芥菜型油菜种皮转录组De Novo拼接及类黄酮生物...（二）
COG分类：将49,758个能比对到 Nr蛋白数据库的基因进行COG分类，结果显示25,140个基因聚成25种功能组。其中最大的COG分类组为信号转导机制组（10,471个基因大约占41.6%），详见Figure 5。
Figure 5. COG function classification of transcriptome.
KEGG分类：通过Kyoto Encyclopedia of Genes and Genomes (KEGG)分析69,605个基因，显示14,998个基因能比对到258条信号通路。最主要的信号通路为代谢通路（3,506个基因，大约占23.37%），其次为次生代谢物的合成 (1,785,11.9%)，不同环境中微生物的代谢（802，5.35%)，RNA降解（538, 3.59%）以及核糖体(535, 3.57%)。研究者将目光聚焦到了与荠菜型油菜种皮着色相关的次生代谢物合成通路，发现154个基因与苯丙素生物合成相关，114个基因与苯丙氨酸、酪氨酸、色氨酸生物合成相关，46个基因与类黄酮生物合成相关，9个基因与黄酮以及黄酮醇生物合成相关。
种皮转绿组中转录因子的鉴定：将所有拼接得到的基因通过Blastx比对到AGRIS (Arabidopsis Gene Regulatory Information Server)数据库，E-value值小于10-5，identity大于70%，2,347个基因被推定属于48个转录因子家族，其中MYB（100个基因）以及bHLH（190个基因）两个家族在植物中与类黄酮生物合成相关。
黄色与棕色种皮中不同表达的转录本:为了观察两种不同颜色的种皮中基因的表达水平，通过RPKM分析它们各自被拼接出的69,605个基因。其中1,304个基因在两者中的表达水平有差异，棕色种皮与黄色种皮相比较，有802个基因上调，502个基因下调，在这些基因中，170 (12.8%)个基因表达水平有15倍的差异，471 (36.4%)个有2-3倍的差异，详见Figure 6。对差异表达的基因进行注释发现455个基因属于28个GO组，849个基因不能进行归类，详见Figure 7。
Figure 6. The fold change distribution of differentially expressed between the yellow- and brown-seeded testa of Brassica juncea.
Figure 7. Functional categoried of unigenes differentially expressed between the yellow- and brown-seeded testa of Brassica juncea.
与类黄酮生物合成信号通路相关的种皮转绿组基因：在拟南芥中，PA的合成显示种子内皮细胞在授粉后3天（days after pollination，DAP)会有其生物合成基因的表达。在芥菜型油菜种皮中10 DAP才会出现PA的积累，芯片结果显示在埃塞俄比亚芥棕籽形成时， 22 DAP（形成角果）时，6个类黄酮基因（CHS、F3H、FOMT、DFR、GST以及TTG1）发生上调，2个基因（F39H、FLS）发生下调，该现象在黄籽形成时未发现。将次生壁丰富的甘蓝型油菜种皮及其下胚轴进行对比发现，类黄酮生物合成转录本的基因：ANR、FLS 以及 CHS在种皮中的含量更为丰富，这就意味着类黄酮生物合成基因在种皮中高表达，与PA在种皮中的沉积相一致。
Figure 8呈现了芥菜型油菜与类黄酮生物合成相关基因的表达情况。过去的研究表明DFR, LDOX 及ANR基因与PA合成相关，且DFR与LDOX基因在黄籽的芸苔属植物中不表达，本研究发现，DFR, ANR基因在黄籽中几乎不表达，在棕籽中高表达，LDOX在棕籽中的表达量也高于黄籽，结果表明，与PA合成相关的基因不表达或者低表达导致了黄籽的芥菜型油菜中没有PA的积累。
Figure 8. Unigenes involved in the flavonoid biosynthesis pathway in seed coat of Brassica juncea. Abbreviation: ANR, anthocyanidin reductase; CHS, chalcone synthase; CHI, chalcone isomerase; DFR, dihydroflavonol 4-reductase; F3H, flavanone 3-hydroxylase; F39H, flavonoid 39-hydroxylase; FLS, flavonol synthase; LDOX, leucoanthocyanidin dioxygenase.
类黄酮生物合成通路中基因的RT-PCR分析：为了对RPKM的分析结果进一步确证，选取了类黄酮生物合成过程中的8个基因（Figure 9）进行qRT-PCR分析，发现在棕色种皮中Unigene_920 (CHS), Unigene_29246 (CHI), Unigene_7597 (DFR), Unigene_7701 (LDOX), Unigene_16036(ANR)发生上调，Unigene_28310 (FLS)下调，Unigene_682 (F3H) 及Unigene_396 (F39H)未发生明显变化。该结果与RPKM分析结果相一致。
Figure 9. qRT-PCR validation of RPKM analysis of the eight unigenes involved in flavonoid biosynthesis of Brassica juncea seed coat.
原文出处：De Novo Transcriptome of Brassica juncea Seed Coat and Identification of Genes for the Biosynthesis of Flavonoids
Abstract：Brassica juncea, a worldwide cultivated crop plant, produces seeds of different colors. Seed pigmentation is due to the deposition in endothelial cells of proanthocyanidins (PAs), end products from a branch of flavonoid biosynthetic pathway.
To elucidate the gene regulatory network of seed pigmentation in B. juncea, transcriptomes in seed coat of a yellow-seeded inbred line and its brown-seeded near- isogenic line were sequenced using the next-generation sequencing platform
Illumina/Solexa and de novo assembled. Over 116 million high-quality reads were assembled into 69,605 unigenes, of which about 71.5% (49,758 unigenes) were aligned to Nr protein database with a cut-off E-value of 1025. RPKM analysis showed
that the brown-seeded testa up-regulated 802 unigenes and down-regulated 502 unigenes as compared to the yellow seeded one. Biological pathway analysis revealed the involvement of forty six unigenes in flavonoid biosynthesis. The unigenes encoding dihydroflavonol reductase (DFR), leucoantho-cyanidin dioxygenase (LDOX) and anthocyanidin reductase (ANR) for late flavonoid biosynthesis were not expressed at all or at a very low level in the yellow-seeded testa, which implied that these genes for PAs biosynthesis be associated with seed color of B. juncea, as confirmed by qRT-PCR analysis of these genes. To our knowledge, it is the first time to sequence the transcriptome of seed coat in Brassica juncea. The unigene sequences obtained in this study will not only lay the foundations for insight into the molecular mechanisms
underlying seed pigmentation in B.juncea, but also provide the basis for further genomics research on this species or its allies.
