罗汉果转录组、表达谱的高通量测序及甜苷V生物合成关键酶的克隆
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摘要
罗汉果Siraitia grosvenorii (Swingle) C. Jeffrey是我国特有珍贵药用和甜料植物,为葫芦科(Cucurbitaceae)罗汉果属(Siraitia)多年生藤本植物,具有止咳祛痰、凉血舒胃、润肠通便等作用。其主要成分甜苷V是世界上最强的非糖甜味物质之一,为蔗糖甜度的300-400倍,是一种低热量、纯天然的甜味剂及理想的保健品,此外还有抗氧化、免疫调节、抗癌、降血糖等作用。甜苷V目前还不能化学合成,主要依靠提取获得。罗汉果对生境要求苛刻,只适宜在广西生长,且栽培中不能连作,果实中甜苷V含量低,无法满足市场需求,因此亟待探索提高甜苷V含量的新途径、新方法。本论文利用高通量测序技术对罗汉果果实的转录组和表达谱进行了研究,从分子水平阐释罗汉果甜苷V生物合成的分子机理,大规模寻找罗汉果功能基因,并在此基础上克隆了甜苷V生物合成相关基因的全长。主要研究结果如下：
1.罗汉果授粉后30天到50天之间主要以苦味成分苷ⅡE为主,70天时含量急剧下降,检测不到苷ⅡE。苷Ⅲ只在50天果实中出现。苷V含量在50天之前含量低,在50天至70天之间急剧增长了25.9倍,至85天增长到最高含量。葡萄糖含量在30天和50天变化不明显,而70天下降了3.24倍。葡萄糖含量与苷ⅡE、苷V含量的变化存在一定的消长关系。
2.应用Solexa高通量测序技术对罗汉果果实进行转录组测序,得到3.41G的原始数据,通过拼接得到43891条Unigenes。与次生代谢相关的unigenes有739条,其中涉及萜类骨架合成的Unigenes有60条。利用转录组数据发现了罗汉果甜苷V生物合成通路中几乎所有的基因,涉及到20种基因。另外还得到了80条P450基因Unigenes、72条glycosyltransferase基因的Unigenes和90条glucosyltransferase基因的Unigenes。这些Unigenes是今后对罗汉果三萜皂苷生物合成进行全面调控的重要基因资源。
3.在3d、50d、70d表达谱中均得到了300万条以上Clean tags,去冗余后得到85000条以上不同种类的Tags。通过分析发现占mRNA总量近73-76%的是种类不到5.1-5.9%的少数mRNA,而占种类56-59%的mRNA全部加起来不到mRNA,总量的3.9-4.1%。对差异基因进行统计发现在3d/50d中,有2119个基因上调,2642个基因下调；3d/70d中,2150个基因上调,2797个基因下调；50d/70d中,1078个基因上调,1422个基因下调。
4.利用表达谱对罗汉果甜苷V生物合成途径涉及的20种基因在各个时期的表达水平进行分析发现除AACT、MVD、CMK、HDS、SQS基因表达量在3个时期中未见明显变化外,其余16个基因都有各自的变化规律。授粉后3天到50天之间,DXS、DXR、MCS、IDS下调,其余12种基因均上调。50天到70天之间,HMGR、MCS、IDS、IPP, GPS、FPS、CAS下调外,其余9种个基因均上调。从3天幼果到70天接近成熟果实,各个基因表达的总体趋势是：除DXR、MCS、IDS、IPP-I表现下调外,HMGS、HMGR、MK, PMK、MCT、IPI-Ⅱ、GPS、FPS、SQS、CAS和CS基因均上调。其中SQE和CS变化最大,70天比3d的表达量分别提高了8.89倍和9.69倍。
5.通过对80条Cytochrome P450Unigenes和90条glucosyltransferase Unigenes基因表达水平变化规律进行研究,推测Unigene23541、Unigene24189、Unigene26598和Unigene43109等4条候选的P450基因和Unigene4016、Unigene8672、 Unigene13633、Unigene15400、Unigene35056和Unigene38974等6条候选的UDPG基因可能参与甜苷V骨架生物合成。
5.利用RACE技术首次克隆了14条罗汉果甜苷V生物合成相关的基因全长和4条5’或3’片段。其中MVA途径有6条：AACT、HMGS、HMGR、MK、PMK、MVD；共同代谢途径中IPI、GPS、FPS、SQS、CAS、CS等6条,以及SQE5’端；MEP途径中MCT和IDS基因的全长、DXS和HDS的5’端以及MCS的3’端。此外还得到了2条糖基转移酶的全长。并对所有全长基因进行了序列分析,它们均为首次从罗汉果中报道的全长或部分片段序列。
本论文首次利用Solexa高通量测序技术对罗汉果果实的转录组及不同时期的表达谱进行分析,得到了大量的罗汉果基因信息,并克隆了涉及罗汉果甜苷V生物合成相关的基因全长。这为罗汉果功能基因组学以及甜苷V生物合成分子机理研究打下了坚实的基础。
Summary
The fruit of Siraitia grosvenorii, which belongs to the family Cucurbitaceae, has long been used in traditional Chinese medicine as a pulmonary demulcent and emollient for the treatment of dry cough, sore throat, dire thirst and constipation. The main ingredient is mogroside V, it is one of the most sweet material in non-sugar sweet in the world, as300-400times the sweetness as that of sucrose, and it is a low-calorie, natural sweetener and an ideal health care products, in addition to have antioxidant, immunomodulatory, anticancer, lowering blood sugar effect. In order to reveal the mechanism of mogroside V biosynthesis from molecular level, Look for function genes in large-scale and full-length cDNA cloning, a transcriptom and three expression profiles with the high-throughput sequencing technology in Siraitia grosvenorii are studied. The major findings are as follows:
1. The mainly content is mogroside Ⅱ E between30d and50d. It sharp declined on70d, no mogroside ⅡE is detected. Mogroside Ⅲ only appears in50d. Mogroside V is very low before50d, but it is dramatically increased25.9times from50d to70d, and it comes up to the highest levels till85d. The content of glucose did not change significantly in30d and50d, but decreased3.24times at70d. There shall be a certain degree of fluctuation relations between the content of glucose and that of mogroside ⅡE, and mogroside V.
2. With Solexa high-throughput sequencing technology for transcriptome studying,3.41G of the raw data is obtained, and also obtained43,891Unigenes. There are739Unigenes, of which60Unigenes involve in the synthesis of terpenoid skeleton, associated with secondary metabolism. With the usage of transcriptome data, it found that almost all genes are related to the mogrosides V biosynthesis pathway. Also discovered80P450s,72glycosyltransferases, and90glucosyltransferases. These Unigenes are important genetic resources in the regulation for mogrosides biosynthesis in the future.
3. In the3d,50d,70d expression profiles obtained more than3million clean tags, after removing the redundancy, more than85,000different types of tags were obtained. The analysis shows that mRNA, which is less than5.1-5.9%of the types of mRNA, accounts for nearly73-76%of the total ammount, but all which accounts for56-59%of types, is less than3.9-4.1%of total amount mRNA. Statistics on the differential expression genes found that, in the3d/50d genes, there are2119genes up-regulated,2642genes were down-regulated; in the3d/70d, the2150genes gone up-regulated,2797genes were down-regulated; in the50d/70d, the1078genes up-regulated,1422Genes down-regulated.
4. Analysis on21genes expression levels involved in mogroside V skeleton biosynthesis through three expression profiles, it finds that there are no significant change on AACT, MVD, CMK, HDS, SQS gene expression in the three period, the remaining16genes are all have their own variation. In3d and50d, DXS, DXR, MCS, IDS come down, the remaining12genes go up-regulated. In50d and70d, except HMGR, MCS, IDS, IPP, GPS, FPS, CAS that come down, the other9genes go up. From3d to70d, the overall trend of each gene is:in addition to DXR, MCS, IDS, IPP-I come down in the expression, HMGS, HMGR, MK, PMK, MCT, IPI-II, GPS, FPS, SQS, CAS, and CS genes are up-regulated. Among these, SQE and CS genes changes mostly, the expression of70d increased8.89times and9.69times respectively than the3d.
5. Studying on the gene expression of80cytochrome P450and90glucosyltransferases, it suggests that the four candidate P450genes:Unigene23541, Unigene24189, Unigene26598and Unigene43109and6candidate UDPG genes: Unigene4016, Unigene8672, Unigene13633, Unigene15400, Unigene35056and Unigene38974may be involved in mogroside V skeleton biosynthesis.
6. Using RACE,12full-length cDNAs related to mogroside V biosynthesis were cloned for the first time, of which there are6in MVA pathway:AACT, HMGS, HMGR, MK, PMK, MVD, and6in common pathway:IPI, GPS, FPS, SQS, CAS, CS, etc, and the5'end of SQE. Also obtained MEP pathway of MCT and the IDS gene in full-length, the5'end of DXS and HDS, and3'end of MCS. Two full-length glycosyltransferase are obtained in another experiment.These full-lenth and partial genes were all first reported in Siraitia grosvenorii.
By applying high-throughput Solexa sequencing technology on studying transcriptom and expression profiles in Siraitia grosvenorii firstly, a large number of genes information were abtained, and many genes related to secondary metabolite were cloned. This is a solid foundation for the study of Siraitia grosvenorii functional genomics and mogrosides V molecular mechanism of biosynthesis.
1.罗汉果授粉后30天到50天之间主要以苦味成分苷ⅡE为主,70天时含量急剧下降,检测不到苷ⅡE。苷Ⅲ只在50天果实中出现。苷V含量在50天之前含量低,在50天至70天之间急剧增长了25.9倍,至85天增长到最高含量。葡萄糖含量在30天和50天变化不明显,而70天下降了3.24倍。葡萄糖含量与苷ⅡE、苷V含量的变化存在一定的消长关系。
2.应用Solexa高通量测序技术对罗汉果果实进行转录组测序,得到3.41G的原始数据,通过拼接得到43891条Unigenes。与次生代谢相关的unigenes有739条,其中涉及萜类骨架合成的Unigenes有60条。利用转录组数据发现了罗汉果甜苷V生物合成通路中几乎所有的基因,涉及到20种基因。另外还得到了80条P450基因Unigenes、72条glycosyltransferase基因的Unigenes和90条glucosyltransferase基因的Unigenes。这些Unigenes是今后对罗汉果三萜皂苷生物合成进行全面调控的重要基因资源。
3.在3d、50d、70d表达谱中均得到了300万条以上Clean tags,去冗余后得到85000条以上不同种类的Tags。通过分析发现占mRNA总量近73-76%的是种类不到5.1-5.9%的少数mRNA,而占种类56-59%的mRNA全部加起来不到mRNA,总量的3.9-4.1%。对差异基因进行统计发现在3d/50d中,有2119个基因上调,2642个基因下调；3d/70d中,2150个基因上调,2797个基因下调；50d/70d中,1078个基因上调,1422个基因下调。
4.利用表达谱对罗汉果甜苷V生物合成途径涉及的20种基因在各个时期的表达水平进行分析发现除AACT、MVD、CMK、HDS、SQS基因表达量在3个时期中未见明显变化外,其余16个基因都有各自的变化规律。授粉后3天到50天之间,DXS、DXR、MCS、IDS下调,其余12种基因均上调。50天到70天之间,HMGR、MCS、IDS、IPP, GPS、FPS、CAS下调外,其余9种个基因均上调。从3天幼果到70天接近成熟果实,各个基因表达的总体趋势是：除DXR、MCS、IDS、IPP-I表现下调外,HMGS、HMGR、MK, PMK、MCT、IPI-Ⅱ、GPS、FPS、SQS、CAS和CS基因均上调。其中SQE和CS变化最大,70天比3d的表达量分别提高了8.89倍和9.69倍。
5.通过对80条Cytochrome P450Unigenes和90条glucosyltransferase Unigenes基因表达水平变化规律进行研究,推测Unigene23541、Unigene24189、Unigene26598和Unigene43109等4条候选的P450基因和Unigene4016、Unigene8672、 Unigene13633、Unigene15400、Unigene35056和Unigene38974等6条候选的UDPG基因可能参与甜苷V骨架生物合成。
5.利用RACE技术首次克隆了14条罗汉果甜苷V生物合成相关的基因全长和4条5’或3’片段。其中MVA途径有6条：AACT、HMGS、HMGR、MK、PMK、MVD；共同代谢途径中IPI、GPS、FPS、SQS、CAS、CS等6条,以及SQE5’端；MEP途径中MCT和IDS基因的全长、DXS和HDS的5’端以及MCS的3’端。此外还得到了2条糖基转移酶的全长。并对所有全长基因进行了序列分析,它们均为首次从罗汉果中报道的全长或部分片段序列。
本论文首次利用Solexa高通量测序技术对罗汉果果实的转录组及不同时期的表达谱进行分析,得到了大量的罗汉果基因信息,并克隆了涉及罗汉果甜苷V生物合成相关的基因全长。这为罗汉果功能基因组学以及甜苷V生物合成分子机理研究打下了坚实的基础。
Summary
The fruit of Siraitia grosvenorii, which belongs to the family Cucurbitaceae, has long been used in traditional Chinese medicine as a pulmonary demulcent and emollient for the treatment of dry cough, sore throat, dire thirst and constipation. The main ingredient is mogroside V, it is one of the most sweet material in non-sugar sweet in the world, as300-400times the sweetness as that of sucrose, and it is a low-calorie, natural sweetener and an ideal health care products, in addition to have antioxidant, immunomodulatory, anticancer, lowering blood sugar effect. In order to reveal the mechanism of mogroside V biosynthesis from molecular level, Look for function genes in large-scale and full-length cDNA cloning, a transcriptom and three expression profiles with the high-throughput sequencing technology in Siraitia grosvenorii are studied. The major findings are as follows:
1. The mainly content is mogroside Ⅱ E between30d and50d. It sharp declined on70d, no mogroside ⅡE is detected. Mogroside Ⅲ only appears in50d. Mogroside V is very low before50d, but it is dramatically increased25.9times from50d to70d, and it comes up to the highest levels till85d. The content of glucose did not change significantly in30d and50d, but decreased3.24times at70d. There shall be a certain degree of fluctuation relations between the content of glucose and that of mogroside ⅡE, and mogroside V.
2. With Solexa high-throughput sequencing technology for transcriptome studying,3.41G of the raw data is obtained, and also obtained43,891Unigenes. There are739Unigenes, of which60Unigenes involve in the synthesis of terpenoid skeleton, associated with secondary metabolism. With the usage of transcriptome data, it found that almost all genes are related to the mogrosides V biosynthesis pathway. Also discovered80P450s,72glycosyltransferases, and90glucosyltransferases. These Unigenes are important genetic resources in the regulation for mogrosides biosynthesis in the future.
3. In the3d,50d,70d expression profiles obtained more than3million clean tags, after removing the redundancy, more than85,000different types of tags were obtained. The analysis shows that mRNA, which is less than5.1-5.9%of the types of mRNA, accounts for nearly73-76%of the total ammount, but all which accounts for56-59%of types, is less than3.9-4.1%of total amount mRNA. Statistics on the differential expression genes found that, in the3d/50d genes, there are2119genes up-regulated,2642genes were down-regulated; in the3d/70d, the2150genes gone up-regulated,2797genes were down-regulated; in the50d/70d, the1078genes up-regulated,1422Genes down-regulated.
4. Analysis on21genes expression levels involved in mogroside V skeleton biosynthesis through three expression profiles, it finds that there are no significant change on AACT, MVD, CMK, HDS, SQS gene expression in the three period, the remaining16genes are all have their own variation. In3d and50d, DXS, DXR, MCS, IDS come down, the remaining12genes go up-regulated. In50d and70d, except HMGR, MCS, IDS, IPP, GPS, FPS, CAS that come down, the other9genes go up. From3d to70d, the overall trend of each gene is:in addition to DXR, MCS, IDS, IPP-I come down in the expression, HMGS, HMGR, MK, PMK, MCT, IPI-II, GPS, FPS, SQS, CAS, and CS genes are up-regulated. Among these, SQE and CS genes changes mostly, the expression of70d increased8.89times and9.69times respectively than the3d.
5. Studying on the gene expression of80cytochrome P450and90glucosyltransferases, it suggests that the four candidate P450genes:Unigene23541, Unigene24189, Unigene26598and Unigene43109and6candidate UDPG genes: Unigene4016, Unigene8672, Unigene13633, Unigene15400, Unigene35056and Unigene38974may be involved in mogroside V skeleton biosynthesis.
6. Using RACE,12full-length cDNAs related to mogroside V biosynthesis were cloned for the first time, of which there are6in MVA pathway:AACT, HMGS, HMGR, MK, PMK, MVD, and6in common pathway:IPI, GPS, FPS, SQS, CAS, CS, etc, and the5'end of SQE. Also obtained MEP pathway of MCT and the IDS gene in full-length, the5'end of DXS and HDS, and3'end of MCS. Two full-length glycosyltransferase are obtained in another experiment.These full-lenth and partial genes were all first reported in Siraitia grosvenorii.
By applying high-throughput Solexa sequencing technology on studying transcriptom and expression profiles in Siraitia grosvenorii firstly, a large number of genes information were abtained, and many genes related to secondary metabolite were cloned. This is a solid foundation for the study of Siraitia grosvenorii functional genomics and mogrosides V molecular mechanism of biosynthesis.
引文
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