The rice tapetum-specific gene RA39 encodes a type I ribosome-inactivating protein

Abstract

A tapetum-specific cDNA encoded by a rice gene, RA39, was isolated by cDNA subtractive hybridization, differential screening and rapid amplification of cDNA ends. RA39 is a single-copy gene in the rice genome. mRNA in situ hybridization indicates that this gene is a tapetum-specific gene, and highly expressed in the tapetal cells at the meiosis and tetrad stages. The RA39 cDNA is 1,013 bp in length with an open reading frame encoding 298 amino acid residues. This cDNA sequence does not show significant homology to any known sequences in GenBank databases, but its deduced amino acid sequence (RA39) has between 19 and 34% sequence identity to ribosome-inactivating proteins (RIPs). Optimal alignment reveals that the five amino acid residues constituting the active site of the ricin A-chain (Tyr 80 , Tyr 123 , Glu 177 , Arg 180 and Trp 211 ), which are invariant among all RIPs published to date, are conserved in RA39. Recombinant RA39 protein expressed in Escherichia coli was purified to homogeneity. The purified protein exhibits the RNA N-glycosidase activity of RIPs. This demonstrates that RIPs occur in the reproductive organs of rice. The possible function of RA39 in anther development is discussed.

Proteomics Identification of Differentially Expressed Proteins Associated with Pollen Germination and Tube Growth Reveals Characteristics of Germinated Oryza sativa Pollen

“…Primers Pr1 (5Ј-ACG CGC TGA CCA AGG AGA TC-3Ј) and Pf1 (5Ј-CAG GTT GTA GTC GCC GTG TG-3Ј) were used to detect the transcript for ␤-1,4-xylanase (GenBank accession number AAP53220), primers Pr2 (5Ј-ACT TGG CCT GGA CGT TGG AC-3Ј) and Pf2 (5Ј-ATG GCA TCC TCC TCC CTT CT-3Ј) were used for ␤-expansin (NP_91258), primers Pr3 (5Ј-GCA AGC ATG CAG CAA CAC AT-3Ј) and Pf3 (5Ј-GCA GCA ATG GCA TCC TCC T-3Ј) were for major pollen allergen Ory s 1 (Q40638), primers Pr4 (5Ј-TTT GCA TAT GGC TCC ACA GC-3Ј) and Pf4 (5Ј-ATG GCC TCC ATG TCC TCC TTC-3Ј) were for pollen allergen 1 (CAD40508), primers Pr5 (5Ј-TCA GAT CAT GTT GGA GAG G-3Ј) and Pf5 (5Ј-ATG GCG AGA TCA CTG GCG C-3Ј) were for pectin methylesterase inhibitor (NP_912762), and primers Pr6 (5Ј-ACA GAG CCT GCC ACG ACA GA-3Ј) and Pf6 (5Ј-GAC CGT TGC CTT CAA TAG CG-3Ј) were for ␤-galactosidase (NP_920740). The amplified tubulin tubA cDNA (X91806) was used as a constitutive control (23). First strand cDNA was synthesized with SuperScript II RNase H Ϫ reverse transcriptase (200 units/l, Invitrogen) according to the manufacturer's protocol.…”

OsRAF is an ethylene responsive and root abundant factor gene of rice

“…PCR was performed in a 50-ll mixture containing 5 ll of firststrand cDNA, 20 pmol each of the gene-specific primers 5 0 -GACAGATCTGATGTGTGGAGGCGC CATC-3 0 and 5 0 -TCAACTAGTGAAAAGGGCGC CGTCGATTG-3 0 , 0.4 lM dNTPs, 1· GC buffer (Takara) and 2.5 U Taq DNA polymerase (5 U/ll, Takara) for 35 cycles. Rice Tubulin A cDNA (Tub A) was amplified for 25 cycles as a constitutive control (Ding et al 2002).…”

“…Endogenous OsRAF transcript in OsRAF RNAi lines was checked by semi-quantitative RT-PCR with the gene specific primer pair. Rice Tubulin A (Tub A) cDNA was amplified as a constitutive control (Ding et al 2002).…”

Development of male sterile transgenic lines in rice by tapetum specific expression of barnase gene

“…OsC6 (Tsuchiya et al 1994;Yokoi et al 1997), OsRA8 (Jeon et al 1999), OsRA39 (Ding et al 2002), OsYY2 (Kuriakose et al 2009), OsRTS (Luo et al 2006;Deveshwar et al 2011), OsIPP3, OsbHLH, OsIPK, OsFbox, OsIPA, (Swapna et al 2011;Khurana et al 2012;Khurana et al 2013), OsSCP1, OsSCP2, and OsSCP3 (Park et al 2006). Of these, the OsRTS promoter from rice cultivar IR54 has been used to demonstrate the development of male sterile line by expression of the barnase gene under the control of this promoter (Luo et al 2006).…”