COG分類：將49,758個能比對到 Nr蛋白數據庫的基因進行COG分類，結果顯示25,140個基因聚成25種功能組。其中最大的COG分類組為信號轉導機制組（10,471個基因大約占41.6%），詳見Figure 5。
Figure 5. COG function classification of transcriptome.
KEGG分類：通過Kyoto Encyclopedia of Genes and Genomes (KEGG)分析69,605個基因，顯示14,998個基因能比對到258條信號通路。最主要的信號通路為代謝通路（3,506個基因，大約占23.37%），其次為次生代謝物的合成 (1,785,11.9%)，不同環境中微生物的代謝（802，5.35%)，RNA降解（538, 3.59%）以及核糖體(535, 3.57%)。研究者將目光聚焦到了與薺菜型油菜種皮著色相關的次生代謝物合成通路，發現154個基因與苯丙素生物合成相關，114個基因與苯丙氨酸、酪氨酸、色氨酸生物合成相關，46個基因與類黃酮生物合成相關，9個基因與黃酮以及黃酮醇生物合成相關。
種皮轉綠組中轉錄因子的鑒定：將所有拼接得到的基因通過Blastx比對到AGRIS (Arabidopsis Gene Regulatory Information Server)數據庫，E-value值小于10-5，identity大于70%，2,347個基因被推定屬于48個轉錄因子家族，其中MYB（100個基因）以及bHLH（190個基因）兩個家族在植物中與類黃酮生物合成相關。
黃色與棕色種皮中不同表達的轉錄本:為了觀察兩種不同顏色的種皮中基因的表達水平，通過RPKM分析它們各自被拼接出的69,605個基因。其中1,304個基因在兩者中的表達水平有差異，棕色種皮與黃色種皮相比較，有802個基因上調，502個基因下調，在這些基因中，170 (12.8%)個基因表達水平有15倍的差異，471 (36.4%)個有2-3倍的差異，詳見Figure 6。對差異表達的基因進行注釋發現455個基因屬于28個GO組，849個基因不能進行歸類，詳見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.
與類黃酮生物合成信號通路相關的種皮轉綠組基因：在擬南芥中，PA的合成顯示種子內皮細胞在授粉后3天（days after pollination，DAP)會有其生物合成基因的表達。在芥菜型油菜種皮中10 DAP才會出現PA的積累，芯片結果顯示在埃塞俄比亞芥棕籽形成時， 22 DAP（形成角果）時，6個類黃酮基因（CHS、F3H、FOMT、DFR、GST以及TTG1）發生上調，2個基因（F39H、FLS）發生下調，該現象在黃籽形成時未發現。將次生壁豐富的甘藍型油菜種皮及其下胚軸進行對比發現，類黃酮生物合成轉錄本的基因：ANR、FLS 以及 CHS在種皮中的含量更為豐富，這就意味著類黃酮生物合成基因在種皮中高表達，與PA在種皮中的沉積相一致。
Figure 8呈現了芥菜型油菜與類黃酮生物合成相關基因的表達情況。過去的研究表明DFR, LDOX 及ANR基因與PA合成相關，且DFR與LDOX基因在黃籽的蕓苔屬植物中不表達，本研究發現，DFR, ANR基因在黃籽中幾乎不表達，在棕籽中高表達，LDOX在棕籽中的表達量也高于黃籽，結果表明，與PA合成相關的基因不表達或者低表達導致了黃籽的芥菜型油菜中沒有PA的積累。
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.
類黃酮生物合成通路中基因的RT-PCR分析：為了對RPKM的分析結果進一步確證，選取了類黃酮生物合成過程中的8個基因（Figure 9）進行qRT-PCR分析，發現在棕色種皮中Unigene_920 (CHS), Unigene_29246 (CHI), Unigene_7597 (DFR), Unigene_7701 (LDOX), Unigene_16036(ANR)發生上調，Unigene_28310 (FLS)下調，Unigene_682 (F3H) 及Unigene_396 (F39H)未發生明顯變化。該結果與RPKM分析結果相一致。
Figure 9. qRT-PCR validation of RPKM analysis of the eight unigenes involved in flavonoid biosynthesis of Brassica juncea seed coat.
原文出處：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.