苹果花粉中与花柱S-RNase互作的γ-硫堇筛选及鉴定

Screen and Identification of Pollen γ-thionin Interacting with Style S-RNase in Apple

以‘国光’苹果（S1S2）花粉为材料,成功构建了酵母pGADT7-cDNA文库,转化效率约为1.2×106.μg-1,插入片段大小在300~2000bp之间。通过酵母双杂交方法,用苹果S2-RNase的C2HVC3区筛选文库获得一个长505bp的cDNA片段,经分析发现该cDNA序列上有一个261bp的开放阅读框（ORF）,该ORF编码一个具N端信号肽的多肽,该成熟多肽由64个氨基酸组成,富含带正电荷的赖氨酸和精氨酸,其C端有8个半胱氨酸和一个γ-硫堇功能域,结构预测显示其具有一个α螺旋,3个β折叠,4个二硫键,由此,推断其为苹果γ-硫堇,命名为MdD1。RT-PCR分析发现：MdD1基因在‘国光’苹果叶片、萼片、花瓣、子房、花药和花柱等组织中都有表达,但花药中表达量最高。酵母双杂交结果表明MdD1除了与苹果S2-RNase的C2HVC3区互作外,还与S1-RNase的C2HVC3区,S1、S2-RNase的成熟多肽区存在互作。初步认为：苹果γ-硫堇可能通过与S-RNase非特异性互作,作为花粉非S因子参与自交不亲和反应。

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.