紫苏ω-3-脂肪酸去饱和酶8基因的克隆与功能研究

Cloning and Function Analysis ofω-3-Fatty Acid Desaturase 8 Gene from Perilla frutescens

ω-3-脂肪酸去饱和酶8(fatty acid desaturase 8,FAD8)作为一种脱氢酶,在饱和脂肪酸(saturated fatty acid,SFA)向不饱和脂肪酸(unsaturated fatty acid,UFA)的转化中发挥关键作用,有利于提高植物的抗寒性。然而,目前仍不清楚FAD8在紫苏中的表达水平是否受低温调节。基于转录物组数据,1个FAD8基因被克隆与鉴定,并在烟草中成功表达。该基因被命名为PfFAD8,其全长编码序列为为1317 bp,编码438个氨基酸,预测分子量为50 kD,理论等电点为9.13。我们的研究表明,紫苏茎10℃低温处理4 h,PfFAD8的表达量是常温时的5.6倍,37℃高温处理4 h,其表达量与常温时相比减少了9.1倍,说明PfFAD8是一个低温诱导型基因,低温促进其表达,高温抑制其表达。为了进一步证实这一结果,我们构建了35S::PfFAD8载体,并通过农杆菌介导的叶盘法转化到烟草中。过度表达PfFAD8基因的转基因烟草叶片表现出明显较高的UFA(如亚油酸(C18:2)和棕榈酸(C16:0))含量和较高的抗寒性。此外,PfFAD8在转基因烟草叶片中的过量表达增加了丙二醛(MDA)和脯氨酸(PRO)的含量,并在一定程度上增强了超氧化物歧化酶(SOD)和过氧化氢酶(CAT)的防御酶活性,这可能会防止UFA的下降。综上所述,PfFAD8可能参与了脂质的去饱和过程,导致膜的稳定性增加或诱导了其他与抗寒性有关的基因,如十八烷酸途径或脂质过氧化产物。因此,PfFAD8的过表达可能有助于生产抗寒品种。

ω-3-Fatty acid desaturase 8(FAD8),as a dehydrogenase enzyme,plays a key role in the transformation of saturated fatty acids into unsaturated fatty acids,which is helpful to enhance the freezing tolerance of plants.However,it remains unclear whether the expression level of FAD8 in Perilla frutescens is regulated by low temperature.Based on transcriptome data,the FAD8 gene was cloned,characterized and then successfully expressed in tobacco Nicotiana tabacum.The gene was designated as Pf-FAD8 and has a full-length coding sequence of 1317 bp coding for 438 amino acids with a predicted molecular weight of 50 kD and a theoretical isoelectric point of 9.13.Our research indicated that the expression of PfFAD8 in Perilla frutescens was increased under the freezing stress.To further confirm this result,a 35S::PfFAD8 vector were constructed and transformed into N.tabacum by Agrobacterium tumefaciens-mediated transformation.Transgenic tobacco leaves that over-expressed the PfFAD8 gene exhibited significantly higher unsaturated fatty acids(UFA)such as linoleic(C18:2)and palmitic acid(C16:0)content and advanced freezing tolerance.Moreover,PfFAD8 overexpression in transgenic tobacco leaves increases malondialdehyde(MDA)and proline(PRO)content,and enhances defense enzymes activities of superoxide dismutase(SOD)and catalase(CAT)to some extent under the cold condition,which might prevent the decline of UFA.Taken together,PfFAD8 overexpression in Perilla frutescens might be involved in the desaturation process of lipids leading to increased membrane stability and/or induction of other genes related to freezing tolerance by octadecanoid pathway or lipid peroxidation products.Thus,PfFAD8 overexpression could be useful in the production of freeze-tolerant varieties of N.tabacum.