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Xiao G.,Institute of Oil Crops National Oil Crops Improvement Center | Xiao G.,Pre State Key Laboratory for Germplasm Innovation and Resource Utilization of Crops | Zhang Z.-Q.,Institute of Oil Crops National Oil Crops Improvement Center | Wu X.-M.,Institute of Oil Crops National Oil Crops Improvement Center | And 3 more authors.
Acta Agronomica Sinica | Year: 2010

FAD2 gene is essential for converting oleic acid to linoleic acid in plant seeds. From Xiangyou 15, a cultivar of Brassica napus, 56 FAD2 DNA clones and 47 FAD2 cDNA clones were obtained including 6 new copies of FAD2, which were designated FAD2P1 to FAD2P6. These new FAD2 copies ranged from 1141 bp to 1157 bp in length and contained no introns in their open reading frames. They had 96.1% homology in nucleotide sequence and shared more than 87.0% of nucleotides identity to the published FAD2 gene (AY577313). The deduced amino acid sequences of the 6 FAD2 copies presented 1-12 stop codons within the coding regions, which prevented from coding functional proteins. These copies were transferred into Saccharomyces cerevisiae through vector pYES2.0, and the expression products showed no functions to synthesize 16:2 and 18:2 fatty acids. These results indicate that they are pseudogenes of FAD2. Copyright © 2010, Crop Science Society of China and Institute of Crop Sciences, Chinese Academy of Agricultural Sciences. Source


Zhenqian Z.,China Agricultural University | Gang X.,China Agricultural University | Gang X.,Pre State Key Laboratory for Germplasm Innovation and Resource Utilization of Crops | Ruiyang L.,China Agricultural University | And 5 more authors.
African Journal of Agricultural Research | Year: 2011

The cDNA library normalized by reassociation is a newly developed, effective platform for gene discovery and function analysis. In order to screen the genes related to high oleic synthesis, a normalized cDNA library was constructed. Total RNAs were prepared from high oleic acid rapeseed seeds using TRIzol reagent. CDS III/3' primer was used to synthesize the first-strand cDNA with SMART technique; and the double strand (ds) cDNA fraction formed by abundant transcripts is degraded by 1/2 duplex specific nuclease (DSN) dilution. Then amplified with twice the PCR cycles, which was 11 and 12 respectively. Finally, the cDNAs longer than 0.5 kb were collected and ligated to λTriplEx2 vector. The titer of the unamplified cDNA library was 2.61×10 6 cfu/ml with a recombinant rate of 100%, and the titer of the amplified library was 9.24×10 9 cfu/ml. Electrophoresis gel results fragments ranged from 800 bp to 2 kb, with an average size of 1000 bp. These results indicated that the normalized full length cDNA library has been constructed successfully, which is convenient for further studying the oleic synthesis mechanism in rapeseed. © 2011 Academic Journals. Source


Xiao G.,Chinese Academy of Agricultural Sciences | Xiao G.,Pre State Key Laboratory for Germplasm Innovation and Resource Utilization of Crops | Xiao G.,The Oil Crops Research Institute National Oil Crops Improvement Center | Zhang Z.Q.,The Oil Crops Research Institute National Oil Crops Improvement Center | And 9 more authors.
Gene | Year: 2014

In the present study, we characterized the transcriptional regulatory region (KF038144) controlling the expression of a constitutive FAD2 in Brassica napus. There are multiple FAD2 gene copies in B. napus genome. The FAD2 gene characterized and analyzed in the study is located on chromosome A5 and was designated as BnFAD2A5-1. BnFAD2A5-1 harbors an intron (1192. bp) within its 5'-untranslated region (5'-UTR). This intron demonstrated promoter activity. Deletion analysis of the BnFAD2A5-1 promoter and intron through the β-glucuronidase (GUS) reporter system revealed that the 220 to 1. bp is the minimum promoter region, while 220 to 110. bp and +. 34 to +. 285. bp are two important regions conferring high-levels of transcription. BnFAD2 transcripts were induced by light, low temperature, and abscisic acid (ABA). These observations demonstrated that not only the promoter but also the intron are involved in controlling the expression of the BnFAD2A5-1 gene. The intron-mediated regulation is an essential aspect of the gene expression regulation. © 2014 Elsevier B.V. Source


Xiao G.,Chinese Academy of Agricultural Sciences | Xiao G.,Pre State Key Laboratory for Germplasm Innovation and Resource Utilization of Crops | Zhang Z.-Q.,Chinese Academy of Agricultural Sciences | Zhang Z.-Q.,Pre State Key Laboratory for Germplasm Innovation and Resource Utilization of Crops | And 7 more authors.
Journal of Integrative Agriculture | Year: 2013

Based on the sequence of a novel expressed sequence tag (EST), the full-length cDNA of 1 017 nucleotides was cloned from Brassica napus cv. Xiangyou 15 through rapid amplification of cDNA ends (RACE). The gene was designated as Bnhol34 (HQ585980), encoding a protein of 338 amino acids. BLAST analysis showed no high degree of sequence identity to any known gene. The calculated molecular weight of the Bnhol34 protein was 36.23 kDa, and the theoretical isoelectric point was 8.74. The Bnhol34 was also cloned from a high oleic acid mutant 854-1 through homologous cloning. There was no difference between the two Bnhol34 genes. Bnhol34 was localized in a tissue-specific manner in B. napus, and its expression level was about eight-fold greater in Xiangyou 15 seeds than in 854-1. The promoter region sequences of Bnhol34 were then isolated from Xiangyou 15 and 854-1, and a 93-bp deletion was found to occur in the Bnhol34 promoter region of 854-1. Three abscisic acid-responsive cis-elements (ABRE) were identified in the promoter region of Xiangyou 15. Real-time PCR analyses revealed that exogenous abscisic acid increased Bnhol34 expression by about four-fold in Xiangyou 15 seeds, yet did not change Bnhol34 expression in 854-1. It appeared that Bnhol34 might be abscisic acid insensitive in 854-1. © 2013 Chinese Academy of Agricultural Sciences. Source

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