Wasaki et al. 2000: Molecular cloning and root specific expression of secretory acid phosphatase from phosphate deficient lupin (Lupinus albus L.). Soil Sci. Plant Nutr., 46, in press.

RNA extraction and 1st stranded cDNA synthesis.

Total RNA of lupin tissues was isolated by the SDS-phenol method (Palmiter 1974) and 1 mg of the total RNA was used for single stranded cDNA synthesis using a first strand cDNA synthesis kit (Takara Shuzo), according to the manufacturer's instructions. One twenty-fifth of the first strand cDNA synthesized solution was used as template for RT-PCR.
 

Primers.

The primer sets and the reaction steps for amplifying lasap1, lasap2, and actin fragments were as follows;

for amplification of lasap1 transcripts,

SAP1-RT5S primer (sense, 5'- ATGGG TTATT ATTCA ATTTA TTGTT TGATA GTG -3')

LUAPAS02 primer (antisense, 5'- CATGT TCCAA CTACA ACAGA AGCGG C -3')

for amplification of lasap2 transcripts,

SAP2-RT5S primer (sense, 5'- AGTAG TTTTG TTGCA ATAGC TT -3')

LUAPAS02 primer (antisense, 5'- CATGT TCCAA CTACA ACAGA AGCGG C -3')

for the amplification of actin transcripts,

ActSBS1 primer* (sense, 5'- TGTGT TGGAT TCTGG GGATG GTG -3')

ActSBA1 primer* (antisense, 5'- AGAGA TTCGT GGAAG GTCGT CCA -3')

*These sequense were designated based on a cDNA sequence for an actin isolated from soybean (Shah et al. 1983).
 

PCR reaction.

PCR reaction was performed using a DNA Thermal Cycler (MP2000) and Elongase enzyme mix. The number of PCR cycles for lasap1, lasap2, and actin fragments was determined when the difference between +P and -P treatments became clearest. It was confirmed that product of PCR reaction was not saturated at this number of cycles employed.

for amplification of lasap1 transcripts,

94｡C for 1 min, at 55｡C for 3 min, and at 72｡C for 5 min for 25 cycles.

for amplification of lasap2 transcripts,

94｡C for 1 min, at 55｡C for 3 min, and at 72｡C for 5 min for 30 cycles.

for the amplification of actin transcripts,

94｡C for 30 s, at 57｡C for 1 min, and at 72｡C for 1 min for 30 cycles.
 

Analysis.

Amplified fragments were separated on a 2% agarose gel and stained with ethidium bromide. Image analysis of the detected bands was performed using NIH image software (Rasband and Bright 1995).
 

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Results

Fig.1 Expression pattern of acid phosphatase isozymes in the phosphate sufficient germinating lupin and phosphate treated mature lupin.

B, C, H, and R: blank lane, cotyledons, hypocotyls, and roots of phosphate sufficient lupin seedlings before treatment, respectively. +L, -L, +R, and -R: +P treated leaves, -P treated leaves, +P treated lateral roots, and -P treated lateral roots, respectively.
 
 

Fig.2 Relative accumulation of acid phosphatase mRNAs.

The expression level is estimated by multipliying the value of the area and the mean of depth of bands based on the data of RT-PCR (Fig.1). Panels A, B, and C indicate accumulation of LASAP1, LASAP2, and actin mRNA, respectively. Cotyledon, Hypocotyl, and Root are organs from lupin seedlings; +P, Leaf, -P, Leaf, +P, Root, and -P, Root: the phosphate treated leaves and roots, respectively.