Page 9 - gabVol4No1

Genomics and Applied Biology

, 2013, Vol. 4 No.1 1-7

http://gab.sophiapublisher.com

- 6 -

AATGTTCTTTCTGCCTTGTGACCAG

-

3' (

Bam

HI

site is underlined). The PCR products were digested

by

Eco

RI and

Bam

HI, and cloned into the

Eco

RI/

Bam

HI site of pGADT7-rec, generating

pGAD-

RD21a

.

Yeast transformation using yeast

strain AH109 and

screening were performed by Matchmaker Gold Yeast

Two-Hybrid System (Clontech). At least 5 colonies

grown on the SD media lacking leucine and

tryptophan (SD/-Leu/-Trp), were streaked on the

SD/-Leu/-Trp and the SD media lacking leucine,

tryptophan, adenine, and histidine (SD/-Trp/-Leu/-His/

-Ade). Photos were taken after the yeasts were

cultured for 3-5 days. The experiments were

performed for three times.

3.3

In vitro

Coimmunoprecipitation (Co-IP)

Proteins were expressed by TNT Quick Coupled

Transcription/Translation Systems (Promega). Plasmid

DNA pGBK-

AGB1

,

pGAD-

AtPP2C52,

pGAD

-AtPP2

C52

G99D

,

pGAD

-AtPP2C52

G105D

, pGAD

-AtPP2C52

DGH102-104ERN

and

pGADT7-rec vector were used to

synthesize proteins Myc-tagged AGB1, HA-tagged

AtPP2C52 and HA epitope tag, respectively. Co-IP

was carried out using anti-HA antibody

(

MBL),

anti-Myc antibody

(

MBL) and protein G Sepharose

(

GE Healthcare) following the MATCHMAKER

Co-IP Kit User Manual (Cat No. 630449, 2003).

3.4 Bimolecular Fluorescence Complementation

(BiFC) assay

The vectors for BiFC assay were constructed by

replacing GFP in the vector pBS-35SMCS-GFP

(Tsugama et al., 2012b) with the N-terminus (154

amino acids) or the C-terminus (80 amino acids) of

YFP, generating pBS-35SMCS-nYFP and pBS-

35SMCS-cYFP, respectively. The pGAD-AtPP2C52

was digested by

Xba

I and

Xho

I, and the resultant ORF

fragments of

AtPP2C52

were inserted into the

Xba

I/

Sal

I site of pBS-35SMCS-cYFP, generating

pBS-35S-AtPP2C52-cYFP. The ORF of

UMP1

without stop codon was amplified by PCR using the

cDNA clone as template and the following primer pair:

5'

-

GAGTCGGTACCATGGGGTTCCTTAAGCCAC

-

3' (

Kpn

I site is underlined) and 5'

-

CTCGA

ACTAGTGGCAATGTTCTTTCTGC

-

3' (

Spe

I site is

underlined). The PCR products were digested by

Kpn

I

and

Spe

I, and cloned into the

Kpn

I/

Spe

I site of

pBS-35SMCS-nYFP, generating pBS-35S-nYFP-

UMP1

. The ORF of

RD21a

without stop codon

was

amplified by PCR using the cDNA clone as template

and the following primer pair: 5'

-

GAGTC

GGTACCATGGAGTCTGAGAAAAAGATAGC

-

3'

(

Kpn

I site is underlined) and 5'

-

CTCGAACTAGT

CATGAAACTTGGGTAAATCGG-3' (

Spe

I site is

underlined). The PCR products were digested by

Kpn

I

and

Spe

I, and cloned into the

Kpn

I/

Spe

I site of

pBS-35SMCS-nYFP, generating pBS-35S-nYFP

-

RD21a

. Arabidopsis protoplast isolation and transfor-

mation were conducted as described (Wu et al., 2009).

Plasmids DNA were introduced into onion epidermal

cells by a bombardment system (Bio Red, PDS-1000).

Images were processed using Canvas X software

(ACD Systems) and enhanced using Photoshop CS4

software (Adobe).

3.5 Preparation of chimeric constructs

An approximately 2 kb upstream promoter sequence

of

AtPP2C52

(P

AtPP2C52

)

was

cloned from an

Arabidopsis genetic DNA by using primer pair: 5'

-

GGATCCCGGGATGAATCATGTAGGTGAC

-

3'

(

Sma

I site is underlined) and 5'

-

CCCTCTAGA

TGTTTAATCCCAGCCTAGA

-

3' (

Xba

I site is

underlined). The PCR products were double-digested

with

Sma

I/

Xba

I and used to replace the CaMV 35S

promoter of pBI121 vector (Clontech, Bevan, 1984),

generating pBI121-P

AtPP2C5

2

::

GUS

.

3.6 Plant transformation

Arabidopsis Col-0 plants were transformed by

Agrobacterium tumefaciens

-mediated transformation

using the floral-dip method (Clough and Bent, 1998).

The

Agrobacterium

strain used was EHA105

harboring binary vector pBI121 carrying the

P

AtPP2C52

::GUS fusion gene.

3.7 GUS histochemical analysis

For GUS histochemical characterization of the

P

AtPP2C52

::GUS lines, several developmental stages

were examined. Samples were treated with 90%

acetone for 30 min at 4

℃

, immersed in a staining

solution (5 mmol/L X-gluc, 0.5 mmol/L K

3

Fe(CN)

6

,

0.1% Triton X-100, 0.5 mmol/L K

4

Fe(CN)

6

,

10 mmol/L Na

2

EDTA and 50 mmol/L sodium

phosphate buffer, pH 7.0), vacuum infiltrated for more

than 1 hour, then incubated at 37

℃

. After staining,

samples were cleared by several changes of 70%