Summer Project

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Summer Project
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• Speaker ??? 901673

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Nitrate transporter in Arabidopsis
Nitrate is the major nitrogen source for
plants. The uptake and transport are important
for plant growth.
There are 52 NRT1 genes in Arabidopsis.
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1. small genome size7 x 107 bp(10E. coli)
2. short life span2 months
3. small size
4. complete genetic and physical map

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• NTL7
• transformant screening
• Examine gene expression
• Oocyte functional test
• NTL4
• Oocyte functional test

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Transfromant screening-ntl7
By the result of PCR, one can recognize whether
the line is homozygous, heterozygous mutants, or
wild type. PrimerFR FLB RLB
with T-DNA?1.52.0kb without T-DNA?3.5kb
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Transformant screening
By the result of Southern Hybridization, one
can tell how many copies of T-DNA the mutant
possesses. Probe Kan

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4 1-D3 is wild-type
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1,2 or 3
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• 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 .

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• Homozygous transformants
• NTL7Gus-A, NTL7Gus-O, NTL7GFP3-H,
• NTL7GFP4-B, NTL7GFP4-J
• As to distinguish the copy number of T-DNA
  inserted in each line, Southern Hybridization
  must be performed.

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Expression of Gus-stainingGrow in MsKan plate
For 7 days

• Group 1 no expression

NTL7Gus-V
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Expression of Gus-stainingGrow in MsKan plate
For 7 days
Group2Strongly express in new leaves but it
shows decreased expression in old ones. It also
expresses in lateral roots and leaf-stem.

• NTL7Gus-B

NTL7Gus-O
NTL7Gus-A
NTL7Gus-S
NTL7Gus-?
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Expression of Gus-stainingGrow in MsKan plate
For 7 days
Group2 express in new leaves and in lateral
roots and leaf-stem.
NTL7GUS-?
NTL7GUS-?
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Expression of Gus-stainingGrow in MsKan plate
For 7 days
Group3Express in new leaves close to the
leaf-stem, but distribute to the edge of the old
leaves.

• NTL7Gus-?

NTL7Gus-P
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Expression of Gus-stainingGrow in MsKan plate
For 7 days
Group4Express only in new leaves.
NTL7Gus-G
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Expression of Gus-stainingGrow in MsKan plate
For 12 days
Group1No expression
NTL7Gus-V
NTL7Gus-A
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Expression of Gus-stainingGrow in MsKan plate
For 12 days
Group2Express through whole plant but primary
root
NTL7Gus-?
NTL7Gus-?
NTL7Gus-?
NTL7Gus-O
NTL7Gus-Q
NTL7Gus-S
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Expression of Gus-stainingGrow in MsKan plate
For 12 days
Group3Express only on the edge of the leaves.
NTL7Gus-?
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Expression of Gus-stainingGrow in MsKan plate
For 12 days
Group4Express only in new leaves and the
expression decreases when the leaves grow old.
NTL7Gus-G
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Conclusion The photos show that the
NTL7Gus expressions are different in different
lines. Besides, one noticeable thing is that
there seems to exist stage-dependent patterns.
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Gus staining of flower-NTL7
G2
Q3
P3
G3
I-2
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S6
G4
P2
P1
I-1
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Section of NTL7Gus-stained flower
NTL7 is expressed mostly in the calyxes and
anthers.
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• The red fluorescent light reveals the
  auto-fluoresced chloroplast the green
  fluorescence is the GFPNTL7.
• The result shows that NTL7 is a membrane protein.

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Functional test-Xenopus oocyte
1.Operate to obtain the oocyte and then treat the
oocyte with collagenase for purpose of getting
individual ones.
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Inject 0.5µg cRNA into the plant pole of each
oocyte.
cRNA disolved in Rnase-free water
Plant pore
Anamal pore(containing nuclei)
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The graph shows that there seems no uptake in
both NTL7 and NTL4 strains.
20 µM H3-labeled dileucine 20 µM dileucine
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NTL4 Nitrate uptake assay-HPLC
10 mM nitrate buffer
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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.

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Nitrate uptake assay-HPLC
150µM KNO3
As the data shows, it seems no nitrate uptake in
NTL4.
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Thanks for your listening!!