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ISSN 1672-9145 Acta Biochim Biophys Sin 2004, 36(10): 695-700 CN 31-1940/Q

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Construction of the Seed-Coat cDNA Microarray and Screening of Differentially Expressed Genes in Barley

Jin-Song PANG*, Meng-Yuan HE, and Bao LIU

Laboratory of Molecular Epigenetics, Institute of Genetics and Cytology, Northeast Normal University, Changchun 130024, China

Abstract Some barley mutants can synthesize neither anthocyanins nor proanthocyanidins in the seed coat, which is related to several genes in locus Ant13, but the exact model of action remains unknown. We used the cDNA microarray technology with barley transcription-deficient mutant (ant13-152) that does not synthesize proanthocyanidins as the tester, and its wild type genotype (Triumph) as the driver, to study this question. Six-thousand and forty-eight clones from the wild type Morex testa+pericarp cDNA library were amplified using PCR, and the DNA fragments were spotted on commercial amino-modified glass slide as microarray. The mRNAs from the developing seed coat (8-15 days) of both the mutant and the wild-type barley plants were isolated, and labeled respectively with Cy3-dUTP and Cy5-dUTP when reversely trans-cribed to cDNAs. The labeled cDNAs were used as probes, mixed at the same molar concentration, and hybridized with the DNA fragments on the slide. Seventy clones exhibiting marked differential expression (ratio>4) were identified from the microarray. All the 25 cDNA clones that showed an over-expression in wild type in comparison to the mutant ant13-152 were sequenced. It was found that most of these over-expressing clones were transcription/translation and hordein-associated genes. These results have laid a solid material basis for further elucidation of the metabolic pathway in proanthocyanidin synthesis in barley and likely other plants.

Key words barley; seed coat; proanthocyanidin; cDNA microarray

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Received: July 5, 2004 Accepted: August 16, 2004

This work was supported by a grant from the National Science Fund for Distinguished Young Scholars of China (30225003)

*Corresponding author: Tel, 86-431-5099367; E-mail, [email protected]