摘要
以康乃馨 (DianthuscaryophyllusL .)花瓣为材料 ,用改进的异硫氰酸胍一步法提取总RNA ,根据已报道的康乃馨ACC氧化酶 (1 aminocyclopropane 1 carboxylicacidoxidase ,ACO)基因的序列设计并合成一对引物 ,通过RT PCR方法获得一约 1 2kb特异片段 ,把该片段连接到pGEM(R) Teasyvector上进行测序 ,其全长共 115 6bp ,编码区 915bp ,共编码 30 4个氨基酸残基。序列分析结果表明该序列与GenBankL35 15 2中的康乃馨ACC氧化酶基因的cDNA序列完全相符 ,推断该基因在康乃馨种内可能是完全或高度保守的。随后将此片段反向插入植物表达载体pBI12 1的 35S启动子和NOS终止子之间 ,构建了一反义植物表达载体pBO ;又把花特异表达启动子PchsA插入pBI12 1的HindIII +Xbal位点构建中间载体pCHB ,再把康乃馨ACC氧化酶基因反向插入中间载体pCHB的XbaI +Sst1位点构建成另一反义植物表达载体pCBO。
Total RNA was extracted from petals of Carnation (Dianthus caryophyllus L.) and reverse transcripted to first strand of cDNA. For getting the 1-Aminocyclopropane-1-Carboxylic acid Oxidase (ACO) cDNA, a pair of primer was designed and synthesized according to the sequence reported. The PCR product of the ACO cDNA was obtained by using the first strand of cDNA as template. This product was ligased to pGEM (R)-T easy vector and sequenced. The sequencing data showed that the PCR product was 1156bp, which containing a 915bp coding region and encoding 304 predicted amino acid residues. Comparation of the cDNA sequence from this experiment with that reported by GenBank L35152 indicated the homology was 100%. We give the results that this ACO gene among the species of Dianthus caryophyllus L.is highly conserved. The ACO gene antisense sequence was then inserted between the CaMv 35S promoter and NOS terminator into the expression vector pBI121. This expression vector called pBO; Then the flower-specific promoter PchsA was first inserted into the sites of the expression vector pBI121/HindIII+Xbal, This recombinant plasmid called pCHB, then inserted the ACO antisense gene into the sites of the plasmid pCHB/XbaI+SstI. Hence the antisense plant expression vector pCBO was constructed.
出处
《云南植物研究》
CSCD
北大核心
2002年第6期775-780,共6页
Acta Botanica Yunnanica
基金
中国博士后基金资助项目
