罗汉果苷代谢酶基因转录组研究及葡萄糖基转移酶基因克隆与表达
发布时间：2019-07-06 16:12
【摘要】：罗汉果甜苷Ⅴ在果实中含量低,致使其甜味剂生产与应用成本一直居高不下,限制了产业的发展。提高果实中甜苷Ⅴ含量,降低其生产与应用成本成为研究热点。传统栽培与育种方法提高果实中甜苷Ⅴ含量存在技术难点。罗汉果苷具有共同的苷元罗汉果醇,差异主要是C-3、C-24位连接葡萄糖基数目、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种成为解决其成本问题的新途径。摸清甜苷Ⅴ体内合代谢规律和分离鉴定相关关键酶基因,是有效进行其生物合成研究与基因工程育种的先决条件。因此,本研究对果实发育期罗汉果苷代谢规律,不同调控方式下甜苷V与其底物葡萄糖积累关系,果实发育期罗汉果醇代谢规律及其在葫芦科植物的分布,甜苷V合成积累主要限制因子罗汉果醇与苷ⅡE、苷Ⅲ代谢酶基因表达转录组分析,以及葡萄糖基转移酶基因克隆与表达进行了研究。获得的主要结果如下：1、‘农院B6’品种授粉后10 d至90 d果实罗汉果苷含量测定显示,30 d前幼果主要含苷ⅡE、苷Ⅲ低糖苷,30 d至50 d定形后果实苷ⅡE、苷Ⅲ、苷Ⅳ、苷Ⅴ共存,50d至成熟果实(90 d)主要含苷V高糖苷,表明罗汉果苷V可能是以苷ⅡE为前体,经由苷Ⅲ、苷Ⅳ转化而来的。2、不同品种、遮阴、CO2贮藏调控下罗汉果苷与糖含量变化动态测定显示,无论高含量品种还是低含量品种,遮阴还是不遮阴条件下,空气中还是CO2中贮藏,伴随苷ⅡE、苷Ⅲ减少消失,甜苷V快速合成积累后,果实中葡萄糖含量均会出现明显下降；无论高含量品种还是低含量品种,遮阴还是不遮阴条件下,授粉后30 d至成熟过程中果实中淀粉含量逐渐降低,葡萄糖含量均保持在约10%以上；高甜苷Ⅴ品种光合速率快、葡萄糖含量高,苷Ⅱ E、苷Ⅲ、苷Ⅴ含量也高；30 d后遮阴降低叶片光合速率,减少了可溶性糖、蔗糖含量,但葡萄糖和苷Ⅴ含量仍然增加；40 d果实CO2中贮藏抑制呼吸,减缓了葡萄糖含量下降,但苷Ⅴ含量仍然下降。这些表明苷V合成积累过程中会消耗较多葡萄糖,但是葡萄糖供应充足,不是苷Ⅴ合成积累的主要限制因子。3、罗汉果及其他6种葫芦科植物不同果实发育期、组织部位罗汉果醇含量测定显示,罗汉果醇仅存在于罗汉果中,检测表明30 d前幼果含量基本保持在约300.0 μg/g,30 d后迅速减少至几乎消失,与苷Ⅱ E和苷Ⅲ合成积累规律相似,提示罗汉果醇、苷ⅡE和苷Ⅲ是甜苷Ⅴ合成积累的主要限制因子。4、在甜苷Ⅴ合成积累限制因子罗汉果醇、苷ⅡE和苷Ⅲ合成积累的幼果期,选择0d3d、15d、30 d果实进行基因表达转录组分析,总共获得81940条unigene,其中57107条(69.69%)被功能注释。31436条被比对到KEGG数据库,其中2543条与次生代谢相关,涉及21条次生代谢途径。几乎发现了萜类和甾体两条次生代谢途径所有已知关键酶基因。其中,萜类和甾体代谢途径末端特异的氧化鲨烯环化酶、细胞色素P450单加氧酶和葡萄糖基转移酶基因在不同发育期间表达存在差异。合成罗汉果苷共同前体葫芦二烯醇的SgCS基因0d至15 d表达大幅上调,15 d至30 d则表达大幅下调。合成甾体共同前体环尔乔醇的SgCAS基因3d至15d表达上调后,15d至30d保持较高水平表达。11个候选细胞色素P450单加氧酶基因与SgCS协同表达,表达规律与罗汉果醇合成积累规律一致。6个候选葡萄糖基转移酶基因表达规律与苷ⅡE合成积累规律一致。2个候选葡萄糖基转移酶基因表达规律与苷Ⅲ合成积累规律一致。转录组测序分析获得的这些丰富基因信息为分离克隆甜苷Ⅴ合成相关基因研究提供了一定的科学依据。5、采用RACE技术,从5d和70 d果肉中克隆了SgUGT4、SgUGT6和SgUGT7三个葡萄糖基转移酶基因。SgUGT4编码蛋白含有植物次生代谢葡萄糖基转移酶特征结构域PSPG-box,与糖基化罗汉果醇的UGT73家族葡萄糖基转移酶遗传关系最近,主要在甜苷Ⅴ进入快速合成积累的50 d左右的果实表达,且在高甜苷Ⅴ含量品种中表达也相对较高,提示其有可能参与了甜苷V的生物合成。在大肠杆菌和毕赤酵母中重组表达,获得了SgUGT4、SgUGT6和SgUGT7可溶性重组蛋白,为进一步通过体外活性筛选实验进行蛋白功能和结构分析奠定了基础。
文内图片：
图片说明：图２－１转录组测序流程逡逑Ｆｉｇ．邋２－１邋Ｅｘｐｅｒｉｍｅｎｔ邋ｐｉｐｅｌｉｎｅ邋ｏｆ邋ｔｒａｎｓｃｒｉｐｔｏｍｅ逡逑
[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，
本文编号：2511151
文内图片：
图片说明：图２－１转录组测序流程逡逑Ｆｉｇ．邋２－１邋Ｅｘｐｅｒｉｍｅｎｔ邋ｐｉｐｅｌｉｎｅ邋ｏｆ邋ｔｒａｎｓｃｒｉｐｔｏｍｅ逡逑
[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，
本文编号：2511151
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