001), serial images were
obtained every 1mm using a fluorescence microscope
(Olympus, Japan) equipped with 40oil-immersion
objectives and a confocal unit, CSU10 (Yokogawa
Electric Corporation, Japan). Images and movies were
digitally processed with Image-Pro Plus 4.1 (Media
Cybernetics, L.P.), Adobe Photoshop 4.0 (Adobe Corp.,
Mountain View, CA) and VideoMach 2.7.2.
Drug treatments and FM4-64 staining
Growing root apices were exposed to the following
drugs: 2,3-butanedione monoxime (10 mM), latrunculin
B(1mM forA. thalianaand 10mM forM. truncatula),
jasplakinolide (1mM/5mM), brefeldin A(10mM for
A. thalianaand 100mM forM. truncatula), wortmannin
(10mM), and oryzalin (1mM). The drugs were diluted in
culture medium and directly added to the roots of
transgenic M. truncatulaor A. thaliana. Regarding
FM4-64, stock solution (1 mg/ml) was prepared in
DMSO and used at 17.5mM forM. truncatula. Before
FM4-64 treatment, plants were incubated for 25 min at
61C to slow down endocytosis.
Results
A tandem FYVE construct recognizes plant
endosomes
In both yeast and mammals, the phosphoinositide
PI(3)P accumulates preferentially in endosomal membranes (Gillooly et al., 2000), and binding of FYVEdomain proteins to PI(3)P is sufficient to target
endosomal proteins to these subcellular compartments.
ForA. thalianait is known that the classical FYVE
domain binds specifically to PI(3)P in vitro (Jensen et
al., 2001). Therefore, we were interested to know if the
tandem FYVE domain would be sufficient for targeting
to endocytic compartments in plants. To this end, the
FYVE domain from the mouse Hrs protein was
การแปล กรุณารอสักครู่..
PI (3) P accumulates preferentially in endosomal membranes (Gillooly et al, 2000), and binding of FYVEdomain proteins to. PI (3) P is sufficient to target
endosomal proteins to these subcellular compartments.
ForA. Thalianait is known that the. Classical FYVE
domain binds specifically to PI (3) P in vitro (Jensen et
AL, 2001). Therefore we were, interested to know. If the
.แปลงพืช
เปลี่ยนราก OFM . truncatulacv . jemalong
ได้รับ usingagrobacterium rhizogenesarqua1
ตามขั้นตอนของ boisson dernier et al .
( 2001 ) ประมาณ 3 – 6 สัปดาห์ต่อมา เป็นพืชที่มีรากเป็นใส่แบบเปลี่ยน
จานพลาสติกสี่เหลี่ยม 12 เซนติเมตร ( ไกรเนอร์ labortechnik kremsmu , ตั้ง nster ออสเตรีย )
บน fahraeus ขนาดกลางที่มี 1% วุ้น thaliana
Aพืช ( พันธุ์โคลัมเบีย ) ถูกเปลี่ยนใช้
. tumefaciens โดยวิธีจุ่มดอกไม้ การเพาะปลูกของพืช Arabidopsis
คือใช้ครึ่ง MS
อุดมไปด้วยวิตามิน ซูโครส 1% และ 0.4% phytagel .
/
) truncatularoots เติบโตบนอาหารวุ้นที่ถูกปกคลุม
25 biofolie ( Sartorius AG , vivascience สนับสนุน
ศูนย์ไปตั้ง ttingen , เยอรมนี ) และสังเกตกับ
leicatcs 4D ด้วยกล้องจุลทรรศน์ ( Leica , เยอรมนี )
ใช้ 63 น้ำแช่วัตถุประสงค์ สี่วันเก่า
. thalianaseedlings ติดในน้ำครึ่ง MS
1 % อาหารที่มีซูโครสโดยใช้ spacer 1
ชั้นของพาราฟิล์มระหว่างภาพนิ่งและปิดสไลด์ และสังเกตด้วยกล้องจุลทรรศน์ด้วย
. พืชดัดแปลง
น้ำกลางคืนเพื่อให้ใช้ยา
.สำหรับการสังเกตของ transgenica . thalianaexpressing ara6 GFP ( อุเอดะ et al . , 2001 ) , อนุกรมภาพ
ได้ทุก 1 mm ใช้กล้องจุลทรรศน์เรืองแสง
( โอลิมปัส ญี่ปุ่น ) ซึ่งมี 40 น้ำมันแช่
วัตถุประสงค์และหน่วยด้วย csu10 ( , โยโกกาวา
ไฟฟ้า Corporation , Japan ) ภาพและภาพยนตร์ดิจิตอลประมวลผลกับภาพ
โปรพลัส 4.1 ( สื่อ
ไซเบอร์เนติกส์ , L.P ) , Adobe Photoshop 40 ( Adobe .
Mountain View , CA ) และการรักษาด้วยยา และ videomach 2.7.2 .
fm4-64 staining ปลูกราก apices ได้รับยาต่อไปนี้
: 2,3-butanedione monoxime ( 10 มม. ) , latrunculin
b ( 1mm ได้แล้ว thalianaand 10mm แบบฟอร์ม truncatula )
jasplakinolide ( 1 mm / 5 ) , brefeldin ( 10mm . thalianaand 100mm สำหรับ
แบบฟอร์ม truncatula ) wortmannin
( 10mm ) และริซาลิน ( 1 mm ) ยาลดใน
สื่อวัฒนธรรมและโดยตรงเพิ่มรากของต้นม. truncatulaor
. thaliana . เกี่ยวกับ
fm4-64 Stock สารละลาย ( 1 mg / ml ) ถูกเตรียมใน DMSO และใช้ใน 17.5mm
แบบฟอร์ม truncatula . ก่อน
fm4-64 รักษาพืชบ่ม 25 นาที
61c ช้าลงเอนโดไซโทซิส .
a ผลควบคู่ fyve สร้างโรงงาน endosomes
จำทั้งยีสต์ และสัตว์เลี้ยงลูกด้วยนม , phosphoinositide
พาย ( 3 ) P ที่สะสมใน endosomal preferentially เมมเบรน ( gillooly et al . , 2000 ) และการจับของโปรตีน fyvedomain กับพาย ( 3 ) P เป็นเพียงพอที่จะ endosomal กับตําแหน่งภายในเซลล์โปรตีนเป้าหมาย
ช่องเหล่านี้ได้แล้ว thalianait เป็นที่รู้จักกันว่าโดเมน fyve
คลาสสิกผูกเฉพาะเพื่อพาย ( 3 ) P ในหลอดทดลอง ( เจนเซ่น et
al . , 2001 ) ดังนั้นเราจึงมีความสนใจที่จะทราบว่าถ้า
การแปล กรุณารอสักครู่..