薯蕷皂素合成基因富集、挖掘及其功能分析系統(tǒng)建立
發(fā)布時間：2018-07-24 10:39
【摘要】：薯蕷皂素是甾體激素類藥物的重要合成前體,以其為原料可以生產(chǎn)皮質(zhì)激素、性激素等300種以上的藥物,因此薯蕷皂素有"藥用黃金"之稱,然而目前其生物合成路徑還不清楚。葫蘆巴是薯蕷皂素源植物之一,可以其為實驗材料研究薯蕷皂素的生物合成途徑。本實驗以中國山西省葫蘆巴植物為實驗材料,采用不同濃度的茉莉酸甲酯(MeJA)誘導(dǎo)薯蕷皂素的合成,以富集薯蕷皂素合成相關(guān)基因,建立了經(jīng)過0.01%(v/v)MeJA處理和未經(jīng)過MeJA處理的葫蘆巴轉(zhuǎn)錄組文庫,通過比較轉(zhuǎn)錄組學(xué)的方法篩選可能參與薯蕷皂素合成的相關(guān)基因,并建立了葫蘆巴發(fā)根體系,研究了發(fā)根農(nóng)桿菌菌液濃度與超聲波輔助處理兩個因素對葫蘆巴發(fā)根轉(zhuǎn)化率的影響,為薯蕷皂素合成相關(guān)基因的功能鑒定提供分析平臺。主要得到以下結(jié)果:(1)本研究采用不同濃度MeJA(0%,0.005%,0.01%,0.02%,v/v)處理葫蘆巴幼苗,發(fā)現(xiàn)0.01%MeJA處理對葫蘆巴中薯蕷皂素的富集最為顯著,含量為13.38 mg/g,是對照(0%MeJA)的1.76倍。膽固醇和β-谷甾醇是參與薯蕷皂素合成兩個可能前體,通過GC-MS檢測0.01%MeJA處理幼苗后膽固醇和β-谷甾醇含量的變化,發(fā)現(xiàn)經(jīng)過0.01%MeJA處理的幼苗中膽固醇和β-谷甾醇含量分別為0.03 mg/g和3.04 mg/g,分別下降了 1.41倍和1.65倍,我們推測膽固醇和β-谷甾醇均可能是薯蕷皂素的生物合成前體。(2)基于0.01%(v/v)MeJA處理對葫蘆巴中薯蕷皂素的富集最為顯著,我們建立了經(jīng)過0.01%MeJA處理和未經(jīng)過MeJA處理的2個葫蘆巴轉(zhuǎn)錄組文庫,共獲得了 86,291,104個高質(zhì)量reads,拼接組裝了 69669條Unigenes,經(jīng)過功能注釋分析,其中有57579條Unigenes得到功能注釋,并對Unigene利用公共數(shù)據(jù)庫進(jìn)行了功能分類統(tǒng)計。篩選了 2,3-氧化鯊烯下游參與植物中膽固醇和β-谷甾醇合成的13個酶,共得到36個Unigene,挖掘到差異表達(dá)的CYP450酶基因14個,糖基轉(zhuǎn)移酶基因12個,甲基轉(zhuǎn)移酶基因11個,轉(zhuǎn)錄因子16個。這為薯蕷皂素合成途徑的解析提供了充足的分子基礎(chǔ)。(3)我們以中國山西省葫蘆巴植物為實驗材料,研究了發(fā)根農(nóng)桿菌菌液濃度與超聲波輔助處理對葫蘆巴發(fā)根轉(zhuǎn)化率的影響,為研究薯蕷皂素合成路徑提供一個功能分析平臺。結(jié)果發(fā)現(xiàn)隨著菌液濃度升高,發(fā)根數(shù)和發(fā)根轉(zhuǎn)化率均逐漸升高,中濃度菌液(OD600=2.2)與低濃度菌液(OD600=1.1)相比,發(fā)根數(shù)量和發(fā)根轉(zhuǎn)化率分別升高了 2.58倍和3.90倍,利用高濃度菌液(OD600=4.4)侵染獲得的發(fā)根數(shù)量和發(fā)根轉(zhuǎn)化率最高,分別是低濃度菌液的7.33倍和4.32倍。超聲波處理實驗中,超聲波(工作頻率40 KHz,超聲功率180 W)處理30 s時,較未超聲波處理對照組,發(fā)根數(shù)量及發(fā)根轉(zhuǎn)化率略有下降,而超聲波處理60s時,發(fā)根轉(zhuǎn)化率升高,為對照組的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.
【學(xué)位授予單位】：中國科學(xué)院武漢植物園
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：Q946
本文編號：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.
【學(xué)位授予單位】：中國科學(xué)院武漢植物園
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：Q946
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