论文部分内容阅读
采用RT-PCR和RACE(rapid amplification of cDNA ends)技术,从红花叶片中分离到2个质体类ω3脂肪酸脱氢酶基因(CtFAD7和CtFAD8)的全长cDNA和1个微体类ω3脂肪酸脱氢酶基因(CtFAD3)的部分序列。将其在NCBI中进行序列比对,结果显示均与其他植物质体和微体ω3脂肪酸脱氢酶基因具有较高的相似性。氨基酸序列分析表明红花CtFAD3、CtFAD7和CtFAD8均含有3个富含组氨酸的保守结构域,分别为HDCGH、HXXXXXHRTHH和HVIHH,其中CtFAD7和CtFAD8的N-端分别含有56和27个氨基酸残基的质体信号肽序列。疏水性和跨膜分析表明,3个红花ω3脂肪酸脱氢酶氨基酸序列均包含4个疏水区域,分别跨膜1~3次。蛋白质二级结构预测结果表明,3个ω3脂肪酸脱氢酶蛋白主要由α螺旋和β折叠组成。通过对cDNA和DNA序列比较发现,CtFAD7和CtFAD8的DNA序列中均包含有7个内含子和8个外显子。各内含子的碱基序列和长度在物种间表现出丰富的多态性,在内含子出现的位置,均为该基因的保守区;而从第2#到7#外显子的长度和序列相似性在物种间非常保守。推测ω3脂肪酸脱氢酶基因的内含子对于保证基因在物种进化过程中功能的保守性起着关键作用。
The full-length cDNA of two plastid omega-3 fatty acid dehydrogenase genes (CtFAD7 and CtFAD8) and one microsomal omega-3 fatty acid were isolated from safflower leaves using RT-PCR and RACE (rapid amplification of cDNA ends) Partial sequence of the dehydrogenase gene (CtFAD3). Sequence alignment of these genes in NCBI showed that they all had high similarity with other plant plastids and microzyme ω3 fatty acid dehydrogenase genes. Amino acid sequence analysis showed that safflower CtFAD3, CtFAD7 and CtFAD8 contain three histidine-rich conserved domains, namely HDCGH, HXXXXXHRTHH and HVIHH, respectively, in which the N-terminal of CtFAD7 and CtFAD8 contain 56 and 27 amino acid residues Of the plastid signal peptide sequence. Hydrophobicity and transmembrane analysis showed that the amino acid sequences of the three safflower ω3 fatty acid dehydrogenases all contained four hydrophobic regions, which were transmembrane 1 to 3 times respectively. The prediction of protein secondary structure showed that the three ω3 fatty acid dehydrogenase proteins are mainly composed of α-helices and β-sheets. By comparing the cDNA and DNA sequences, the CtFAD7 and CtFAD8 DNA sequences contain 7 introns and 8 exons. The nucleotide sequence and length of each intron showed rich polymorphism among the species, and all the introns appeared at the conserved region of the gene. However, the length of exon 2 # to 7 # And sequence similarity is very conserved among species. It is speculated that the intron of the ω3 fatty acid dehydrogenase gene plays a key role in ensuring the conservation of the function of the gene in the process of species evolution.