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!!