‘沙糖橘’S蛋白同源基因CrSPH的克隆与表达分析  

Cloning and Expression Analysis of the S-proteinHomologous Gene ( CrSPH ) from ‘Shatangju’

作　　者：吴秀兰 唐文武[2] 徐呈祥[2] 李桂花[3] WU Xiulan;TANG Wenwu;XU Chengxiang;LI Guihua(School of Food and Pharmaceutical Engineering,Zhaoqing University,Zhaoqing,Guangdong 526061,China;School of Life Sciences,Zhaoqing University,Zhaoqing,Guangdong 526061,China;Vegetable Research Institute,Guangdong Academy of Agricultural Sciences,Guangzhou 510640,China)

机构地区：[1]肇庆学院食品与制药工程学院,广东肇庆526061 [2]肇庆学院生命科学学院,广东肇庆526061 [3]广东省农业科学院蔬菜研究所,广州510640

出　　处：《西北植物学报》2023年第8期1261-1267,共7页Acta Botanica Boreali-Occidentalia Sinica

基　　金：国家外专局项目(QN2022030025L);肇庆学院博士科研启动基金项目(611-612281)。

摘　　要：柑橘自交不亲和由S位点基因控制,前期SSH文库中筛选出1个自交不亲和相关的S蛋白同源基因(S-protein homologous gene,SPH),但其功能尚不明确。研究以‘无籽沙糖橘’和‘沙糖橘’为材料,利用反转录PCR技术克隆CrSPH基因,通过qPCR技术分析该基因表达特性,并进行原核表达和花粉萌发实验。结果表明,(1)CrSPH的CDS长度为417 bp,编码138个氨基酸,两材料间的CDS区存在3个碱基替换,引起2个氨基酸突变。(2)Southern杂交实验表明,CrSPH基因在两材料基因组中均以单拷贝形式存在。(3)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,子房中表达量最大,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。(4)CrSPH基因在异交授粉第6,7天表达量最高,与其他时段差异均达到显著水平,第6天表达量是第1天表达量的12.4倍。(5)原核表达实验成功诱导出CrSPH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。研究表明,CrSPH基因具有表达特异性,可能与柑橘自交不亲和有关。Self-incompatibility in Citrus was controlled by the S locus gene,one self-incompatibility related S-protein homologous gene(SPH)was screened from SSH library in the early stage,but its function is still unclear.In the study,the SPH gene was cloned from‘Wuzishatangju’and‘Shatangju’by reverse transcription PCR,named CrSPH.The expression level of the CrSPH gene was analyzed by qPCR.The prokaryotic expression and pollen germination experiments were also conducted.The results showed,(1)the CDS of SPH gene was successfully cloned from‘Wuzishatangju’and‘Shatangju’,and the CDS was 417 bp encoding a protein of 138 amino acids.There were three base pairs difference between‘Wuzishatangju’and‘Shatangju’,which resulted in two amino acids mutation.(2)The southern hybridization experiment showed there was one copy of CrSPH in the genomic DNA both Wuzishatangju’and‘Shatangju’.(3)The qPCR showed that the expression level of CrSPH gene was significantly different 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’.(4)The highest expression of the CrSPH gene was detected at the 6 th and 7 th d after outcrossing(‘Wuzishatangju’בShatangju’)pollination,and there were significant difference than the expression of the CrSPH gene at other periods,the relative expression level of CrSPH gene at 6 d was 16 times higher than that at 1 d.(5)The prokaryotic expression experiment successfully induced the CrSPH heterologous protein,and the pollen germination experiment in vitro showed that the pollen germination rate of‘Wuzishatangju’was significantly inhibited with increasing CrSPH protein concentrations from‘Wuzishatangju’.The above results showed that the CrSPH gene had tissue expression specificity.We speculated that the CrSPH gene may be related to self-in

关 键 词：‘沙糖橘’ CrSPH 表达分析 花粉萌发 自交不亲和 

分 类 号：Q943.2[生物学—植物学] S666[农业科学—果树学]