利用抑制差减杂交技术构建了20d和35d甘蓝型油菜湘油15发育种子特异表达基因的SSH文库。随机挑取单菌落进行PCR表明,文库质量较好。在20d和35 d SSH文库中随机挑选489个阳性克隆进行测序,获得452条高质量的表达序列标签(EST)。对序列进...利用抑制差减杂交技术构建了20d和35d甘蓝型油菜湘油15发育种子特异表达基因的SSH文库。随机挑取单菌落进行PCR表明,文库质量较好。在20d和35 d SSH文库中随机挑选489个阳性克隆进行测序,获得452条高质量的表达序列标签(EST)。对序列进行Blast比对及功能注释,比较20d和35 d SSH文库的基因表达谱,发现在20 d SSH库中参与糖代谢的基因出现频率较高,而在35 d SSH库中与脂肪酸储存有关的油体蛋白家族、与脂肪酸转运有关的柠檬酸合酶、与脂肪酸合成有关的酰基载体蛋白去饱和酶等,参与油脂代谢相关基因出现频率较高。该结果对进一步研究油菜脂肪酸代谢调控的分子机制打下了基础。展开更多
The molecular basis of color polymorphism in the shells of the pearl oyster Pinctada fucata is largely unknown. We developed a red-shelled family line and used suppression subtractive hybridization （SSH） to screen f...The molecular basis of color polymorphism in the shells of the pearl oyster Pinctada fucata is largely unknown. We developed a red-shelled family line and used suppression subtractive hybridization （SSH） to screen for differentially expressed genes in red- and non-red-shelled pearl oysters. We constructed forward and reverse cDNA subtractive libraries consisting of 2 506 and 797 clones, respectively. Among 343 randomly selected clones in the forward library, 304 sequences were identified in GenBank using BLASTx and BLASTn. Of the 304 sequences, 13 showed no similarity to known sequences and 291 were matched with known genes of the pearl oyster, including shematrin-1, shematrin-2, shematrin-6, shematrin-7, nacrein, nacrein-like protein, aspein for shell matrix protein, glycine-rich protein, mantle gene 5, 28S, EST00031, EST00036, 16S, and COl. In the reverse library, 7 clones were sequenced and analyzed by BLAST. Two sequences shared similarity with EST00036 from the P. fucata subtraction cDNA library, four with the P. fucata mitochondrial gene for 16S rRNA and 1 with P. fucata shematrin-2. We evaluated the expression of 12 genes from the forward library using RT PCR. Two sequences matched with 16S and CO1 so were considered to be false positives. The remaining 10 sequences were differentially expression in the red-shelled pearl oysters. Our results suggest that differential expression of these genes may be related to color variation in the red-shelled family line of the pearl oyster.展开更多
文摘利用抑制差减杂交技术构建了20d和35d甘蓝型油菜湘油15发育种子特异表达基因的SSH文库。随机挑取单菌落进行PCR表明,文库质量较好。在20d和35 d SSH文库中随机挑选489个阳性克隆进行测序,获得452条高质量的表达序列标签(EST)。对序列进行Blast比对及功能注释,比较20d和35 d SSH文库的基因表达谱,发现在20 d SSH库中参与糖代谢的基因出现频率较高,而在35 d SSH库中与脂肪酸储存有关的油体蛋白家族、与脂肪酸转运有关的柠檬酸合酶、与脂肪酸合成有关的酰基载体蛋白去饱和酶等,参与油脂代谢相关基因出现频率较高。该结果对进一步研究油菜脂肪酸代谢调控的分子机制打下了基础。
基金Supported by National High Technology Research and Development Program of China (863 Program) (No. 2006AA10A409)the Knowledge Innovation Program of the South China Sea Institute of Oceanology, Chinese Academy of Sciences (No. SQ200906)+2 种基金the Science and Technology Program of Guangdong Province (No. 2008A020100004)the National Key Technology Research and Development Program (No. 2007BAD29B01-8)the Key Plan for Marine Development of Guangdong Province (No. A200708C01)
文摘The molecular basis of color polymorphism in the shells of the pearl oyster Pinctada fucata is largely unknown. We developed a red-shelled family line and used suppression subtractive hybridization （SSH） to screen for differentially expressed genes in red- and non-red-shelled pearl oysters. We constructed forward and reverse cDNA subtractive libraries consisting of 2 506 and 797 clones, respectively. Among 343 randomly selected clones in the forward library, 304 sequences were identified in GenBank using BLASTx and BLASTn. Of the 304 sequences, 13 showed no similarity to known sequences and 291 were matched with known genes of the pearl oyster, including shematrin-1, shematrin-2, shematrin-6, shematrin-7, nacrein, nacrein-like protein, aspein for shell matrix protein, glycine-rich protein, mantle gene 5, 28S, EST00031, EST00036, 16S, and COl. In the reverse library, 7 clones were sequenced and analyzed by BLAST. Two sequences shared similarity with EST00036 from the P. fucata subtraction cDNA library, four with the P. fucata mitochondrial gene for 16S rRNA and 1 with P. fucata shematrin-2. We evaluated the expression of 12 genes from the forward library using RT PCR. Two sequences matched with 16S and CO1 so were considered to be false positives. The remaining 10 sequences were differentially expression in the red-shelled pearl oysters. Our results suggest that differential expression of these genes may be related to color variation in the red-shelled family line of the pearl oyster.
