【摘要】：黃瓜是一種水蘇糖轉(zhuǎn)運(yùn)型植物,而水蘇糖合成酶是合成水蘇糖的關(guān)鍵酶,它催化棉子糖和肌醇半乳糖苷生成水蘇糖,在棉子糖系列寡糖(RFOs)的生理生化過程中起著重要的作用。水蘇糖合成酶及其編碼基因曾有相關(guān)研究和報(bào)道,但是對于該基因在葉片韌皮部裝載、碳水化合物分配以及響應(yīng)非生物逆境脅迫過程中的功能鮮有研究。本論文從黃瓜中克隆了水蘇糖合成酶基因(CsSTS),并且對該基因的表達(dá)、定位以及功能進(jìn)行了分析,并獲得了以下主要的結(jié)果:1.黃瓜水蘇糖合成酶基因?qū)儆趩慰截惢颉?shí)時(shí)熒光定量PCR的結(jié)果表明,CsSTS在黃瓜所有被檢測的組織中都有表達(dá),在葉片中,特別是成熟葉片的表達(dá)量明顯高于其它組織,包括根、莖、幼葉、雄花、雌花和果實(shí)。隨著葉片不同發(fā)育時(shí)期,CsSTS表達(dá)量持續(xù)增加,至展葉12天時(shí)達(dá)最高值,之后有所下降。在一天中,CsSTS表達(dá)存在晝夜節(jié)律,取樣時(shí)間節(jié)點(diǎn)在15:00時(shí)表達(dá)量最高。2.組織定位的研究結(jié)果顯示,CsSTS主要在成熟葉片的小葉脈韌皮部伴胞中表達(dá),說明CsSTS參與了碳水化合物的韌皮部裝載。亞細(xì)胞定位結(jié)果表明,CsSTS在細(xì)胞膜、細(xì)胞核和細(xì)胞質(zhì)中都有表達(dá)。3.當(dāng)干擾CsSTS基因之后,在干擾株系中,其表達(dá)量明顯下調(diào),水蘇糖合成酶的活性和水蘇糖的含量明顯低于野生型,淀粉在葉片中累積,說明CsSTS參與了黃瓜葉片的韌皮部裝載。與野生型植株相比較,干擾株系中黃瓜蔗糖轉(zhuǎn)運(yùn)蛋白(CsSUT)的表達(dá)量明顯升高,并且葉柄中蔗糖和棉子糖的輸出比例均有一定程度升高;而在過表達(dá)株系中,蔗糖轉(zhuǎn)運(yùn)蛋白的表達(dá)水平與野生型相當(dāng)。以上說明黃瓜韌皮部在共質(zhì)體裝載(主要途徑)的同時(shí),也存在質(zhì)外體裝載。從這個(gè)角度講,黃瓜也屬于韌皮部混合裝載的模式植物。4.為進(jìn)一步研究CsSTS的功能,對黃瓜幼苗進(jìn)行低溫處理(6℃, 72h)。在低溫處理過程中,野生型和轉(zhuǎn)基因株系中CsSTS表達(dá)量、STS酶活性、水蘇糖含量以及抗氧化酶系統(tǒng)活性均表現(xiàn)出上調(diào)和增加趨勢,特別是在過表達(dá)株系中,但干擾株系上調(diào)和增加最小。干擾株系的葉片在低溫條件72h后明顯萎蔫和下垂,但是低溫對過表達(dá)株系的生長影響較小。這些結(jié)果說明CsSTS參與黃瓜幼苗對于低溫脅迫的響應(yīng),并且低溫下STS酶活性的上升和水蘇糖含量的增加有助于減輕低溫脅迫對于幼苗自身的傷害。
[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，

本文編號：2151731