Transcriptome Sequencing and Flavonoid Biosynthesis-related Genes of Scutellaria barbata D. Don
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摘要: 半枝莲为我国传统中药，具有重要的经济价值，但分子基础研究滞后，在一定程度上限制了其资源利用与开发。本研究利用Illumina HiSeq X Ten技术平台对半枝莲进行了转录组测序，通过de novo组装，获得125 823个unigene，N50长度826 bp、平均长度586 bp，并有74 185个unigene得到注释。半枝莲地上部分对地下部分差异表达unigene有14 832个，其中上调表达6 941个，下调表达7 891个；推测差异unigene多集中于植物-病原菌互作、淀粉和蔗糖代谢以及苯丙素生物合成等代谢途径。利用KEGG数据库挖掘出89个半枝莲黄酮类生物合成途径相关unigene，其中差异表达unigene 36个。半枝莲地上部分与地下部分差异基因分析中，黄酮类化合物合成途径的重要酶都有明显的下调表达，推测半枝莲的黄酮类化合物合成器官是地上部分，其主要的药用部位是茎叶。黄酮类的关键酶研究结果为半枝莲分子生物学研究奠定基础，为半枝莲药用活性成分的生物合成调控、品质改良、品种选育等提供参考依据。Abstract: As a traditional Chinese medicine, Scutellaria barbata D. Don has an important economic value. However, little molecular-based study was reported that limits the utilization and development of the resource. In this study, Illumina HiSeq X Ten was used to sequence the transcriptomes of S. barbata to obtain 125 823 unigenes assembled by the de novo DNA synthesis. The average length of N50 was 826 bp averaging 586 bp with 74 185 unigenes annotated. There were 14 832 differentially expressed unigenes in the above- and under-ground parts of the plants, of which 6 941 were up-regulated and 7 891 down-regulated. According to the KEGG database, 89 unigenes related to the flavonoid biosynthesis pathway. Among them 36 were differentially expressed. By comparing the genes in the above- and the under-ground parts, it was found the important enzymes associated with the flavonoid synthesis to be significantly down-regulated in the parts grown below the ground level. In other words, the stems and leaves were the major parts on a plant that contained the medicinally active components.
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图 2 半枝莲unigene在GO二级水平上的功能分类
Figure 2. Functional classification of S. barbata unigenes at GO level 2
图 3 半枝莲unigene在KOG的功能分类
Figure 3. Functional classification of S. barbata unigenes in KOG
图 5 半枝莲地上部分与地下部分差异unigene的GO显著性富集分析
注：图中列出的为FDR值正向排序前50个，图 6同。
Figure 5. GO significant enrichment analysis of unigenes in above- and under-ground parts of S.barbata
图 6 半枝莲地上部分与地下部分差异unigene的KEGG显著性富集分析
Figure 6. KEGG significant enrichment analysis of unigenes in above- and under-ground parts of S.barbata
表 1 半枝莲unigene在各数据库注释比例
Table 1. Statistics of annotated proportion of S. barbata unigenes cited in various databases
注释数据库类型 注释unigenes数量 占比/% 保守结构域数据库(CDD) 59 452 47.25 欧洲分子生物学实验室蛋白质数据库(TrEMBL) 58 383 46.40 基因本体数据库(GO) 56 712 45.07 真核生物蛋白质同源簇数据库(KOG) 47 506 37.76 美国国立生物技术信息中心核苷酸序列数据库(NT) 25 380 20.17 以上数据库均注释成功 12 004 9.54 美国国立生物技术信息中心非冗余蛋白库(NR) 59 265 47.10 瑞士蛋白质序列及注解数据库(SwissProt) 57 240 45.49 蛋白质家族数据库(PFAM) 51 839 41.20 京都基因与基因组百科全书(KEGG) 30 883 24.54 以上数据库至少一个注释成功 74 185 58.96 
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表 2 半枝莲unigene在NR数据库中匹配的物种分布
Table 2. Species distribution of Scutellaria barbata D.Don unigene in NR database
物种 匹配unigene数量 占比/% 芝麻 16091 27.15 大麦 3970 6.70 朝鲜蓟 1810 3.05 旋蒴苣苔 1203 2.03 蓝藻 1111 1.87 白梨 773 1.30 盘藻 665 1.12 团藻 643 1.08 微胞藻 600 1.01 蓖麻 466 0.79 其他 17930 30.25 猴面花 5398 9.11 软克里藻 2377 4.01 胶球藻 1221 2.06 小立碗藓 1120 1.89 玉米 938 1.58 江南卷柏 692 1.17 衣藻 662 1.12 小球藻 637 1.07 地钱 508 0.86 葡萄 450 0.76
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表 3 半枝莲黄酮类生物合成途径相关unigene
Table 3. unigenes in S.barbata related to flavonoids biosynthesis pathway
合成酶 unigene KO编号 unigene地址 unigene长度/bp 地上部分FPKM值 地下部分FPKM值 P 表达模式 柚皮素3′-双加氧酶 K00475 c25376_g1 1278 7.59 0.67 1.98E-31 下调 肉桂酸-4′-羟化酶 K00487 c25043_g1 456 3.42 3.07 8.58E-01 - K00487 c25043_g2 1281 3.66 2.63 7.12E-02 - K00487 c25401_g1 814 94.67 24.04 4.93E-108 下调 K00487 c25401_g2 1131 95.08 25.35 9.04E-162 下调 K00487 c63011_g1 1739 0.11 4.73 0 上调 K00487 c65486_g1 362 1.92 0 5.26E-02 - K00487 c70975_g1 452 1.34 0.24 1.04E-01 - K00487 c78549_g1 1803 122.69 113.93 3.53E-03 - K00487 c80896_g1 263 - - - - K00487 c86218_g1 526 0.98 0 2.54E-02 - 咖啡酰辅酶A-O-甲基转移酶 K00588 c123541_g1 257 3.59 0 2.25E-01 - K00588 c18444_g2 992 51.63 121.47 0 上调 K00588 c212_g1 463 1.53 0.91 4.75E-01 - K00588 c26754_g2 1054 5.69 54.32 0 上调 K00588 c26754_g3 788 6.94 95.11 0 上调 K00588 c30409_g1 2011 3.76 9.14 9.81E-13 上调 K00588 c40919_g1 375 0 1.91 3.82E-02 - K00588 c4595_g1 440 21.42 61.65 0 上调 K00588 c50713_g1 249 4.21 0 2.25E-01 - K00588 c81350_g1 394 1.13 1.67 5.77E-01 - K00588 c9229_g1 206 - - - - 查尔酮合成酶 K00660 c34103_g1 1793 0 2.43 0 上调 K00660 c63409_g1 1510 178.46 47.52 0 下调 无色花色素双加氧酶 K05277 c24664_g1 1242 1.98 0 1.96E-12 下调 K05277 c49906_g1 1452 29.4 5.41 7.47E-95 下调 类黄酮3′-单加氧酶 K05280 c100531_g1 218 9.01 0 2.25E-01 - K05280 c113021_g1 296 2.9 0 1.09E-01 - K05280 c124721_g1 259 5.19 0 1.09E-01 - K05280 c19717_g1 241 - - - - K05280 c23751_g1 1742 53.93 4.09 0 下调 K05280 c3869_g1 508 1.46 0 5.92E-03 - K05280 c4214_g1 259 1.73 1.46 9.49E-01 - K05280 c51219_g1 442 0.57 0 2.25E-01 - K05280 c51230_g1 462 0.77 0 1.09E-01 - K05280 c55685_g1 261 3.34 0 2.25E-01 - K05280 c63894_g1 399 1.09 0 1.09E-01 - K05280 c64689_g1 316 2.26 0 1.09E-01 - K05280 c76542_g1 212 10.84 0 2.25E-01 - K05280 c7769_g1 350 - - - - K05280 c80303_g1 402 3.57 0.32 4.46E-03 - K05280 c87571_g1 227 0 5.79 2.76E-01 - K05280 c95490_g1 1692 48.86 0.68 0 下调 聚酮还原酶 K08243 c57733_g1 306 2.55 0 1.09E-01 - 花青素还原酶 K08695 c89413_g1 398 1.1 0.33 3.34E-01 - 香豆酰奎宁酸-3′-单加氧酶 K09754 c100091_g1 441 0.85 0 1.09E-01 - K09754 c23424_g1 1739 0.04 1.85 1.78E-15 上调 K09754 c84184_g1 556 0.88 0 2.54E-02 - K09754 c94965_g1 1823 30.76 40.49 5.95E-11 - K09754 c97716_g1 1834 43.23 57.84 0 - 类黄酮3-O-葡萄糖基转移酶 K10757 c113626_g1 1569 11.58 1.98 4.84E-44 下调 奎宁酸香豆酰转移酶 K13065 c102484_g1 330 14.23 0 1.08E-07 下调 K13065 c110857_g1 767 2.79 0.28 1.94E-06 下调 K13065 c118457_g1 528 1.16 0 1.23E-02 - K13065 c22053_g1 1684 8.73 16.83 2.00E-12 - K13065 c30772_g1 541 2.59 0.17 3.18E-04 - K13065 c30772_g2 861 2.53 0.24 5.45E-07 下调 K13065 c30772_g3 815 2.74 0 6.60E-10 下调 K13065 c30772_g4 519 2.2 0.18 2.32E-03 - K13065 c31143_g1 1459 5.88 33.96 1.21E-12 上调 K13065 c32082_g1 482 3.5 0.21 1.63E-04 下调 K13065 c33961_g1 2119 17.74 1.37 5.71E-135 下调 K13065 c34282_g1 1601 254.05 8.49 0 下调 K13065 c35688_g1 376 1.29 0 1.09E-01 - K13065 c4274_g1 1104 8.4 0.06 1.49E-41 下调 K13065 c5404_g1 1402 4.15 0.09 4.05E-26 下调 K13065 c55487_g1 379 2.1 0 2.54E-02 - K13065 c5589_g1 329 135.34 3.4 8.92E-57 下调 K13065 c5589_g2 213 89.24 0 4.09E-06 下调 K13065 c58620_g1 603 0.61 0 5.26E-02 - K13065 c63378_g1 1279 2.06 0.24 1.25E-08 下调 K13065 c68513_g1 988 1.08 0.53 1.09E-01 - K13065 c70167_g1 631 0.98 0 5.92E-03 - K13065 c7571_g1 264 3.16 0 2.25E-01 - K13065 c78553_g1 1785 13.78 34.75 8.08E-12 上调 K13065 c79068_g1 1627 45.09 6.4 4.49E-194 下调 K13065 c80218_g1 1475 1.39 0.08 1.25E-08 下调 K13065 c94800_g1 1985 5.34 115.03 6.07E-13 上调 K13065 c96068_g1 2064 21.84 45.88 0 上调 二氢黄酮还原酶 K13082 c35752_g1 669 0 0.94 5.28E-03 - K13082 c44823_g1 204 - - - - 类黄酮3′, 5′-羟化酶 K13083 c116693_g1 360 0.49 1.29 4.19E-01 - K13083 c124994_g1 542 0.18 0.67 2.52E-01 - 异黄酮2′-羟化酶 K13260 c23780_g1 1835 141.91 156.3 1.03E-06 - K13260 c33762_g1 668 19.59 38.01 1.46E-12 - 异黄酮7-O-葡萄糖基转移酶 K13263 c33206_g4 345 3.91 13.15 1.04E-03 - K13263 c52889_g1 213 - - - - K13263 c900_g1 616 24.03 0.4 1.55E-47 下调
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