summer project
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Summer Project
Speaker : 陳泓志 901673
中研院分生所蔡宜芳老師實驗室 (N317)
Nitrate transporter in ArabidopsisNitrate is the major nitrogen source for plants.The uptake and transport are important for plant growth.
There are 52 NRT1 genes in Arabidopsis.
1. small genome size—7 x 107 bp(10×E. coli)2. short life span—2 months3. small size4. complete genetic and physical map
NTL71. transformant screening2. Examine gene expression3. Oocyte functional test
NTL41. Oocyte functional test
Transfromant screening-ntl7 By the result of PCR, one can recognize whether the line is homozygous, heterozygous mutants, or wild type.Primer:F+R ; F+LB ; R+LB
—with T-DNA→1.5~2.0kb—without T-DNA→3.5kb
Transformant screening
By the result of Southern Hybridization, one can tell how many copies of T-DNA the mutant possesses. Probe: Kan
Individual Genotype Number of T-DNA
2A Homozygous mutant (E-coR )Ⅰ : 6
2B
Homozygous mutant(Bgl )Ⅱ : 6(E-coR )Ⅰ : 6(BstB )Ⅰ : ?
2C Homozygous mutant
2D Homozygous mutant
2E Homozygous mutant
2F Homozygous mutant
3A Wild-type (E-coR )Ⅰ : 0
3B Heterozygous mutant (E-coR )Ⅰ : 5
3C Wild-type (E-coR )Ⅰ : 0
3D
Homozygous mutant(E-coR )Ⅰ : 5(Bgl )Ⅱ : 4(BstB )Ⅰ : ?
3E Heterozygous mutant
3F Heterozygous mutant
Individual抗 KAN(R) 不抗 Kan
(S)R/S ratio Copy number
of T-DNA
NTL7::Gus-A All 4~7
NTL7::Gus-B 32 24 4 : 3 0 or5
NTL7::Gus-G 40 8 5 : 1 1
NTL7::Gus-O All 4
NTL7::Gus-P 1
NTL7::Gus-Q 68 20 3.4 : 1 0,1,3
NTL7::Gus-S 67 6 11.1 : 1 1
NTL7::Gus-V 68 3 22.6 : 1 3,5,7
NTL7::Gus-Ⅰ 60 10 6 : 1 1 or 5
NTL7::Gus-Ⅱ 113 13 8.7 : 1 3
NTL7::Gus-Ⅴ 65 5 13 : 1 2 or 4
NTL7::Gus-Ⅹ 79 7 11.2 : 1 3
Individual抗 KAN(R) 不抗 Kan(S) R/S ratio Copy number
of T-DNANTL7::GFP 1-D 65 21 3.1 : 1
NTL7::GFP 1-E 91 15 6.06 : 1
NTL7::GFP 2-D 69 30 2.3 : 1
NTL7::GFP 3-G 130 6 21.6 : 1
NTL7::GFP 3-H All
NTL7::GFP 3-L 100 13 7.6 : 1
NTL7::GFP 3-M 74 11 6.7 : 1
NTL7::GFP 3-P 100 2 50 : 1
NTL7::GFP 4-B All
NTL7::GFP 4-J All
NTL7::GFP 4-K 137 7 16.5 : 1
41-D3 is wild-type
1
4
1,2 or 3
The transformants caused by T-DNA insertion contain the Kanamycine selection marker, therefore with the R/S ratio, one can distinguishes the genotype of the mutant. For those all alive lines, it could mean they are homozygous transformant.For those all sensitive lines, it means they are wild type.For those having special R/S ratio lines, it could mean they are heterozygous transformant .
Homozygous transformants: NTL7::Gus-A, NTL7::Gus-O, NTL7::GFP3-H, NTL7::GFP4-B, NTL7::GFP4-J
As to distinguish the copy number of T-DNA inserted in each line, Southern Hybridization must be performed.
Expression of Gus-stainingGrow in Ms+Kan plate
For 7 daysGroup 1 : no expression
NTL7::Gus-V
Expression of Gus-stainingGrow in Ms+Kan plate
For 7 daysGroup2:Strongly express in new leaves but it shows decreased expression in old ones. It also expresses in lateral roots and leaf-stem.
NTL7::Gus-O
NTL7::Gus-S
NTL7::Gus-B
NTL7::Gus-Ⅰ
NTL7::Gus-A
Expression of Gus-stainingGrow in Ms+Kan plate
For 7 days
NTL7::GUS-Ⅱ NTL7::GUS-Ⅹ
Group2: express in new leaves and in lateral roots and leaf-stem.
Expression of Gus-stainingGrow in Ms+Kan plate
For 7 daysGroup3:Express in new leaves close to the leaf-stem, but distribute to the edge of the old leaves.
NTL7::Gus-Ⅴ NTL7::Gus-P
Expression of Gus-stainingGrow in Ms+Kan plate For 7 daysGroup4:Express only in new leaves.
NTL7::Gus-G
Expression of Gus-stainingGrow in Ms+Kan plate
For 12 daysGroup1:No expression
NTL7::Gus-A NTL7::Gus-V
Expression of Gus-stainingGrow in Ms+Kan plate For 12 daysGroup2:Express through whole plant but primary root
NTL7::Gus-Ⅰ
NTL7::Gus-O NTL7::Gus-Q
NTL7::Gus-Ⅱ NTL7::Gus-Ⅹ
NTL7::Gus-S
Expression of Gus-stainingGrow in Ms+Kan plate For 12 daysGroup3:Express only on the edge of the leaves.
NTL7::Gus-Ⅴ
Expression of Gus-stainingGrow in Ms+Kan plate For 12 daysGroup4:Express only in new leaves and the expression decreases when the leaves grow old.
NTL7::Gus-G
Conclusion: The photos show that the NTL7::Gus expressions are different in different lines. Besides, one noticeable thing is that there seems to exist stage-dependent patterns.
Gus staining of flower-NTL7
G3
G2 Q3
P3
I-2
G4S6 P2
I-1
P1
Section of NTL7::Gus-stained flower
NTL7 is expressed mostly in the calyxes and anthers.
•The red fluorescent light reveals the auto-fluoresced chloroplast; the green fluorescence is the GFP::NTL7.•The result shows that NTL7 is a membrane protein.
Functional test - Xenopus oocyte
1.Operate to obtain the oocyte and then treat the oocyte with collagenase for purpose of getting individual ones.
Inject 0.5μg cRNA into the plant pole of each oocyte.
cRNA disolved in Rnase-free water
Anamal pore(containing nuclei)Plant pore
peptide uptake assay
8.922
63.918
9.26 13.708
01020304050607080
Rnase-freeH2O
AtPTR2 NTL4 NTL7
H3 dileu
cine(1
0-4 pmole
/oocy
te)
The graph shows that there seems no uptake in both NTL7 and NTL4 strains.
×20 μM H3-labeled dileucine+ 20 μM dileucine8001
Low affinity nitrate uptake
0.07
1.36
0.12
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
RNase-free H2O CHL1 NTL4
nitrat
e upta
ke(nm
ole/oo
cyte)
NTL4 Nitrate uptake assay-HPLC
10 mM nitrate buffer
Conclusion:
It seems that there is no peptide uptake in NTL4. But one thing interesting is that we got one result shows 1.36 nmole nitrate uptake / oocyte in NTL4. Maybe we should increase the concentration of RNA injected into oocytes.
Nitrate uptake assay-HPLCHigh affinity nitrate uptake
-0.03
0.34
0.00
-0.1
-0.05
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
RNase-free H2O CHL1 NTL4
nitrat
e upta
ke(nm
ole/oo
cyte)
As the data shows, it seems no nitrate uptake in NTL4.
150μM KNO3
Thanks for your listening!!
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