| ‘沙糖橘’S蛋白同源基因CrSPH的克隆与表达分析 |
| 投稿时间：2023-03-30 修订日期：2023-05-04 点此下载全文 |
| 引用本文：吴秀兰,唐文武,徐呈祥,等.‘沙糖橘’S蛋白同源基因CrSPH的克隆与表达分析[J].西北植物学报,2023,43(8):1261~1267 |
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| 基金项目:国家外专局项目(QN2022030025L)；肇庆学院博士科研启动基金(611-612281) |
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| 中文摘要:配子体自交不亲和是导致柑橘无籽的重要原因，其自交不亲和是由高度多态性S位点基因控制。S-蛋白同源基因（S-protein homologous gene，SPH）属于S位点基因，然而SPH基因在柑橘自交不亲和反应中的作用尚未报道。本研究以‘无籽沙糖橘’和‘沙糖橘’花器官为试材，克隆到一个SPH基因，其CDS长度为417 bp，编码138个氨基酸，命名CrSPH。CrSPH基因在‘沙糖橘’和‘无籽沙糖橘’CDS区存在3个碱基的替换，引起2个氨基酸突变。Southern杂交实验表明CrSPH基因在‘沙糖橘’和‘无籽沙糖橘’基因组中均以单拷贝形式存在。qPCR实验表明，CrSPH基因在‘沙糖橘’花器官不同部位表达差异显著，在子房表达量最大，在花瓣、花丝等组织中表达量均较低；在高表达的子房中，‘沙糖橘’表达量是‘无籽沙糖橘’的16倍，表明CrSPH基因在‘沙糖橘’中具有高度的组织表达特异性。CrSPH基因在异交（‘无籽沙糖橘’ב沙糖橘’）授粉后0 d开始表达，第2-4 d表达量呈上升趋势，第6 d检测到最高表达量，其表达量是最低表达量（第1d）的12.4倍；自交（‘无籽沙糖橘’ב无籽沙糖橘’）授粉后表达量呈先上升后下降趋势，但表达量均低于异交授粉后的表达量。原核表达实验成功地诱导出CrSPH异源蛋白，离体花粉萌发实验表明随着异源‘无籽沙糖橘’CrSPH蛋白浓度的增加，‘无籽沙糖橘’萌发率受到显著的抑制。 |
| 中文关键词:‘沙糖橘’ CrSPH 表达分析 花粉萌发 自交不亲和 |
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| Cloning and Expression Analysis of the S-Protein Homologous Gene (CrSPH) from ‘Shatangju’ |
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| Abstract:Gametophytic self-incompatibility (GSI) is an important factor that can bring out seedless fruit in Citrus，GSI was regulated by a highly polymorphic locus named the S-locus. S-protein Homologous (SPH) gene belonged to the S-locus gene, but the role of SPH gene in the reaction of self-incompatibility has not been reported. In the study, the CDS of SPH gene, named CrSPH, was cloned from ‘Wuzishatangju’ and ‘Shatangju’. the CDS was 417 bp encoding a protein of 138 amino acids. There were three base pairs difference between ‘Wuzishatangju’ and ‘Shatangju’, and two amino acids ware substituted between ‘Wuzishatangju’ and ‘Shatangju’. The Southern hybridization experiment showed there was one copy of CrSPH in the gene genomic DNA both Wuzishatangju’ and ‘Shatangju’.The qPCR showed that the expression level of CrSPH gene was significantly difference in flower organs of ''Shatangju'' , the highest expression level was found in ovary, and it was hardly expressed in petals, filaments, anthers, stigmas, styles. the relative expression level of CrSPH gene was 16 times higher in the ovaries of ‘Shatangju’ than that in ovaries of ‘Wuzishatangju’. CrSPH gene began to express at 0 d after cross-pollination (‘Wuzishatangju’× ‘Shatangju’), the expression level showed an upward trend from 2th to 4th day, the highest expression level was detected at the 6th day in pistil. The expression level was in pistil first up and then down after self-pollination (‘Wuzishatangju’בWuzishatangju’), but the expression level of self-pollination was far lower than that of cross-pollination. The prokaryotic expression experiment successfully induced the CrSPH heterologous protein, and the pollen germination experiment in vitro showed that the germination rate of ‘Wuzishatangju’ was significantly inhibited with increasing CrSPH protein concentrations from ‘Wuzishatangju’. |
| keywords:‘Shatangju’ CrSPH expression analysis pollen germination self-incompatibility |
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