16. Available full length cDNA

 

The following eight full length cDNA libraries and full length cDNA clones were made and will be available from RIKEN BioResource Center (http://www.brc.riken.jp/lab/epd/Eng/).

 

Strain: Physcomitrella patens (Hedw.) Bruch and Schimp subsp. patens

We used an original strain instead of Gransden2004 used for whole genome shotgun sequence by JGI and ESTs may include SNPs.

 

Distribution: Full length cDNA clones of pph, pphb, and pphn libraries are distributed from RIKEN BioResourceCenter (http://www.brc.riken.jp/lab/epd/Eng/species/moss.shtml). Clones of other libraries will be distributed from the same center after publication of the corresponding paper. Before the publication, those clones will be shared as a collaboration work and contact to Dr. Mitsuyasu Hasebe (mhasebe [at mark] nibb.ac.jp).

 

 

(1) pph (Nishiyama et al. 2003 PNAS 100: 8007-8012)

Protonemata and young gametophore without exogenous phytohormones

 

The tissue from which RNA was extracted contained chloronemata and young gametophores with 2 to 5 leaves.

 

Protonemata were blended by the POLYTRON, and then cultivated on the BCDATG medium for 13-14 days under the continuous light.

 

Full length cDNA library. A backbone of the vector is basically from pBluescript(KS),  that was in vivo excised from a modified lPS phage vector (Mo bi Tec, Germany). 5' end of the cDNA that was digested with XhoI was ligated to SalI site of the vector and the 3' end including polyA tail was ligated to BamHI site of the vector.  cDNA instert could be amplified with conventional T7 and T3 primers.  This full-length cDNA library was generated basically according to the method described in Seki M. et al. (The Plant J 15, 707-720 (1998)).

vector information

 

(2) pphb (Nishiyama et al. 2003 PNAS 100: 8007-8012)

Protonemata treated with benzylaminopurine

 

Protonemata were blended by the POLYTRON, and then cultivated on the BCD medium containing 0.5uM BA (benzylaminopurine) for 8 to 13 days under the continuous light. 

 

The tissue from which RNA was extracted contained chloronemata, caulonemata and malformed buds.

 

Normalized full length cDNA library. A backbone of the vector is pBluescript II, that was in vivo excised from a modified lPS phage vector (Mo bi Tec, Germany). XhoI digested-5' end of cDNA is ligated to SalI site of the vector, and the BamHI digested-3' end including poly-A tail is ligated to BamHI site of the vector. cDNA instert could be amplified with conventional T7 and T3 primers. This normarized full-length cDNA library was generated basically according to the method described in Genome Research 10, 1617-1630 (2000), Carninci, P. et al.

 

vector information

 

(3) pphn (Nishiyama et al. 2003 PNAS 100: 8007-8012)

Protonemata treated with NAA

 

Protonemata were blended by the POLYTRON, and then cultivated on the BCD medium containing 1uM NAA (naphthalene acetic acid) for 8 to 11 days under the continuous light. 

 

The tissue from which RNA was extracted contained chloronemata, caulonemata and rhizoid-like protonemata.

 

Normalized full length cDNA library. A backbone of the vector is pBluescript II, that was in vivo excised from a modified lPS phage vector (Mo bi Tec, Germany). XhoI digested-5' end of cDNA is ligated to SalI site of the vector, and the BamHI digested-3' end including poly-A tail is ligated to BamHI site of the vector.  cDNA instert could be amplified with conventional T7 and T3 primers. This normarized full-length cDNA library was generated basically according to the method described in Genome Research 10, 1617-1630 (2000), Carninci, P. et al.

 

vector information

 

(4) pphf (Nishiyama et al. submitted)

Protoplasts at the first cell division

 

Physcomitrella patens protonemata were inoculated on BCDATG medium for every ca. 5 days.  Protoplasts were isolated from the protonemata, further incubated at 25C under continuous light for 2-3 days.  The regenerated cells, which were rich in cells at the stage during the first asymmetric cell division, were collected.  Total RNA was extracted for constructing a full-length cDNA library.

 

cDNA library was prepared by the vector-capping method (Kato et al. 2005. DNA Res. 12: 53-62).

 

vector information

 

(5) ppsp: Sporophytes before meiosis (Nishiyama et al. submitted)

The gametophores harboring gametangia were further cultivated at 15°C under the short day conditions for 21 to 28 days to collect sporophyte tissue before meiosis. Archegonial tissue was included in the sample.

 

cDNA library was prepared by the oligo-capping method (Maruyama and Sugano 1994. Gene 138: 171-174).

 

vector information

 

(6) ppls: Upper halves of gametophores (Nishiyama et al. submitted)

Gametophores were cultivated on sterile peat pellets (Jiffy-7; Jiffy Products International AS, Kristansand, Norway) for 1 to 1.5 months at 25°C under continuous light.

 

cDNA library was prepared by the oligo-capping method (Maruyama and Sugano 1994. Gene 138: 171-174).

 

vector information

 

(7) ppaa: Gametophore tips including the antheridia and archegonia (Nishiyama et al. submitted)

Gametophores cultivated under the same conditions for the same period were moved to 15°C under 8-h light/16-h dark conditions to induce gametangia and cultivated for 11 to 26 days. Those archegonia that were brown in color, which is an indicator of fertilization, were discarded.

 

cDNA library was prepared by the oligo-capping method (Maruyama and Sugano 1994. Gene 138: 171-174).

 

vector information

 

(8) ppgs: Sporophytes during meiosis (Nishiyama et al. submitted)

The gametophores harbouring gametangia were further cultivated at 15°C under the short day conditions for 31 to 53 days to collect sporophyte tissue during meiosis. Archegonial tissue was removed and only sporophytic tissue was collected.

cDNA library was prepared by the oligo-capping method (Maruyama and Sugano 1994. Gene 138: 171-174).

vector information