红花ω3脂肪酸脱氢酶基因的分离及结构分析

Isolation and Structural Analysis of Safflower ω3 Fatty Acid Desaturase

采用RT—PCR和RACE（rapidamplificationofcDNAends）技术，从红花叶片中分离到2个质体类（1）3脂肪酸脱氢酶基因（CtFAD7和CtFAD8）的全长cDNA和1个微体类∞3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‘1）3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问非常保守。推测∞3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。

One microsomal ω3 fatty acid desaturase gene fragment and two plastidial ω3 fatty acid desaturase cDNA and genomic sequences, designatedCtFAD3, CtFAD7 and CtFAD8 have been isolated from safflower （Cartha- mus tinctorius L. ）. The results of sequence alignment with other plant plastid and microbody ω3 fatty acid dehydro- genase gene in the NCBI have showed high similarity. The deduced amino acid sequences of these genes showed three histidine boxes （ HDCGH, HXXXXXHRTHH and HVIHH） characteristic of all membrane-bound desaturases, CtFAD7 and CtFAD8 possessed a putative N-termianl signal peptide,56 and 27 aa respectively. Hydropathy and transmembrane analysis showed that the encoded polypeptides contain four putative hydropathy regions and trans- menmbrane one to three times. The putative protein second-structure indicated that safflower ω3 fatty acid desatu- rase were mainly composed by α-helix and β-sheet. Comparing the genome structures of safflower CtgFAD7 and Ct- gFAD8 with other plant plastidial ω3 desaturase genes showed that all the sequences contain 8 extons and 7 introns. The base sequences and sizes of introns exhibited a wealth polymorphism between species, while the sizes of the in- ternal 6 exons （from 2nd to 7th） were conservative. Comparing the location of the introns revealed that the intron oc- curs were the conserved regions of the enzyme. We speculated that these introns play an important role in keeping the gene function in the species evolutionary process.