Summer Project Speaker : 陳泓志 中研院分生所 蔡宜芳老師實驗室 (N317)

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.

1.small genome size — 7 x 10 7 bp(10×E. coli) 2.short life span — 2 months 3.small size 4.complete genetic and physical map

NTL7 1. transformant screening 2. Examine gene expression 3. Oocyte functional test NTL4 1. 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

IndividualGenotypeNumber 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 : 30 or5 NTL7::Gus-G 4085 : 11 NTL7::Gus-OAll 4 NTL7::Gus-P 1 NTL7::Gus-Q : 10,1,3 NTL7::Gus-S : 11 NTL7::Gus-V : 13,5,7 NTL7::Gus- Ⅰ : 11 or 5 NTL7::Gus- Ⅱ : 13 NTL7::Gus- Ⅴ : 12 or 4 NTL7::Gus- Ⅹ : 13

Individual 抗 KAN(R) 不抗 Kan(S) R/S ratioCopy number of T-DNA NTL7::GFP 1-D : 1 NTL7::GFP 1-E : 1 NTL7::GFP 2-D : 1 NTL7::GFP 3-G : 1 NTL7::GFP 3-H All NTL7::GFP 3-L : 1 NTL7::GFP 3-M : 1 NTL7::GFP 3-P : 1 NTL7::GFP 4-B All NTL7::GFP 4-J All NTL7::GFP 4-K : 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-staining Grow in Ms+Kan plate For 7 days Group 1 : no expression NTL7::Gus-V

Expression of Gus-staining Grow in Ms+Kan plate For 7 days Group2: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-staining Grow 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-staining Grow in Ms+Kan plate For 7 days Group3: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-staining Grow in Ms+Kan plate For 7 days Group4:Express only in new leaves. NTL7::Gus-G

Expression of Gus-staining Grow in Ms+Kan plate For 12 days Group1:No expression NTL7::Gus-A NTL7::Gus-V

Expression of Gus-staining Grow in Ms+Kan plate For 12 days Group2: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-staining Grow in Ms+Kan plate For 12 days Group3:Express only on the edge of the leaves. NTL7::Gus- Ⅴ

Expression of Gus-staining Grow in Ms+Kan plate For 12 days Group4: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

G4 S6 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

The graph shows that there seems no uptake in both NTL7 and NTL4 strains. ×20 μM H 3 -labeled dileucine+ 20 μM dileucine

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-HPLC As the data shows, it seems no nitrate uptake in NTL4. 150μM KNO 3

Thanks for your listening!!