Construction of a specialized cDNA library from plant cells isolated by laser capture microdissection: toward comprehensive analysis of the genes expressed in the rice phloem.
Laser capture microdissection (LCM) is a powerful system which allows the isolation of selectively targeted cells from a tissue section for the analysis of gene-expression profiles of individual cells. The technique has been successfully used for the isolation of specific mammalian cells, mainly cancer cells. However, LCM has never been reported to be applied to the gene expression analysis of plant cells. We used a modified LCM system and successfully applied it to target and isolate phloem cells of rice leaf tissue whose morphology is apparently different from the surrounding cells. Total RNA was extracted from microdissected (approximately 150) phloem cells and the isolated RNA was used for the construction of a cDNA library following the T7 RNA polymerase amplification. Sequence analysis of 413 randomly chosen clones from the library revealed that there was a high level of redundancy in the population and the clones could be subclassified into 124 different groups that contained related sequences. Approximately 37% of both the redundant population and the non-redundant subgroups had novel components while approximately 63% were either homologues to the known genes reported to be localized in phloem of different plant species, or were homologues to other known genes. In situ hybridization revealed that putative amino acid permease, one of the non-redundant clones, was specifically expressed in the phloem. The results proved the effectiveness of construction of a specialized cDNA library from the specific plant cells.
Asano
Takayuki
T
National Institute of Agrobiological Sciences, Kannondai 2-1-2, Tsukuba, Ibaraki 305-8602, Japan.
Masumura
Takehiro
T
Kusano
Hiroaki
H
Kikuchi
Shoshi
S
Kurita
Akihiro
A
Shimada
Hiroaki
H
Kadowaki
Koh-Ichi
K
D026905:Amino Acid Transport Systems / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics* / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism; D020869:Gene Expression Profiling / Q000379:methods; D018506:Gene Expression Regulation, Plant; D015723:Gene Library; D017403:In Situ Hybridization; D007834:Lasers; D012275:Oryza / Q000166:cytology / Q000235:genetics*; D018515:Plant Leaves / Q000166:cytology / Q000235:genetics; D018514:Plant Structures / Q000166:cytology / Q000235:genetics*; D018749:RNA, Plant / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid
D026905:Amino Acid Transport Systems; D018076:DNA, Complementary; D018749:RNA, Plant; D012321:DNA-Directed RNA Polymerases