RNA-Seq Analysis Of Transcriptome And Glucosinolate Metabolism In Seeds And Sprouts Of Broccoli(Brassica Oleracea Var. Italica)

Broccoli (Brassica oleracea var. italica), a member of Cruciferae, is an important vegetable containing high concentration of various nutritive and functional molecules especially the anticarcinogenic glucosinolates. The sprouts of broccoli contain10-100times higher level of glucoraphanin, the main contributor of the anticarcinogenesis than the edible florets. Despite the broccoli sprouts’functional importance, current studies using the available genetic and genomic tools are very limited, which greatly restricts the development of this functionally important vegetable.A total of-85million251bp reads were obtained. After de novo assembly and searching the assembled transcripts against the Arabidopsis thaliana and Nr databases,19441top-hit transcripts were clustered as unigenes with an average length of2133bp. These unigenes were classified according to their putative functional categories. Cluster analysis of total unigenes with similar expression patterns and differentially expressed unigenes among different tissues, as well as transcription factor analysis were performed.We identified25putative glucosinolate metabolism genes sharing62.04-89.72%nucleotide sequence identity with the Arabidopsis orthologs. This established a broccoli glucosinolate metabolic pathway with high colinearity to Arabidopsis. Many of the biosynthetic and degradation genes showed higher expression after germination than in seeds; especially the expression of the myrosinase TGG2was20-130times higher. These results along with the previous reports that glucosinolate concentration decreased exponentially once after germination indicate the breakdown products of glucosinolates may play important roles in broccoli seed germination and sprout development.Then we selected16genes from the25predicted glucosinolates related genes in broccoli to do further RT-PCR validation. Finally we got11partial CDS sequences after RT-PCR amplification, the success ratio was nearly70%. The CYP83A1sequence we got was full CDS sequence for that gene and most of the amplified sequences had more than95%identity when blasted to assembled unigenes. This high identity proved that our sequencing results were authentic. What’s more, we got the actin gene in broccoli ACT8. All these amplified accurate genes including the partial sequence of actin gene would greatly facilitate further functional study at the expression level.Our study provides the largest genetic resource of broccoli to date. These data will pave the way for further studies and genetic engineering of broccoli sprouts to develop functional vegetables containing high levels of the anticarcinogenic glucosinolates. They will also provide new insights into the genomic research of this species and its relatives.