【摘要】 甜瓜(Cucumis melo)主要在世界温带到热带地区栽培。为从转录组水平上挖掘甜瓜雄性不育的发生过程与超氧代谢和相关氧化酶活性的相关性,本研究利用雄性不育两用系对甜瓜可育株与不育株5mm的花蕾雄蕊进行转录组测序,筛选氧化酶相关差异表达基因。此外,本研究在花蕾雄蕊5 mm时期对其进行qRT-PCR分析以鉴定差异基因表达量,并对雄性不育株与可育株的花蕾酶活性进行测定。结果显示,转录组测序共有1 364个差异基因表达,其中表达上调的基因共有834个,表达下调的基因共有530个。根据相同或相似的表达模式对上述筛选得到的差异表达基因进行聚类分析,共将这些差异表达基因分为28类,其中第8类中有18个差异表达基因,这些基因参与超氧代谢途径相关性极其显著(P>0.001)。对差异表达基因进行基因本体论(Gene Ontology,GO)功能分析显示,共有1 118个基因归属于生物合成、分子功能和细胞组分3大分支,过氧化氢酶参与的催化过程中有576个差异表达基因,其中17个基因差异表达极显著。京都基因与基因组百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)注释结果显示,与氧化还原酶相关的差异基因为10个,其中9个为过氧化物酶同源基因,1个为过氧化氢酶相关表达基因。在苯丙素的生物合成途径中,与过氧化物酶相关基因MELO3C012183在雄性不育株与雄性可育株相比基因表达显著上调,过氧化物酶相关基因MELO3C014656表达显著下调。在色氨酸代谢途径中,与过氧化氢酶同工酶相关基因MELO3C017024在雄性不育株与雄性可育株相比基因表达显著上调。qRT-PCR验证表明,与过氧化物酶(peroxidase,POD)相关基因peroxidase 2-like precursor和peroxidase 11表达量可育株都低于不育株,而过氧化氢酶(cata-lase,CAT)相关表达基因catalase isozyme1-like表达量可育株高于不育株,而雄性不育株中POD和CAT酶活性均高于可育株。研究结果表明,过氧化物酶POD和过氧化氢酶CAT活性在不育株系中的差异表达是由植物自我保护激发诱导产生,氧自由基的积累使不育株系的膜脂过氧化水平异常升高,此现象有可能对小孢子发育造成伤害,从而导致花粉败育。本研究为甜瓜花粉发生败育的内在原因和作用机理提供了一定的科学依据。更多还原

【Abstract】 Melon(Cucumis melo) was mainly cultivated in the temperate zone to the tropics of the world. In order to explor in the relationship between the occurrence of male sterility in melon and the metabolism of superoxide and the related oxidase activities from the transcriptional level, this study used the male sterile AB line of stamens of the fertile and sterile plants of 5 mm in melon to do RNA sequencing(RNA-seq), and screened differentially expressed genes which related to the oxidase. In addition, the study carried out the real time fluorescence quantitative analysis in 2 stages of microspore development to identify the expression quantities of differential genes, and determined enzymatic activities of the buds of male sterile and fertile plants. The results showed that there were total of 1 364 differentially expressed genes in transcriptome sequencing, including 834 up-regulated genes and 530 down regulated genes. Then, it did cluster analysis to the screened differentially expressed genes according to the same or similar expression patterns, it showed that these differentially expressed genes divided into 28 categories, and eighth of them have 18 differentially expressed genes, these genes had extremely significant correlation(P>0.001) with pathway of superoxide metabolism. Gene Ontology(GO) function analysis of differentially expressed genes showed that a total of 1118 genes were involved in the 3 branches including the biosynthesis, molecular function and cell component,576 differentially expressed genes were involved in the catalytic process of catalase, of which 17 genes were differentially expressed significantly. Kyoto Encyclopedia of Genes and Genomes(KEGG) annotation results showed that there were 10 genes related to the oxidoreductase among them including 9 peroxidase homologous genes and a catalase homologous gene. In the biosynthetic pathway of phenylpropanoidand, compared with the male fertile plants, the gene expression of peroxidase(POD) related gene MELO3C012183 was significantly up regulated in male sterile plants, and the expression of gene MELO3C014656 was down regulated. Furthermore,in the pathway of tryptophan metabolism, compared with the male fertile plants, the gene expression of catalase(CAT) related gene MELO3C017024 was significantly up regulated in male sterile plants. Quantitative real time polymerase chain reaction(qRT-PCR) validation showed that fertile plants’ expression quantities of peroxidase2-like precursor gene and peroxidase 11 gene which related to POD were lower than sterile plants, on the contrary, fertile plants’ expression quantities of catalase isozyme 1-like gene which related to CAT were higher than sterile plants, the enzymatic activities of POD and CAT in male sterile plants were higher than that of fertile plants. The results supposed that the differential performances of enzymatic activities of POD and CAT in the male sterile line were caused by the self protection and inducing of plants, the accumulations of oxygen free radicals caused the abnormal increase in the level of membrane lipid peroxidation in male sterile lines, this phenomenon was likely to cause damage to microspore development and pollen abortion. This study would provid a scientific basis for the internal cause and mechanism of the pollen abortion in melon.更多还原