摘要
自交不亲和性是高等植物通过抑制自花授粉来阻止近亲繁殖的一种繁殖机制。在以茄科、蔷薇科、车前草科为代表的配子体自交不亲和机制中,花粉和雌蕊之间的自我/非自我识别由多态性S位点决定,其中SLF(S-locus F-box)/SFB(S-haplotype-specific F-box)基因是配子体自交不亲和花粉S决定因子。本研究通过生物信息学方法从菠萝基因组中筛选鉴定出21个SLF/SFB家族基因(AcSLF1~AcSLF21),各AcSLF基因在N端具有F-box保守结构域,其氨基酸长度、理论等电点、分子量存在较大差异,编码氨基酸序列大小范围为242~612 aa,分子量为27.778~69.070 kDa,其中18个AcSLFs蛋白偏碱性,总体蛋白稳定性较差,预测18个AcSLFs蛋白亚细胞定位于细胞核中,蛋白二级结构60%以上由α-螺旋和无规则卷曲组成。通过染色体定位分析发现其不均匀地分布在14条染色体上,其中AcSLF21未定位到具体染色体位置,并发现AcSLF4、AcSLF5、AcSLF6、AcSLF8、AcSLF14和AcSLF15与菠萝RNaseT2家族基因发生连锁。从进化关系中发现,菠萝SLF/SFB家族与蔷薇科、车前科、茄科植物SLF/SFB进化关系较远。利用qRT-PCR对AcSLFs基因在菠萝雌蕊、雄蕊、叶片、果肉、茎和根的表达量进行分析,结果发现AcSLFs基因在菠萝不同组织中具有明显的表达差异性,其中AcSLF1、AcSLF2、AcSLF4、AcSLF9、AcSLF10、AcSLF11、AcSLF15、AcSLF19、AcSLF20、AcSLF21基因在菠萝雄蕊中呈高表达量,AcSLF3、AcSLF7在果肉中表达量最高,AcSLF5、AcSLF12、AcSLF18在根中表达量显著高于其他组织,总体来说,大部分菠萝AcSLFs基因主要在菠萝雄蕊或叶片中高表达。同时分析了雌蕊自花、异花授粉前后的表达变化,发现AcSLF1、AcSLF2、AcSLF15三个基因在授粉雌蕊中的表达量较未授粉显著上调,尤其在授粉6h表达量急剧上调,随着花粉管的伸长其表达量降低,但在授粉24h仍具有较高的表达量,推测这些基因可能在菠萝自交不亲和中起到重要作用。该研究为菠萝SLF/SFB家族基因的克隆提供参考,并为菠萝自交不亲和机制研究奠定良好基础。
Self-incompatibility is a genetic mechanism to prevent self-fertilization and the consequent inbreeding and fitness decay in plant populations.In the gametophyte self-incompatibility mechanism represented by Solanaceae,Rosaceae and Plantaginaceae,the self/non-self recognition between pollen and pistil is determined by the polymorphic S locus.SLF(S-locus F-box)/SFB(S-haplotype-specific F-box)gene is the pollen S determinant of gametophyte self-incompatibility.In this study,21 SLF/SFB family genes were screened and identified based on the genome-wide of the pineapple.Each AcSLF gene had an F-box conserved domain at the N-terminal.There were great differences in amino acid length,theoretical isoelectric point and molecular weight.The size range of the encoded amino acid se-quence was 242-612 aa,and the molecular weight was 27.778-69.070 kDa.The isoelectric point ranged from 5.70 to 9.57.18 AcSLF proteins were alkaline and most proteins were unstability,only AcSLF19,AcSLF21 were stability.It was predicted that 18 AcSLF proteins were located in the nucleus,one AcSLF protein in chloroplast,two AcSLF protein in both chloroplast and nucleus,and more than 60%of the secondary structure of the protein was composed of alpha helix and random coil.Bioinformatics analysis showed that the pineapple SLF/SFB family genes,which were unevenly distributed on 14 chromosomes,and the chromosome location of AcSLF21 was undetermined.Furthermore,AcSLF4,AcSLF5,AcSLF6,AcSLF8,AcSLF14 and AcSLF15 were linked with RNase T2 family genes,this was one of the main characteristics of pollen SLF gene.AcSLF genes contained different numbers of introns,most of which contained 1~2 introns,and AcSLF3 contained 4 introns.Based on the above analysis results,it showed that AcSLF proteins had dif-ferent characteristics.Phylogenetic analysis indicated that the evolutionary relationship of AcSLFs in pineapple was far away from the SLF/SFB in Rosaceae,Plantaginaceae and Solanaceae,the degree of clustering was low on the branches of evolutionary tree.The expression levels of the AcSLF genes in pistil,stamen,leaf,sarcocarp,stem and root were analyzed by qRT-PCR.The results showed that AcSLFs had obvious expression differences in different tissues of pine-apple.AcSLF1,AcSLF2,AcSLF4,AcSLF9,AcSLF10,AcSLF11,AcSLF15,AcSLF19,AcSLF20 and AcSLF21 genes were highly expressed in pineapple stamens.The expression of AcSLF3 and AcSLF7 in sarcocarp was higher than that of other tissue,and AcSLF5,AcSLF12,AcSLF18 were higher expression in root than other tissues.In general,most pine-apple AcSLF genes are mainly expressed in pineapple stamens or leaves.It was found that the expression of AcSLF1,AcSLF2,AcSLF15 genes in pollinated pistil were significantly up-regulated than that in non-pollinated pistil.Especially at 6 h after cross-pollination,the expression level was significantly higher than that of non-pollinated pistils.With the extension of the pollen tube in the pistil,the expression level decreased,but it still had a high expression at 24 h after pollination.And the expression trend of AcSLF1,AcSLF2 and AcSLF15 in cross-pollination was consistent with the growth of pollen tube in the pistil.Therefore,it is speculated that AcSLF1,AcSLF2 and AcSLF15 play an important role in pineapple self-incompatibility.This is the first time to analyze the expression pattern of SLF/SFB family genes in pineapple.This study would provide a reference for the cloning of pineapple SLF/SFB family genes,and basis for the study of pineapple self-incompatibility mechanism.
作者
井敏敏
李小针
李栋梁
戴小红
顾帅磊
马朝明
陈志辉
陈晶晶
JING Minmin;LI Xiaozhen;LI Dongliang;DAI Xiaohong;GU Shuailei;MA Chaoming;CHEN Zhihui;CHEN Jingjing(South Subtropical Crops Research Institute,Chinese Academy of Tropical Agricultural Sciences/National Field Genebank for Tropical Fruit(Zhanjiang)/Key Laboratory of Tropical Fruit Tree Biology,Ministry of Agriculture and Rural Affairs,Zhanjiang,Guangdong 524091,China)
出处
《热带作物学报》
CSCD
北大核心
2023年第7期1317-1327,共11页
Chinese Journal of Tropical Crops
基金
国家重点研发计划专项(No.2019YFD1001104)
海南省自然科学基金项目(No.322QN377)
农业农村部物种品种资源保护项目(No.125163006000160001)。
