MITE insertion-dependent expression of CitRKD1 with a RWP-RK domain regulates somatic embryogenesis in citrus nucellar tissues

Somatic embryogenesis in nucellar tissues is widely recognized to induce polyembryony in major citrus varieties such as sweet oranges, satsuma mandarins and lemons. This capability for apomixis is attractive in agricultural production systems using hybrid seeds, and many studies have been performed to elucidate the molecular mechanisms of various types of apomixis. To identify the gene responsible for somatic embryogenesis in citrus, a custom oligo-DNA microarray including predicted genes in the citrus polyembryonic locus was used to compare the expression profiles in reproductive tissues between monoembryonic and polyembryonic varieties. The full length of CitRKD1, which was identified as a candidate gene responsible for citrus somatic embryogenesis, was isolated from satsuma mandarin and its molecular function was investigated using transgenic ‘Hamlin’ sweet orange by antisense-overexpression. The candidate gene CitRKD1, predominantly transcribed in reproductive tissues of polyembryonic varieties, is a member of the plant RWP-RK domain proteins. CitRKD1 of satsuma mandarin comprised two alleles (CitRKD1-mg1 and CitRKD1-mg2) at the polyembryonic locus controlling embryony type (mono/polyembryony) that were structurally divided into two types with or without a miniature inverted-repeat transposable element (MITE)-like insertion in the upstream region. CitRKD1-mg2 with the MITE insertion was the predominant transcript in flowers and young fruits where somatic embryogenesis of nucellar cells occurred. Loss of CitRKD1 function by antisense-overexpression abolished somatic embryogenesis in transgenic sweet orange and the transgenic T1 plants were confirmed to derive from zygotic embryos produced by self-pollination by DNA diagnosis. Genotyping PCR analysis of 95 citrus traditional and breeding varieties revealed that the CitRKD1 allele with the MITE insertion (polyembryonic allele) was dominant and major citrus varieties with the polyembryonic allele produced polyembryonic seeds. To identify the gene responsible for somatic embryogenesis in citrus, a custom oligo-DNA microarray including predicted genes in the citrus polyembryonic locus was used to compare the expression profiles in reproductive tissues between ‘Kiyomi’ and ‘Harumi’ as materials for monoembryonic and polyembryonic varieties, respectively. Eight independent experiment performed at 15, 30, 45 and 60 DAF (Day After Flowering) using whole young fruits in each variety.

珠心组织体细胞胚胎发生（somatic embryogenesis）被广泛认为可诱导甜橙、温州蜜柑、柠檬等主要柑橘品种产生多胚现象（polyembryony）。这种无融合生殖（apomixis）特性在杂交种子农业生产体系中极具应用价值，因此已有诸多研究旨在阐明各类无融合生殖的分子机制。为鉴定调控柑橘体细胞胚胎发生的关键基因，本研究设计了包含柑橘多胚位点预测基因的定制寡聚DNA微阵列（oligo-DNA microarray），以单胚和多胚柑橘品种的生殖组织为材料，对比二者的基因表达谱。研究人员从温州蜜柑中分离得到柑橘体细胞胚胎发生候选基因CitRKD1的全长序列，并通过反义过表达（antisense-overexpression）技术转化‘哈姆林’甜橙，对其分子功能进行验证。该候选基因CitRKD1属于植物RWP-RK结构域蛋白（RWP-RK domain proteins）家族，在多胚品种的生殖组织中高丰度转录。温州蜜柑的CitRKD1存在两个等位基因（CitRKD1-mg1和CitRKD1-mg2），位于调控胚胎发生类型（单/多胚）的多胚位点，二者的结构差异在于上游区域是否存在微型反向重复转座元件（MITE）样插入序列。携带MITE插入的CitRKD1-mg2是花以及发生珠心体细胞胚胎发生的幼果中的主要转录本。通过反义过表达敲除CitRKD1功能可消除转基因甜橙的体细胞胚胎发生能力，且经DNA诊断证实，转基因T1代植株源自自花授粉产生的合子胚（zygotic embryos）。对95份柑橘地方品种和育成品种开展基因分型PCR（genotyping PCR）分析发现，携带MITE插入的CitRKD1等位基因（多胚等位基因）呈显性遗传特性，多数携带该多胚等位基因的主栽柑橘品种均可产生多胚种子。为进一步鉴定柑橘体细胞胚胎发生的关键基因，本研究以单胚品种‘清见’和多胚品种‘晴姬’为材料，利用包含柑橘多胚位点预测基因的定制寡聚DNA微阵列，对比二者生殖组织的基因表达谱。实验共开展8次独立重复实验，分别采集开花后15、30、45、60天的各品种完整幼果作为样本（DAF为开花后天数，Day After Flowering）。