抑制黄瓜水苏糖合成酶基因CsSTS降低韧皮部装载和低温胁迫耐受性
发布时间：2018-07-29 05:51
【摘要】：黄瓜是一种水苏糖转运型植物,而水苏糖合成酶是合成水苏糖的关键酶,它催化棉子糖和肌醇半乳糖苷生成水苏糖,在棉子糖系列寡糖(RFOs)的生理生化过程中起着重要的作用。水苏糖合成酶及其编码基因曾有相关研究和报道,但是对于该基因在叶片韧皮部装载、碳水化合物分配以及响应非生物逆境胁迫过程中的功能鲜有研究。本论文从黄瓜中克隆了水苏糖合成酶基因(CsSTS),并且对该基因的表达、定位以及功能进行了分析,并获得了以下主要的结果:1.黄瓜水苏糖合成酶基因属于单拷贝基因。实时荧光定量PCR的结果表明,CsSTS在黄瓜所有被检测的组织中都有表达,在叶片中,特别是成熟叶片的表达量明显高于其它组织,包括根、茎、幼叶、雄花、雌花和果实。随着叶片不同发育时期,CsSTS表达量持续增加,至展叶12天时达最高值,之后有所下降。在一天中,CsSTS表达存在昼夜节律,取样时间节点在15:00时表达量最高。2.组织定位的研究结果显示,CsSTS主要在成熟叶片的小叶脉韧皮部伴胞中表达,说明CsSTS参与了碳水化合物的韧皮部装载。亚细胞定位结果表明,CsSTS在细胞膜、细胞核和细胞质中都有表达。3.当干扰CsSTS基因之后,在干扰株系中,其表达量明显下调,水苏糖合成酶的活性和水苏糖的含量明显低于野生型,淀粉在叶片中累积,说明CsSTS参与了黄瓜叶片的韧皮部装载。与野生型植株相比较,干扰株系中黄瓜蔗糖转运蛋白(CsSUT)的表达量明显升高,并且叶柄中蔗糖和棉子糖的输出比例均有一定程度升高;而在过表达株系中,蔗糖转运蛋白的表达水平与野生型相当。以上说明黄瓜韧皮部在共质体装载(主要途径)的同时,也存在质外体装载。从这个角度讲,黄瓜也属于韧皮部混合装载的模式植物。4.为进一步研究CsSTS的功能,对黄瓜幼苗进行低温处理(6℃, 72h)。在低温处理过程中,野生型和转基因株系中CsSTS表达量、STS酶活性、水苏糖含量以及抗氧化酶系统活性均表现出上调和增加趋势,特别是在过表达株系中,但干扰株系上调和增加最小。干扰株系的叶片在低温条件72h后明显萎蔫和下垂,但是低温对过表达株系的生长影响较小。这些结果说明CsSTS参与黄瓜幼苗对于低温胁迫的响应,并且低温下STS酶活性的上升和水苏糖含量的增加有助于减轻低温胁迫对于幼苗自身的伤害。
[Abstract]:Cucumber is a kind of water threonose transport plant, and threonose synthase is the key enzyme in the synthesis of threonose. It catalyzes the formation of threonose from cottonseed sugar and inositol galactoside, which plays an important role in the physiological and biochemical process of cottonseed sugar series oligosaccharide (RFOs). Threonose synthase and its coding gene have been studied and reported, but the function of the gene in phloem loading, carbohydrate allocation and response to abiotic stress has been rarely studied. In this paper, the threonose synthase gene (CsSTS), was cloned from cucumber and its expression, localization and function were analyzed, and the following main results were obtained: 1. Cucumber threonose synthase gene belongs to single copy gene. The results of real-time fluorescence quantitative PCR showed that CsSTS was expressed in all tissues of cucumber, and the expression of CSSTS in leaves, especially in mature leaves, was significantly higher than that in other tissues, including roots, stems, young leaves, male flowers, female flowers and fruits. The expression of CsSTS increased continuously at different developmental stages of leaves and reached its highest value at 12 days of leaf extension and then decreased. There was a circadian rhythm in the expression of CsSTS in one day, and the highest expression of CsSTS was observed at 15:00 in the sampling time node. The results of tissue localization showed that CsSTS was mainly expressed in the phloem of small veins of mature leaves, indicating that CsSTS was involved in the loading of carbohydrates in phloem. Subcellular localization showed that CsSTS was expressed in cell membrane, nucleus and cytoplasm. After interfering with CsSTS gene, the expression of CsSTS was down-regulated, the activity of threonose synthase and the content of threonose were significantly lower than that of wild type, and starch accumulated in the leaves, indicating that CsSTS was involved in the phloem loading of cucumber leaves. Compared with wild-type plants, the expression of sucrose transporter (CsSUT) was significantly increased in interference lines, and the proportion of sucrose and cottonseed sugar output in petiole was increased to a certain extent, but in over-expressed lines, the expression of sucrose transporter (CsSUT) was significantly higher than that of wild-type plants. The expression level of sucrose transporter was similar to that of wild type. The results showed that there was also hypoplast loading in the phloem of cucumber at the same time as the symplast loading (main pathway). From this point of view, cucumber also belongs to phloem mixed loading model plant. 4. In order to further study the function of CsSTS, cucumber seedlings were treated at low temperature (6 鈩，
本文编号：2151731
[Abstract]:Cucumber is a kind of water threonose transport plant, and threonose synthase is the key enzyme in the synthesis of threonose. It catalyzes the formation of threonose from cottonseed sugar and inositol galactoside, which plays an important role in the physiological and biochemical process of cottonseed sugar series oligosaccharide (RFOs). Threonose synthase and its coding gene have been studied and reported, but the function of the gene in phloem loading, carbohydrate allocation and response to abiotic stress has been rarely studied. In this paper, the threonose synthase gene (CsSTS), was cloned from cucumber and its expression, localization and function were analyzed, and the following main results were obtained: 1. Cucumber threonose synthase gene belongs to single copy gene. The results of real-time fluorescence quantitative PCR showed that CsSTS was expressed in all tissues of cucumber, and the expression of CSSTS in leaves, especially in mature leaves, was significantly higher than that in other tissues, including roots, stems, young leaves, male flowers, female flowers and fruits. The expression of CsSTS increased continuously at different developmental stages of leaves and reached its highest value at 12 days of leaf extension and then decreased. There was a circadian rhythm in the expression of CsSTS in one day, and the highest expression of CsSTS was observed at 15:00 in the sampling time node. The results of tissue localization showed that CsSTS was mainly expressed in the phloem of small veins of mature leaves, indicating that CsSTS was involved in the loading of carbohydrates in phloem. Subcellular localization showed that CsSTS was expressed in cell membrane, nucleus and cytoplasm. After interfering with CsSTS gene, the expression of CsSTS was down-regulated, the activity of threonose synthase and the content of threonose were significantly lower than that of wild type, and starch accumulated in the leaves, indicating that CsSTS was involved in the phloem loading of cucumber leaves. Compared with wild-type plants, the expression of sucrose transporter (CsSUT) was significantly increased in interference lines, and the proportion of sucrose and cottonseed sugar output in petiole was increased to a certain extent, but in over-expressed lines, the expression of sucrose transporter (CsSUT) was significantly higher than that of wild-type plants. The expression level of sucrose transporter was similar to that of wild type. The results showed that there was also hypoplast loading in the phloem of cucumber at the same time as the symplast loading (main pathway). From this point of view, cucumber also belongs to phloem mixed loading model plant. 4. In order to further study the function of CsSTS, cucumber seedlings were treated at low temperature (6 鈩，
本文编号：2151731
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