Cell Cycle Nucleic Acids, Polypeptides and Uses Thereof
a cell cycle and nucleic acid technology, applied in the field of plant molecular biology, can solve the problems of difficult and time-consuming transformation of some agronomically important crop plants, difficult to obtain a culture response from some maize varieties, and serious genotype limitations still exis
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example 1
Isolation of Maize CKI Genes
[0163] Total RNA was isolated from corn tissues with TRIzol Reagent (Life Technology Inc. Gaithersburg, Md.) using a modification of the guanidine isothiocyanate / acid-phenol procedure described by Chomczynski and Sacchi [Chomczynski, P., and Sacchi, N., Anal. Biochem. 162:156 (1987)]. In brief, plant tissue samples were pulverized in liquid nitrogen before the addition of the TRIzol Reagent, and then were further homogenized with a mortar and pestle. Addition of chloroform followed by centrifugation was conducted for separation of an aqueous phase and an organic phase. The total RNA was recovered by precipitation with isopropyl alcohol from the aqueous phase.
Poly(A)+ RNA Isolation:
[0164] The selection of poly(A)+ RNA from total RNA was performed using PolyATact system (Promega Corporation. Madison, Wis.). In brief, biotinylated oligo(dT) primers were used to hybridize to the 3′ poly(A) tails on mRNA. The hybrids were captured using streptavidin couple...
example 2
Using CKI in a Two-Hybrid System to Identify Maize Genes Involved in Control of Cell Division
[0167] The CKI genes and their encoded proteins can be used to identify other proteins involved in the above processes. This can be done using the CKI gene as bait (the target fused to the DNA-binding domain) in a yeast two-hybrid screen. Methods for two-hybrid library construction, cloning of the reporter gene, cloning of the DNA-binding and activation domain hybrid gene cassettes, yeast culture, and transformation of the yeast are all done according to well-established methods (see Sambrook et al., 1990; Ausubel et al., 1990; Hannon and Bartels, 1995). When maize CKI is used as bait in such a two-hybrid screen, proteins that interact with CKI such as cyclin dependent kinases and cyclins per se may be identified.
example 3
Transient CKI-Antisense Expression Stimulates Cell Division and Enhances Transgene Integration
[0168] A CKI-antisense sequence is cloned into a cassette with a constitutive promoter (i.e. either a strong maize promoter such as the ubiquitin promoter including the first ubiquitin intron, or a weak constitutive promoter such as nos). Delivery of the CKI-antisense DNA in an appropriate plant expression cassette (for example, in a UBI::ZmCKI-antisense::pinII-containing plasmid) along with UBI::bar::pinII can be accomplished through numerous well-established methods for plant transformation. Using one of these methods, DNA is introduced into maize cells capable of growth on suitable maize culture medium. Such competent cells can be from maize suspension culture, callus culture on solid medium, freshly isolated immature embryos or meristem cells. Immature embryos of the Hi-II genotype are used as the target for co-delivery of these two plasmids.
[0169] Cytological methods can be used to v...
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