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番茄N-聚糖酶基因SlaPNGase1的功能分析

Functional Analysis of N-glycanase Gene SlaPNGase1 in Tomato
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摘要 番茄(Solanum lycopersicum)作为新疆红色产业,对当地经济发展有重要意义。由于新疆气候特点,无霜期较短,导致番茄被集中采收。加工能力不足,番茄采摘后被搁置数天,伴随高温天气,番茄成熟过度,导致软化变质,造成严重的经济损失和资源浪费。本试验为研究N-聚糖酶基因SlaPNGase1在番茄果实成熟软化过程中的潜在生物学功能,以加工番茄‘里格87-5’为材料,克隆到番茄N-聚糖酶基因SlaPNGase1(Solyc06g051020)及其上游2 220 bp的启动子序列。通过生物信息学初步分析,发现SlaPNGase1含有信号肽序列,不含有跨膜结构域,该启动子中含有多个与果实成熟应答相关的顺式作用元件。通过实时定量PCR检测SlaPNGase1在番茄幼苗根、茎、叶,开花后5 d幼果(DPA5),成熟绿果(DPA33),成熟红果(DPA52)中的转录表达水平(day post-anthesis, DPA)。结果显示,SlaPNGase1在番茄成熟红果中的表达水平最高。构建启动子活性分析载体prPNG1::GUS,对T1代转基因番茄果实进行GUS染色,结果表明,SlaPNGase1启动子在破色果实(DPA39)中的活性强于在未成熟绿果(DPA26)中的活性。构建35S::SlaPNGase1过表达载体,遗传转化番茄后,采摘同时期(DPA52)的过表达番茄果实和野生型番茄果实,将其放置相同光照,温度,湿度环境下。10 d后,转35S::SlaPNGase1基因过表达番茄果实有50%变黑,腐烂,发霉。相反,野生型果实只有轻微表皮失水皱缩,推测基因SlaPNGase1对番茄果实成熟软化具有作用。 Tomato(Solanum lycopersicum), as a red industry in Xinjiang, is of great significance to economic development. Due to the climate in Xinjiang, the frost-free period is short, tomatoes are harvested intensively. Because of insufficient processing capacity, the tomatoes were put on hold for several days after being picked. In high temperature weather, the tomatoes were over-mature, resulting in soften and deteriorate, which caused serious economic losses and waste of resources. In order to study the potential biological function of SlaPNGase1 in the ripening and softening process of tomato fruit, in this research, N-glycanase gene, SlaPNGase1(Solyc06 g051020),and its promoter with 2 220 bp were cloned from tomato ’RIG 87-5’. Preliminary analysis of bioinformatics showed that SlaPNGase1 contained a signal peptide sequence and didn’t contain a transmembrane domain. The promoter contained a plurality of cis-acting elements related to fruit ripening response. Real-time quantitative PCR was used to detect the transcriptional expression level of SlaPNGase1 in various tissues of tomato, including seedling roots,stems, and leaves, young fruit with 5 days after flowering(DPA5), mature green fruit(DPA33), ripe red fruit(DPA52)(day post-anthesis, DPA). The results showed that SlaPNGase1 had the highest expression level in tomato ripe red fruit. prPNG1::GUS fusion expression vector was constructed. GUS staining analysis was performed on T1 generation transgenic tomato fruits. The results showed that SlaPNGase1 promoter had higher activity in the color-breaking fruit(DPA39) than that in the immature green fruit(DPA26). 35 S::SlaPNGase1 overexpression vector was constructed. It was transferred into tomatoes. We picked the overexpressed tomato fruits and wild-type tomato fruits at the same period(DPA52), placing them under the same light, temperature and humidity environment. After 10 days,50% of transfer 35 S::SlaPNGase1 gene to overexpressed tomato fruits turned black, rot, and moldy. On the contrary, wild-type fruits had only a slight epidermal shrinkage. We speculated that SlaPNGase1 played a function in the ripening and softening process of tomato fruit.
作者 马月星 马雪萌 郑银英 向本春 崔百明 Ma Yuexing;Ma Xuemeng;Zheng Yinying;Xiang Benchun;Cui Baiming(School of Life Science,Shihezi University,Shihezi,832003)
出处 《分子植物育种》 CAS 北大核心 2022年第3期697-705,共9页 Molecular Plant Breeding
基金 本研究由国家自然科学基金项目(31460466) 石河子大学科学技术研究发展计划项目(KX0304)共同资助。
关键词 番茄 N-聚糖酶 游离N-聚糖 启动子活性 功能分析 Tomato N-glycanase Free N-glycan Promoter activity Functional analysis
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