沙冬青AmFAD7和AmFAD8基因的克隆与转化及其功能初步分析

Cloning,Transformation and Preliminary Function Analysis of AmFAD7 and AmFAD8 from Ammopiptanthus mongolicus

对强抗逆植物蒙古沙冬青2个脂肪酸去饱和酶基因AmFAD7和AmFAD8的功能进行了初步研究。结果表明:(1)AmFAD7和AmFAD8的编码蛋白分别含455和457个氨基酸残基。(2)半定量RT-PCR分析显示,在野外生长植株的嫩叶中,AmFAD7表达量在夏季很低、冬季较高,春秋两季略低于冬季,而AmFAD8在春秋两季和初冬时表达量高于其他月份;在低温(4℃^-6℃梯度降温)处理2~48h的幼苗中,AmFAD7的表达呈先降低后升高的变化趋势,而AmFAD8的表达比处理前略有上调;在高温(42℃)处理2~48h的幼苗中,2个基因的表达均被抑制,尤其对AmFAD7的抑制较为迅速而且明显;在干燥处理2~48h的幼苗中,AmFAD7的表达量有较明显的升高,而AmFAD8则略有降低。(3)成功构建了2个基因的植物表达载体(p3300-35S-AmFAD7和p3300-35S-AmFAD8)并转化拟南芥,分别获得18和16个转基因株系。RT-PCR检测表明,其中F7-1和F7-2以及F8-1~F8-4转基因株系中目的基因的表达量均较高。(4)相对电导率分析显示,在正常温度下,AmFAD7转基因株系(F7-1和F7-2)的相对电导率与野生型无显著差异;在低温(-1℃)胁迫24h后,F7-1和F7-2的相对电导率(分别为32.8%和36.1%)显著低于野生型(48.5%),而在高温(37℃)胁迫24h后,F7-1和F7-2的相对电导率(分别为44.7%和41.9%)显著高于野生型(33.2%)。研究表明,AmFAD7在转录水平受低温和干燥胁迫的诱导,但被高温胁迫抑制,而AmFAD8的转录受低温诱导但被干燥和高温胁迫抑制;在拟南芥中组成型表达AmFAD7增强了转基因植株在低温胁迫下细胞膜的稳定性及其耐冻性,但却增加了其在高温胁迫下的细胞膜损伤程度和热敏感性。

The functions of two fatty acid desaturase genes,AmFAD7 and AmFAD8,from Ammopiptanthus mongolicus with very strong tolerance to abiotic stresses were analyzed.The results showed that:(1)AmFAD7 and AmFAD8 gene encode 455 and 457 amino acids,respectively.(2)The semi-quantitative RT-PCR results showed that for young leaves of the A.mongolicus plants growing in the wild,the expression level of AmFAD7 was very low in summer and quite high in winter,and it was slightly lower in both spring and autumn than that in winter.The expression levels of AmFAD8 in the leaves were higher in spring,autumn,and early winter than those in other months.The expression level of AmFAD7 reduced firstly and then increased obviously,while the expression of AmFAD8 increased slightly compared with that before the treatment in the A.mongolicus seedlings under cold(4℃to-6℃gradient cooling)treatment for 2 to 48 h.Under heat(42℃)treatment for 2 to 48 h,the expression of both genes was inhibited,and the suppression of AmFAD7 was particularly rapid and marked.During dehydration(25℃)treatment for 2 to 48 h,the expression of AmFAD7 showed an obvious increase,while that of AmFAD8 had a slight decrease.(3)The constructions of plant expression vectors p3300-35S-AmFAD 7 and p3300-35S-AmFAD 8 of AmFAD7 and AmFAD8 genes were introduced into Arabidopsis to obtain eighteen and sixteen independent transgenic lines,respectively.After being examined by semi-quantitative RT-PCR,two(F7-1 and F7-2)and four(F8-1 to F8-4)transgenic lines showed high transcription level of AmFAD7 and AmFAD8,respectively.(4)The relative electrolytic leakage(REL)analysis showed that at normal condition(22℃),the REL of AmFAD7 transgenic lines and wild type had no significant difference.After treatment at-1℃for 24 h,the REL levels in F7-1 and F7-2(32.8%and 36.1%,respectively)were significantly lower than that of wild type(48.5%).And after heat treatment at 37℃for 24 h,the REL levels in F7-1 and F7-2(44.7%and 41.7%,respectively)were significantly higher than that of the wild type(33.2%).In conclusion,the expression of AmFAD7 was induced by cold and dehydration stresses but inhibited by heat stress.The expression of AmFAD8 was induced by cold stress but inhibited by dehydration and heat stresses.Constitutive expression of AmFAD7 in Arabidopsis increased the membrane stability and freezing tolerance under low temperature stress.However,it also increased the membrane damage and heat sensitivity under high temperature stress.