發(fā)布時間：2019-07-06 16:12

【摘要】：羅漢果甜苷Ⅴ在果實(shí)中含量低,致使其甜味劑生產(chǎn)與應(yīng)用成本一直居高不下,限制了產(chǎn)業(yè)的發(fā)展。提高果實(shí)中甜苷Ⅴ含量,降低其生產(chǎn)與應(yīng)用成本成為研究熱點(diǎn)。傳統(tǒng)栽培與育種方法提高果實(shí)中甜苷Ⅴ含量存在技術(shù)難點(diǎn)。羅漢果苷具有共同的苷元羅漢果醇,差異主要是C-3、C-24位連接葡萄糖基數(shù)目、方式和C-7、C-11位氧功能不同。甜苷Ⅴ生物合成研究和基因工程育種成為解決其成本問題的新途徑。摸清甜苷Ⅴ體內(nèi)合代謝規(guī)律和分離鑒定相關(guān)關(guān)鍵酶基因,是有效進(jìn)行其生物合成研究與基因工程育種的先決條件。因此,本研究對果實(shí)發(fā)育期羅漢果苷代謝規(guī)律,不同調(diào)控方式下甜苷V與其底物葡萄糖積累關(guān)系,果實(shí)發(fā)育期羅漢果醇代謝規(guī)律及其在葫蘆科植物的分布,甜苷V合成積累主要限制因子羅漢果醇與苷ⅡE、苷Ⅲ代謝酶基因表達(dá)轉(zhuǎn)錄組分析,以及葡萄糖基轉(zhuǎn)移酶基因克隆與表達(dá)進(jìn)行了研究。獲得的主要結(jié)果如下：1、‘農(nóng)院B6’品種授粉后10 d至90 d果實(shí)羅漢果苷含量測定顯示,30 d前幼果主要含苷ⅡE、苷Ⅲ低糖苷,30 d至50 d定形后果實(shí)苷ⅡE、苷Ⅲ、苷Ⅳ、苷Ⅴ共存,50d至成熟果實(shí)(90 d)主要含苷V高糖苷,表明羅漢果苷V可能是以苷ⅡE為前體,經(jīng)由苷Ⅲ、苷Ⅳ轉(zhuǎn)化而來的。2、不同品種、遮陰、CO2貯藏調(diào)控下羅漢果苷與糖含量變化動態(tài)測定顯示,無論高含量品種還是低含量品種,遮陰還是不遮陰條件下,空氣中還是CO2中貯藏,伴隨苷ⅡE、苷Ⅲ減少消失,甜苷V快速合成積累后,果實(shí)中葡萄糖含量均會出現(xiàn)明顯下降；無論高含量品種還是低含量品種,遮陰還是不遮陰條件下,授粉后30 d至成熟過程中果實(shí)中淀粉含量逐漸降低,葡萄糖含量均保持在約10%以上；高甜苷Ⅴ品種光合速率快、葡萄糖含量高,苷Ⅱ E、苷Ⅲ、苷Ⅴ含量也高；30 d后遮陰降低葉片光合速率,減少了可溶性糖、蔗糖含量,但葡萄糖和苷Ⅴ含量仍然增加；40 d果實(shí)CO2中貯藏抑制呼吸,減緩了葡萄糖含量下降,但苷Ⅴ含量仍然下降。這些表明苷V合成積累過程中會消耗較多葡萄糖,但是葡萄糖供應(yīng)充足,不是苷Ⅴ合成積累的主要限制因子。3、羅漢果及其他6種葫蘆科植物不同果實(shí)發(fā)育期、組織部位羅漢果醇含量測定顯示,羅漢果醇僅存在于羅漢果中,檢測表明30 d前幼果含量基本保持在約300.0 μg/g,30 d后迅速減少至幾乎消失,與苷Ⅱ E和苷Ⅲ合成積累規(guī)律相似,提示羅漢果醇、苷ⅡE和苷Ⅲ是甜苷Ⅴ合成積累的主要限制因子。4、在甜苷Ⅴ合成積累限制因子羅漢果醇、苷ⅡE和苷Ⅲ合成積累的幼果期,選擇0d3d、15d、30 d果實(shí)進(jìn)行基因表達(dá)轉(zhuǎn)錄組分析,總共獲得81940條unigene,其中57107條(69.69%)被功能注釋。31436條被比對到KEGG數(shù)據(jù)庫,其中2543條與次生代謝相關(guān),涉及21條次生代謝途徑。幾乎發(fā)現(xiàn)了萜類和甾體兩條次生代謝途徑所有已知關(guān)鍵酶基因。其中,萜類和甾體代謝途徑末端特異的氧化鯊烯環(huán)化酶、細(xì)胞色素P450單加氧酶和葡萄糖基轉(zhuǎn)移酶基因在不同發(fā)育期間表達(dá)存在差異。合成羅漢果苷共同前體葫蘆二烯醇的SgCS基因0d至15 d表達(dá)大幅上調(diào),15 d至30 d則表達(dá)大幅下調(diào)。合成甾體共同前體環(huán)爾喬醇的SgCAS基因3d至15d表達(dá)上調(diào)后,15d至30d保持較高水平表達(dá)。11個候選細(xì)胞色素P450單加氧酶基因與SgCS協(xié)同表達(dá),表達(dá)規(guī)律與羅漢果醇合成積累規(guī)律一致。6個候選葡萄糖基轉(zhuǎn)移酶基因表達(dá)規(guī)律與苷ⅡE合成積累規(guī)律一致。2個候選葡萄糖基轉(zhuǎn)移酶基因表達(dá)規(guī)律與苷Ⅲ合成積累規(guī)律一致。轉(zhuǎn)錄組測序分析獲得的這些豐富基因信息為分離克隆甜苷Ⅴ合成相關(guān)基因研究提供了一定的科學(xué)依據(jù)。5、采用RACE技術(shù),從5d和70 d果肉中克隆了SgUGT4、SgUGT6和SgUGT7三個葡萄糖基轉(zhuǎn)移酶基因。SgUGT4編碼蛋白含有植物次生代謝葡萄糖基轉(zhuǎn)移酶特征結(jié)構(gòu)域PSPG-box,與糖基化羅漢果醇的UGT73家族葡萄糖基轉(zhuǎn)移酶遺傳關(guān)系最近,主要在甜苷Ⅴ進(jìn)入快速合成積累的50 d左右的果實(shí)表達(dá),且在高甜苷Ⅴ含量品種中表達(dá)也相對較高,提示其有可能參與了甜苷V的生物合成。在大腸桿菌和畢赤酵母中重組表達(dá),獲得了SgUGT4、SgUGT6和SgUGT7可溶性重組蛋白,為進(jìn)一步通過體外活性篩選實(shí)驗(yàn)進(jìn)行蛋白功能和結(jié)構(gòu)分析奠定了基礎(chǔ)。
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[Abstract]:The low content of the Siraitia grosvenorii V in the fruit causes the production and application cost of the sweetener to be high and the development of the industry is limited. Improve that content of the sweet spot V in the fruit, and reduce the production and application cost of the fruit to be a hot spot. The traditional cultivation and breeding method has the technical difficulty in improving the content of the sweet spot V in the fruit. The momordica grosvenori has a common meta-momordica grosvenori alcohol, the difference is mainly that C-3, C-24 are connected with the number of glucose groups, and the function of C-7 and C-11 is different. Biosynthesis and genetic engineering breeding of Stevia V have become a new way to solve the cost problem. It is a prerequisite for the study of biological synthesis and genetic engineering breeding. In that present study, the metabolism of the momordica grosvenori in the fruit development period, the relationship between the sweetness and the substrate glucose in different control modes, the law of the metabolism of the momordica grosvenori alcohol during the development of the fruit and the distribution of the plant in the cucurbitaceae were studied. The analysis of the transcriptome and the cloning and expression of the glucosyltransferase gene were studied. The main results are as follows:1. The determination of the content of the momordica grosvenori from 10 days to 90 days after the pollination of the 6 'varieties of the primary agricultural institute shows that the young fruits of the 30 days are mainly contained in the fruits, 鈪, P鈪ow saccharin,30 days to 50 days, and the fruits of the fruit are coexistent after the shaping. The results showed that the momordica grosvenori was the precursor of the momordica grosvenori, and it was transformed from the momordica grosvenori to the sugar content under the control of different varieties, shade and CO2. In that condition of high content or low content, in the shade or under the condition of no shade, the air is still store in CO2, and the content of glucose in the fruit is decreased obviously with the decrease of the vitamin II E and the vitamin III and the rapid synthesis and accumulation of the stevia V. Under the condition of high content or low content, the content of starch in the fruit of 30 days after pollination and the content of the glucose decreased gradually, and the content of glucose remained above about 10%. The high photosynthetic rate of the high-sweet and high-sweet and high-sweet and high-sweet sorghum was high, and the content of glucose was high. The content of vitamin V was also high; after 30 days, the photosynthetic rate of the leaves was reduced, the content of soluble sugar and sucrose was reduced, but the content of glucose and vitamin V increased; in the 40-d fruit CO2, the respiration was inhibited, and the content of glucose decreased, but the content of vitamin V decreased. The results showed that more glucose was consumed in the process of synthesis and accumulation, but the supply of glucose was sufficient, and it was not the main limiting factor of the synthesis and accumulation of vitamin V.3. The determination of the content of the momordica grosvenori in the tissue was shown by the determination of the content of the momordica grosvenori in the different fruit development stages of the momordica grosvenori and other six cucurbitaceous plants. The fruit of the momordica grosvenori is only present in the momordica grosvenori, and the content of the young fruit before 30 days is basically kept at about 300.0. m The main limiting factors of the synthesis and accumulation of the sweet and the V. 鈪，

本文編號：2511151