关键词: 苹果, S-RNase, 自交不亲和, 花粉cDNA文库, 酵母双杂交, γ–硫堇

Abstract: Yeast pGADT7-cDNA library was successfully constructed for apple‘Ralls Janet’pollen，with a transformation efficiency up to 1.2 × 106 . μg-1 and being inserted by sequences of 300–2 000 bp in size. Through yeast two-hybrid（Y2H）assay，one cDNA of 505 bp in size was screened from the library by C2HVC3 domain of apple S2-RNase as bait. The cDNA was predicted to contain one complete ORF of 216 bp in size，coding an peptide with a signal peptide on its N terminal. The deduced mature peptide was designated apple γ-thionin MdD1 for that it contained both 8 cysteines and γ-thionin motif on its C terminal，was rich in Lys and Arg with positive charge. Structure prediction revealed one α-helix，three β-sheets and four disulfide bones. RT-PCR showed that the gene MdD1 preferentially expressed in anther，though also expressed in other tissues such as leaf，sepal，petal，ovary and style of apple‘Ralls Janet’. Y2H assay showed that MdD1 interacted with S-RNase nonspecifically，because it not only interacted with apple S2-RNase-C2HVC3，but also with S1-RNase-C2HVC3，mature peptide of S1-RNase and S2-RNase. All these results taken together suggest that apple γ-thionin MdD1 may be involved in SI as a pollen non-S factor interacting with S-RNase nonspecifically.

Key words: apple, S-RNase, self-incompatibility, pollen cDNA library, yeast two-hybrid, γ-thionin