根据已公布液泡膜H+-PPase基因家族同源序列保守区设计简并引物,克隆出海滨雀稗中PvVP1基因的中间序列,然后用快速扩增cDNA末端(rapid amplification of cDNA end,RACE)方法,从海滨雀稗中克隆到PvVP15′cDNA序列。该序列ORF长1605bp,编...根据已公布液泡膜H+-PPase基因家族同源序列保守区设计简并引物,克隆出海滨雀稗中PvVP1基因的中间序列,然后用快速扩增cDNA末端(rapid amplification of cDNA end,RACE)方法,从海滨雀稗中克隆到PvVP15′cDNA序列。该序列ORF长1605bp,编码535个氨基酸,其编码序列、氨基酸序列与玉米相应基因的同源性分别为93%和99%。展开更多
Vacuolar H^+-ATPase was regarded as a key enzyme promoting the fiber cell elongation in cotton (Gossypium hirsuturm L.) through regulating turgor-driven pressure involved in polarity expansion of single cell fiber. Th...Vacuolar H^+-ATPase was regarded as a key enzyme promoting the fiber cell elongation in cotton (Gossypium hirsuturm L.) through regulating turgor-driven pressure involved in polarity expansion of single cell fiber. The DET3, a V-ATPase subunit C, plays an important role in assembling subunits and regulating the enzyme activity, and is involved in Brassinosteroid-induced cell elongation. To analyze the function of GhDET3 on the elongation of cotton fibers, seven candidates of ESTs were screened and contigged for a 5'-upstream sequence, and the 3'-RACE technique was used to clone the 3'-downstream sequence for the full length of GhDET3 gene. The full length of the target clone was 1,340 bp, including a 10 bp 5'-UTR, an ORF of 1,134 bp, and a 196 bp 3'-UTR. This cDNA sequence encoded a polypepide of 377 amino acid residues with a predicted molecular mass of 43 kDa and a basic isoelectric point of 5.58. Furthermore, a length of 3,410 bp sequence from genomic DNA of GhDET3 was also cloned by PCR. The deduced amino acid sequence had a high homology with DET3 from Arabidopsis, rice, and maize. Quantitative real-time PCR (qRT-PCR) analysis showed that the GhDET3 expression pattern was ubiquitous in all the tissues and organs detected. The result also revealed that the accumulation of GhDET3 mRNA reached the highest profile at the fiber elongation stage in 12 DPA (days post anthesis) fibers, compared with the lowest level at the fiber initiation stage in 0 DPA ovules (with fibers). The transcript accumulation in fibers and ovules shared the similar variation tendency. In addition, in vitro ovule culture experiment demonstrated that exogenous 24-EBL treatment to 4 DPA ovules (with fibers) was capable of increasing the expression level of GhDET3, and the mRNA accumulation of GhDET3 increased in transgenic FBP7::GhDET2 cotton fibers in vivo. These results indicate that GhDET3 gene plays a crucial role in cotton fiber elongation.展开更多
文摘根据已公布液泡膜H+-PPase基因家族同源序列保守区设计简并引物,克隆出海滨雀稗中PvVP1基因的中间序列,然后用快速扩增cDNA末端(rapid amplification of cDNA end,RACE)方法,从海滨雀稗中克隆到PvVP15′cDNA序列。该序列ORF长1605bp,编码535个氨基酸,其编码序列、氨基酸序列与玉米相应基因的同源性分别为93%和99%。
基金the National Natural Science Foundation of China (No. 30370904 , 30671258) the National High Technology Research and Development Program (863 Project) of China (No. 2006AA10Z121) the Program for New Century Excellent Talents in University (No. NCET-07-0712).
文摘Vacuolar H^+-ATPase was regarded as a key enzyme promoting the fiber cell elongation in cotton (Gossypium hirsuturm L.) through regulating turgor-driven pressure involved in polarity expansion of single cell fiber. The DET3, a V-ATPase subunit C, plays an important role in assembling subunits and regulating the enzyme activity, and is involved in Brassinosteroid-induced cell elongation. To analyze the function of GhDET3 on the elongation of cotton fibers, seven candidates of ESTs were screened and contigged for a 5'-upstream sequence, and the 3'-RACE technique was used to clone the 3'-downstream sequence for the full length of GhDET3 gene. The full length of the target clone was 1,340 bp, including a 10 bp 5'-UTR, an ORF of 1,134 bp, and a 196 bp 3'-UTR. This cDNA sequence encoded a polypepide of 377 amino acid residues with a predicted molecular mass of 43 kDa and a basic isoelectric point of 5.58. Furthermore, a length of 3,410 bp sequence from genomic DNA of GhDET3 was also cloned by PCR. The deduced amino acid sequence had a high homology with DET3 from Arabidopsis, rice, and maize. Quantitative real-time PCR (qRT-PCR) analysis showed that the GhDET3 expression pattern was ubiquitous in all the tissues and organs detected. The result also revealed that the accumulation of GhDET3 mRNA reached the highest profile at the fiber elongation stage in 12 DPA (days post anthesis) fibers, compared with the lowest level at the fiber initiation stage in 0 DPA ovules (with fibers). The transcript accumulation in fibers and ovules shared the similar variation tendency. In addition, in vitro ovule culture experiment demonstrated that exogenous 24-EBL treatment to 4 DPA ovules (with fibers) was capable of increasing the expression level of GhDET3, and the mRNA accumulation of GhDET3 increased in transgenic FBP7::GhDET2 cotton fibers in vivo. These results indicate that GhDET3 gene plays a crucial role in cotton fiber elongation.
