Zhongsong Liu

De novo transcriptome of Brassica juncea seed coat and identification of genes for the biosynthesis of flavonoids.

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 10−5. 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.

Cloning and Expression Analysis of Dihydroflavonol 4-Reductase Gene in Brassica juncea

Abstract Dihydroflavonol 4-reductase (DFR) gene is a key gene of proanthocyanidins biosynthesis pathway in seed coat of Arabidopsis thaliana. The mutation of this gene brings about a transparent testa. To study molecular mechanism of seed coat color in Brassica, DFR gene was cloned from B. juncea using homology-based clone strategy. The cloned gene 1,612 bp in length contains 5 introns. The complementary DNA (cDNA) consists of 1,214 bp and has a 1,158 bp open reading frame encoding a deduced polypeptide of 385 amino acids with a predicted molecular weight of 42,886 Da and an estimated isoelectric point of 5.54. RT-PCR analysis showed that DFR was expressed in leaves, embryos, and seed coats of Purple-Leaf Mustard and 2 black-seeded near-isogenic lines developed from backcross breeding using Sichuan Yellow as a recurrent parent. Gene DFR was expressed only in the leaves and embryos of Sichuan Yellow, but not in seed coats. No expression of DFR blocked the biosynthesis of anthocyanidins and proanthocyanidins in the yellow seed coat, and seeds appeared yellow because of transparent testa. Gene DFR is essential in the formation of seed coat color in B. juncea. This study provided a foundation for understanding the molecular mechanism of seed coat color and developing novel yellow-seeded rapeseed germplasm through antisense expression or RNAi suppression of DFR gene in black-seeded cultivars using a seed- or seed-coat-specific promoter.

Cloning and SNP Analysis of TT1 Gene in Brassica juncea

Abstract TT1 gene encodes a WIP domain protein with Zn-finger, which is essential for seed coat development and organ color in Arabidopsis . The mutation of this gene causes transparent testa. The BjTT1 gene was cloned from B. juncea using homology-based cloning and rapid-amplification of cDNA ends (RACE) strategy. A modified allele-specific PCR procedure was developed for assaying single nucleotide polymorphisms (SNP) of the BjTT1 gene. The full length of BjTT1 sequence was 2197 bp with only one intron. The cDNA sequence of BjTT1 was 1412 bp in length including 903 bp of open reading frames. This gene encodes a deduced polypeptide of 300 amino acids with a predicted molecular weight of 33.97 kD and an isoelectric point of 6.99. The genomic sequence of BjTT1 showed 99% and 85% identity with that of BnTT1 and AtTT1 , respectively. The reverse transcription-polymerase chain reaction (RT-PCR) analysis showed the expressions of BjTT1 in the seed coats of both black-seeded near-isogenic lines NILA and NILB. Based on sequence comparisons between BjTT1 genes from parents with different seed coat colors and between the NILB mutant and its wide type parent, nucleotide variations at 8 sites inside the BjTT1 coding region were detected, but mutations at these sites had no effect on seed coat color. The allele-specific PCR of BjTT1 could distinguish the yellow-seeded and black-seeded parents.

Genome-Wide Identification, Localization, and Expression Analysis of Proanthocyanidin-Associated Genes in Brassica

Proanthocyanidins (PA) is a type of prominent flavonoid compound deposited in seed coats which controls the pigmentation in all Brassica species. Annotation of Brassica juncea genome survey sequences showed 72 PA genes; however, a functional description of these genes, especially how their interactions regulate seed pigmentation, remains elusive. In the present study, we designed 19 primer pairs to screen a bacterial artificial chromosome (BAC) library of B. juncea. A total of 284 BAC clones were identified and sequenced. Alignment of the sequences confirmed that 55 genes were cloned, with every Arabidopsis PA gene having 2–7 homologs in B. juncea. BLAST analysis using the recently released B. rapa or B. napus genome database identified 31 and 58 homologous genes, respectively. Mapping and phylogenetic analysis indicated that 30 B. juncea PA genes are located in the A-genome chromosomes except A04, whereas the remaining 25 genes are mapped to the B-genome chromosomes except B05 and B07. RNA-seq data and Fragments Per Kilobase of a transcript per Million mapped reads (FPKM) analysis showed that most of the PA genes were expressed in the seed coat of B. juncea and B. napus, and that BjuTT3, BjuTT18, BjuANR, BjuTT4-2, BjuTT4-3, BjuTT19-1, and BjuTT19-3 are transcriptionally regulated, and not expressed or downregulated in yellow-seeded testa. Importantly, our study facilitates in better understanding of the molecular mechanism underlying Brassica PA profiles and accumulation, as well as in further characterization of PA genes.

Research and Utilization of Chemical Inducing Male Sterility for Hybrid Seed Production in Canola (Brassica napus)

Zinc methylarsenate (ZMA) has been found to be the best chemical hybridizing agent for canola among the over 40 chemicals investigated. Cytological, physiological, and biochemical processes of induction of male sterility by several chemical hybridizing agents were revealed in canola. The system for hybrid seed production using a chemical hybridizing agent has been established for the first time and used for breeding hybrid varieties in canola. A number of heterotic hybrid canola varieties such as Xiangzayou 1 and Xiangzayou 6 have been developed using the above hybrid seed system and commercially released in the Yangtze Valley, China, with the planting area of over 2.3 million ha and direct economic benefit of 1 billion RMB yuan. Fifty-two scientific papers have been published. These studies were financially supported by the grants from Ministry of Science and Technology, China ( 96-002-02-13 , 85-03-03-02 ) and from the Department of Science and Technology, Hunan Province, China ( NKY 1004-02 ). These scientific achievements won the 2009 State Science and Technology Award of China (2nd prize).