Table 3.
Identification and Cloning Strategy for HSS- and DHS-Coding cDNA Sequences of Various Angiosperm Species
| cDNA | Organ for Total RNA Preparation | Degenerate Primers | Primers for 3′-RACE and 5′-RACE | Full-Length Primers with Included Restriction Sites | Expression Vector with Used Restriction Sites |
|---|---|---|---|---|---|
| HSS H. indicum | Shoot | P9, P4 | P30, P31-33 | P80 (NdeI), P81 (BamHI) | pET3a (NdeI + BamHI) |
| DHS H. indicum | Root | P9, P4 | P34, P35-37 | P82 (XhoI), P83 (BamHI) | pET3a-mod (XhoI + BamHI) |
| HSS C. officinale | Root | I.P5, P3; II.P9, P8 | P49, P50+51 | P74 (NdeI), P75 (BamHI) | pET3a (NdeI + BamHI) |
| DHS C. officinale | Shoot | I.P5, P3; II.P9, P8 | P45, P46-48 | P76 (XhoI), P77 (BamHI) | pET3a-mod (XhoI + BamHI) |
| HSS S. officinale | Root | P9, P4 | P41, P42-44 | P72 (XhoI), P73 (BclI) | pET3a-mod (XhoI + BamHI) |
| DHS S. officinale | Shoot | I.P9, P3; II.P6, P8 | P78, P38-40 | P78 (XhoI), P79 (BamHI) | pET3a mod (XhoI+BamHI) |
| HSS E. cannabinum | Root | P1, P2 | P10, P11+12 | P64 (XhoI), P65 (BamHI) | pET3a-mod (XhoI + BamHI) |
| DHS E. cannabinum | Shoot | P7, P8 | P13, P11+14 | P66 (XhoI), P67 (BamHI) | pET3a-mod (XhoI + BamHI) |
| HSS S. jacobaea | Root | P1, P2 | P19, P20-22 | P70 (NdeI), P71 (BamHI) | pET3a (NdeI + BamHI) |
| HSS1 S. vernalis | Ober and Hartmann (1999a) | ||||
| HSS2 S. vernalis | Root | as for HSS1 of S. vernalis | pET3a (NdeI + BamHI) | ||
| DHS S. vernalis | Ober and Hartmann (1999a) | ||||
| HSS S. vulgaris | Ober et al. (2000) | ||||
| HSS P. hybridus | Root | P5, P4 | P52, P53-55 | P84 (NdeI), P85 (BamHI) | pET3a (NdeI + BamHI) |
| DHS P. hybridus | Shoot | P5, P4 | P56, P57-59 | P86 (XhoI), P87 (BamHI) | pET3a-mod (XhoI + BamHI) |
| DHS I. hederifolia | Shoot tip | P7, P4 | P15, P16-18 | P68 (NdeI), P69 (BamHI) | pET3a (NdeI + BamHI) |
| DHS N. tabacum | Ober and Hartmann (1999b) | ||||
| DHS C. retusa | Root | P7, P4 | P23, P24-26 | P60 (NdeI), P61 (BamHI) | pET3a (NdeI + BamHI) |
| HSS Phalaenopsis sp | Aerial root tip | P9, P8 | P27, P28+29 | P62 (NdeI), P63 (BamHI) | pET3a (NdeI + BamHI) |
Plant organs from which total RNA was extracted, the primers used for identification and cloning, the vectors for heterologuous protein expression in E. coli BL21(DE3), and the restriction enzymes used for cloning of the full-length cDNA into the expression vectors are listed. RACE, rapid amplification of cDNA ends.