薯蓣皂素合成基因富集、挖掘及其功能分析系统建立
发布时间：2018-07-24 10:39
【摘要】：薯蓣皂素是甾体激素类药物的重要合成前体,以其为原料可以生产皮质激素、性激素等300种以上的药物,因此薯蓣皂素有"药用黄金"之称,然而目前其生物合成路径还不清楚。葫芦巴是薯蓣皂素源植物之一,可以其为实验材料研究薯蓣皂素的生物合成途径。本实验以中国山西省葫芦巴植物为实验材料,采用不同浓度的茉莉酸甲酯(MeJA)诱导薯蓣皂素的合成,以富集薯蓣皂素合成相关基因,建立了经过0.01%(v/v)MeJA处理和未经过MeJA处理的葫芦巴转录组文库,通过比较转录组学的方法筛选可能参与薯蓣皂素合成的相关基因,并建立了葫芦巴发根体系,研究了发根农杆菌菌液浓度与超声波辅助处理两个因素对葫芦巴发根转化率的影响,为薯蓣皂素合成相关基因的功能鉴定提供分析平台。主要得到以下结果:(1)本研究采用不同浓度MeJA(0%,0.005%,0.01%,0.02%,v/v)处理葫芦巴幼苗,发现0.01%MeJA处理对葫芦巴中薯蓣皂素的富集最为显著,含量为13.38 mg/g,是对照(0%MeJA)的1.76倍。胆固醇和β-谷甾醇是参与薯蓣皂素合成两个可能前体,通过GC-MS检测0.01%MeJA处理幼苗后胆固醇和β-谷甾醇含量的变化,发现经过0.01%MeJA处理的幼苗中胆固醇和β-谷甾醇含量分别为0.03 mg/g和3.04 mg/g,分别下降了 1.41倍和1.65倍,我们推测胆固醇和β-谷甾醇均可能是薯蓣皂素的生物合成前体。(2)基于0.01%(v/v)MeJA处理对葫芦巴中薯蓣皂素的富集最为显著,我们建立了经过0.01%MeJA处理和未经过MeJA处理的2个葫芦巴转录组文库,共获得了 86,291,104个高质量reads,拼接组装了 69669条Unigenes,经过功能注释分析,其中有57579条Unigenes得到功能注释,并对Unigene利用公共数据库进行了功能分类统计。筛选了 2,3-氧化鲨烯下游参与植物中胆固醇和β-谷甾醇合成的13个酶,共得到36个Unigene,挖掘到差异表达的CYP450酶基因14个,糖基转移酶基因12个,甲基转移酶基因11个,转录因子16个。这为薯蓣皂素合成途径的解析提供了充足的分子基础。(3)我们以中国山西省葫芦巴植物为实验材料,研究了发根农杆菌菌液浓度与超声波辅助处理对葫芦巴发根转化率的影响,为研究薯蓣皂素合成路径提供一个功能分析平台。结果发现随着菌液浓度升高,发根数和发根转化率均逐渐升高,中浓度菌液(OD600=2.2)与低浓度菌液(OD600=1.1)相比,发根数量和发根转化率分别升高了 2.58倍和3.90倍,利用高浓度菌液(OD600=4.4)侵染获得的发根数量和发根转化率最高,分别是低浓度菌液的7.33倍和4.32倍。超声波处理实验中,超声波(工作频率40 KHz,超声功率180 W)处理30 s时,较未超声波处理对照组,发根数量及发根转化率略有下降,而超声波处理60s时,发根转化率升高,为对照组的2.48倍。
[Abstract]:Diosgenin is an important synthetic precursor of steroidal hormone drugs. It can be used as raw material to produce more than 300 kinds of drugs, such as corticosteroids and sex hormones, so diosgenin soap is known as "medicinal gold". However, the biosynthesis pathway is unclear. Cucurbit root is one of the diosgenin source plants, which can be used as experimental materials to study the biosynthesis of diosgenin. In order to enrich diosgenin biosynthesis, diosgenin synthesis was induced by different concentrations of methyl jasmonate (MeJA) in Cucurbita chinensis, Shanxi Province, China, in order to enrich the genes related to diosgenin biosynthesis. The transcriptome library of cucurbita root was established after 0. 01% (v / v) MeJA treatment and without MeJA treatment. The genes involved in diosgenin synthesis were screened by comparative transcriptome method, and the root formation system of Cucurbita chinensis was established. The effects of the concentration of Agrobacterium tumefaciens and ultrasonic assisted treatment on the root transformation rate of Cucurbita chinensis were studied in order to provide an analytical platform for the functional identification of diosgenin biosynthesis related genes. The main results were as follows: (1) in this study, the seedlings of Cucurbita root were treated with different concentrations of MeJA (0. 005) and 0. 02% (v / v). It was found that the concentration of diosgenin in Dioscorea zingiberensis by 0.01%MeJA treatment was 13.38 mg / g, which was 1.76 times of that of the control (0%MeJA). Cholesterol and 尾 -sitosterol were two possible precursors involved in diosgenin synthesis. The changes of cholesterol and 尾 -sitosterol in seedlings treated with 0.01%MeJA were detected by GC-MS. It was found that the contents of cholesterol and 尾 -sitosterol in seedlings treated with 0.01%MeJA were 0.03 mg/g and 3.04 mg / g, respectively, which decreased by 1.41 and 1.65 times, respectively. We speculate that both cholesterol and 尾 -sitosterol may be biosynthetic precursors of diosgenin. (2) the enrichment of diosgenin in Cucurbita chinensis was most significant based on 0.01% (v / v) MeJA treatment. In this paper, we have established two Hulubin transcriptional library, which are processed by 0.01%MeJA and not treated by MeJA. We have obtained 86291104 high-quality readss, and assembled 69669 Unigenes.The functional annotation analysis shows that 57579 Unigenes are functional annotations. The function classification and statistics of Unigene using public database are also given. Thirteen enzymes involved in the synthesis of cholesterol and 尾 -sitosterol in the lower reaches of squalene were screened. A total of 36 Unigeneic enzymes were obtained. Fourteen differentially expressed CYP450 genes, 12 glycosyltransferase genes and 11 methyltransferase genes were found. 16 transcription factors. This provides a sufficient molecular basis for the analysis of diosgenin synthesis pathway. (3) We studied the effects of the concentration of Agrobacterium rhizogenes and ultrasonic assisted treatment on the transformation rate of cucurbita root from Cucurbitum chinensis from Shanxi Province, China. It provides a functional analysis platform for studying the synthetic pathway of diosgenin. The results showed that the number of roots and the rate of root transformation increased gradually with the increase of the concentration of bacterial solution. The number of root and the transformation rate of root in medium concentration of bacteria (OD600=2.2) were 2.58 times and 3.90 times higher than those in low concentration of bacterial solution (OD600=1.1), respectively. The number of hair roots and the transformation rate of hair roots obtained by high concentration bacterial solution (OD600=4.4) were the highest, which were 7.33 times and 4.32 times of that of low concentration solution, respectively. In the experiment of ultrasonic treatment, the number of hair root and the transformation rate of hair root decreased slightly when the ultrasonic (working frequency 40 kHz, ultrasonic power 180 W) was treated for 30 s, but the rate of root transformation increased after 60 seconds of ultrasonic treatment. It was 2.48 times of the control group.
【学位授予单位】：中国科学院武汉植物园
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q946
本文编号：2141111
[Abstract]:Diosgenin is an important synthetic precursor of steroidal hormone drugs. It can be used as raw material to produce more than 300 kinds of drugs, such as corticosteroids and sex hormones, so diosgenin soap is known as "medicinal gold". However, the biosynthesis pathway is unclear. Cucurbit root is one of the diosgenin source plants, which can be used as experimental materials to study the biosynthesis of diosgenin. In order to enrich diosgenin biosynthesis, diosgenin synthesis was induced by different concentrations of methyl jasmonate (MeJA) in Cucurbita chinensis, Shanxi Province, China, in order to enrich the genes related to diosgenin biosynthesis. The transcriptome library of cucurbita root was established after 0. 01% (v / v) MeJA treatment and without MeJA treatment. The genes involved in diosgenin synthesis were screened by comparative transcriptome method, and the root formation system of Cucurbita chinensis was established. The effects of the concentration of Agrobacterium tumefaciens and ultrasonic assisted treatment on the root transformation rate of Cucurbita chinensis were studied in order to provide an analytical platform for the functional identification of diosgenin biosynthesis related genes. The main results were as follows: (1) in this study, the seedlings of Cucurbita root were treated with different concentrations of MeJA (0. 005) and 0. 02% (v / v). It was found that the concentration of diosgenin in Dioscorea zingiberensis by 0.01%MeJA treatment was 13.38 mg / g, which was 1.76 times of that of the control (0%MeJA). Cholesterol and 尾 -sitosterol were two possible precursors involved in diosgenin synthesis. The changes of cholesterol and 尾 -sitosterol in seedlings treated with 0.01%MeJA were detected by GC-MS. It was found that the contents of cholesterol and 尾 -sitosterol in seedlings treated with 0.01%MeJA were 0.03 mg/g and 3.04 mg / g, respectively, which decreased by 1.41 and 1.65 times, respectively. We speculate that both cholesterol and 尾 -sitosterol may be biosynthetic precursors of diosgenin. (2) the enrichment of diosgenin in Cucurbita chinensis was most significant based on 0.01% (v / v) MeJA treatment. In this paper, we have established two Hulubin transcriptional library, which are processed by 0.01%MeJA and not treated by MeJA. We have obtained 86291104 high-quality readss, and assembled 69669 Unigenes.The functional annotation analysis shows that 57579 Unigenes are functional annotations. The function classification and statistics of Unigene using public database are also given. Thirteen enzymes involved in the synthesis of cholesterol and 尾 -sitosterol in the lower reaches of squalene were screened. A total of 36 Unigeneic enzymes were obtained. Fourteen differentially expressed CYP450 genes, 12 glycosyltransferase genes and 11 methyltransferase genes were found. 16 transcription factors. This provides a sufficient molecular basis for the analysis of diosgenin synthesis pathway. (3) We studied the effects of the concentration of Agrobacterium rhizogenes and ultrasonic assisted treatment on the transformation rate of cucurbita root from Cucurbitum chinensis from Shanxi Province, China. It provides a functional analysis platform for studying the synthetic pathway of diosgenin. The results showed that the number of roots and the rate of root transformation increased gradually with the increase of the concentration of bacterial solution. The number of root and the transformation rate of root in medium concentration of bacteria (OD600=2.2) were 2.58 times and 3.90 times higher than those in low concentration of bacterial solution (OD600=1.1), respectively. The number of hair roots and the transformation rate of hair roots obtained by high concentration bacterial solution (OD600=4.4) were the highest, which were 7.33 times and 4.32 times of that of low concentration solution, respectively. In the experiment of ultrasonic treatment, the number of hair root and the transformation rate of hair root decreased slightly when the ultrasonic (working frequency 40 kHz, ultrasonic power 180 W) was treated for 30 s, but the rate of root transformation increased after 60 seconds of ultrasonic treatment. It was 2.48 times of the control group.
【学位授予单位】：中国科学院武汉植物园
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q946
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