Plant polynucleotides encoding novel prenyl proteases
An isoprenyl, polynucleotide technology, applied in the field of new polynucleotides, which can solve problems such as no description
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Embodiment 1
[1096] Example 1 Growth of Physcomitrella patens cultures
[1097] In this study, the plant species Physcomitrellapatens (Hedw.) B.S.G. from the collection of the genetic studies section of the University of Hamburg was used. They were originally from strain 16 / 14 collected by H.L.K. Whitehouse in Gransden Wood, Huntingdonshire (England) obtained from spore subculture by Engel (1968, Am J Bot 55, 438-446). Plants are multiplied by sporulation or gametophyte regeneration. Protonema develops from haploid spores as chloroplast-rich chloronema and chloroplast-low stem filaments, on which shoots are formed approximately 12 days later. They grow gametoves with sperm and egg organs. After insemination, diploid sporophytes and sporangia with short capsules are produced, in which post-meiotic spores mature.
[1098] In an artificial environment room, the air temperature is 25℃, and the light intensity is 55micromols -1m2 (white light; Philips TL 65W / 25 fluorescent tube) and incubat...
Embodiment 2
[1100] Example 2-Isolation of total RNA and poly-(A)+RNA of Physcomitrella patens and construction of cDNA library
[1101] For transcription studies, total RNA and poly-(A)+ RNA were isolated. Total RNA was obtained from wild-type protonemata grown for 9 days according to the GTC-method (Reski et al. 1994, Mol. Gen. Genet., 244:352-359).
[1102] Poly(A)+ RNA was isolated using Dyna BeadsR (Dynal, Oslo, Norway) as recommended by the manufacturer. After the poly(A)+RNA or RNA concentration was determined, it was precipitated by adding 1 / 10 volume of 3M sodium acetate pH 4.6 and 2 volumes of ethanol and stored at -70°C.
[1103] RNA preparation from Arabidopsis seeds - "hot" extraction:
[1104] 1. Buffers, enzymes and solutions
[1105] -2M KCl
[1106] -Protease K
[1107] - Phenol (for RNA)
[1108] - Chloroform:isoamyl alcohol (phenol:chloroform 1:1; pH adjusted for RNA)
[1109] -4M LiCl, DEPC-treated
[1110] -DEPC-treated water
[1111] -3M NaOAc, pH 5, DEPC-tre...
Embodiment 3
[1134] Example 3 - Sequencing and functional annotation of Physcomitrlela patens ESTs
[1135] DNA sequencing of the cDNA library as described in Example 2 was carried out according to standard methods, specifically by the ABI PRISM Big Dye Terminator Cycle Sequencing ReadyReaction Kit (Perkin Elmer, Weiterstadt, Germany) using the chain termination method. Preparative plasmid recovery from the cDNA library was performed by mass excision in vivo, re-transformation, and DH10B plating on agar plates, followed by random sequencing (detailed description of materials and methods from Stratagene, Amsterdam, Netherlands). Plasmid DNA was prepared from E. coli cultures grown overnight in Luria-Broth medium containing ampicillin by the Qiagene DNA Preparation Robot (Qiagen, Hilden) according to the manufacturer's recommendations (see Sambrook et al. (1989) (Cold Spring Harbor Laboratory Press: ISBN 0-87969-309-6)). The nucleotide sequences of the sequencing primers are as follows:
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