菠萝RNase T2家族基因克隆及表达分析

Cloning and expression analysis of RNase T2 family gene in pineapple

核糖核酸酶T2(ribonuclease T2,RNase T2)家族基因中的S-RNase基因,是部分双子叶植物中自交不亲和的雌蕊决定因子。为了解菠萝(Ananas comosus var.comosus)中的RNase T2家族基因中是否存在S-RNase基因,是否在菠萝自交/异交的过程中起作用,对菠萝RNase T2家族基因进行克隆和表达分析。本研究查找并克隆了菠萝基因组中RNase T2家族基因,分析了基因结构、系统进化关系,重点分析了在不同品种菠萝雌雄蕊及授粉花柱中的基因表达情况。菠萝基因组中仅存在3个RNase T2家族基因,其中一个存在可变剪切。基因结构和进化分析表明S-like RNase1和S-like RNase2均含有3个内含子,被归于第Ⅰ类;S-like RNase3有7个内含子,被归于第Ⅱ类。3个S-like RNase基因均不存在明显的组织特异性表达,其中S-like RNase2整体表达水平低于S-like RNase1和S-like RNase3;S-like RNase1和S-like RNase3在花粉和花柱中的表达水平因品种而异,整体上在花粉中的表达水平高于花柱;转录组、蛋白组及实时荧光定量PCR分析结果均表明自交/异交柱头中S-like RNase1和S-like RNase3表达量显著高于未授粉柱头,且异交高于自交。S-like RNase1和S-like RNase3虽然不属于第Ⅲ类S-RNase,但极有可能在菠萝授粉过程中起重要作用。

The S-RNase gene in RNase T2(ribonuclease T2)family is the pistil determinant S gene of self-incompatibility in some dicotyledonous plants.In order to understand the existence of S-RNase gene in RNase T2 family in pineapple(Ananas comosus var.comosus),and whether it plays a role in the process of self-incompatibility/outcrossing,we cloned and analyzed the expression of RNase T2 family genes in pineapple.In this study,the RNase T2 family in pineapple genome was searched and cloned,and its gene structure and phylogeny were analyzed,focusing on the expression of RNase T2 family in different cultivars of pineapple pistil and pollination style.There are only three RNase T2 family genes in pineapple genome,one of which has variable splicing.Structural and evolutionary analyses showed that both S-like RNase1 and S-like RNase2 contain three introns and are classified as ClassⅠ,while S-like RNase3 contains 7 introns and is classified as ClassⅡ.There is no tissue-specific expression in all three S-like RNases,and the overall expression level of S-like RNase2 was lower than those of S-like RNase1 and S-like RNase3.The expressions of S-like RNase1 and S-like RNase3 were different in pollen and style,and higher in pollen than in style.Transcriptomic,proteomic and quantitative reverse transcription PCR analyses showed that S-like RNase1 and S-like RNase3 expression levels in self-pollinating/outcrossing stigmata were significantly higher than those in unpollinated stigmata,and were higher in outcrossing stigmata than in self-crossing stigmata.Although S-like RNase1 and S-like RNase3 do not belong to ClassⅢS-RNase,they may play important roles in pollination of pineapple.