虾青素合成关键基因在烟草中瞬时表达及效应

Transient expression and effects of two key genes for astaxanthin synthesis in tobacco leaves

[目的]本文旨在评估虾青素生物合成的关键基因β-胡萝卜素酮化酶(BKT)和β-胡萝卜素羟化酶(BHY)在烟草中异源表达的活性及效应。[方法]分别构建来自莱茵衣藻(C.reinhardtii)的CrBKT和来自雨生红球藻(H.plu⁃vialis)的HpBHY酶基因的过表达载体。将含有目的基因的超表达载体应用农杆菌介导法在烟草叶组织中进行瞬时表达。PCR和生化测定目的基因表达水平、叶绿素、类胡萝卜素及虾青素含量等参数。[结果]取侵染3 d的烟叶组织,PCR检测显示CrBKT和HpBHY酶基因在异源宿主烟叶组织高效表达。取侵染5 d的烟草组织生化分析表明,HpB⁃HY+CrBKT基因共表达导致叶绿素a、叶绿素b、类胡萝卜素和虾青素含量分别达到10.33、3.012、2.271和0.518μg·mg^(-1),均显著高于对照组(P≤0.05)。单表达HpBHY基因或CrBKT基因的烟叶组织叶绿素a和叶绿素b含量显著高于对照组(P≤0.05),而类胡萝卜素和虾青素的量与对照组没有显著差异(P>0.05)。[结论]异源共表达来自莱茵衣藻(C.reinhardtii)的CrBKT和来自雨生红球藻(H.pluvialis)的HpBHY可促进虾青素和类胡萝卜素在烟叶组织的合成积累。研究为培育富含虾青素烟草新种质以及应用烟叶生物反应器高效生产虾青素提供了科学依据。

[Objective]The current research aimed to assess activities and effects of the key genes of astaxanthin biosynthesis,β-carotene ketolase(BKT)and β-carotene hydroxylase(BHY)via the heterogeneous expression in tobacco.[Methods]Firstly,the overexpression vectors of CrBKT from Chlamydomonas reinhardtii and HpBHY from Haematococcus pluvialis were constructed.Secondly,the overexpression vectors containing the target genes were transiently transformed into tobaccos leaf tissue via Agrobacterium-mediated.Finally,the corresponding molecular and biochemical indexes,such as the expressed level of target genes and the content of Chlorophyll,carotenoid and astaxanthin,were determined by PCR and biochemical determination measures.[Results]RT-PCR assays were conducted with tobaccos leaf tissue on day-3 after transient expression and the results showed that CrBKT and HpBHY were highly expressed in heterologous host tobaccos;The biochemical analysis of tobacco tissues collected 5 days after infection showed that the contents of chlorophyll a,chlorophyll b,carotenoids and astaxanthin in transient expression HpBHY+CrBKT tobaccos reached to 10.33μg·mg^(-1),3.012μg·mg^(-1),2.271μg·mg^(-1) and 0.518μg·mg^(-1),respectively,which were significantly higher than those of control group(P≤0.05);The contents of chlorophyll a and chlorophyll b in tobacco leaf tissue expressing HpBHY gene or CrBKT gene were significantly higher than that of the control group(P≤0.05),while the amount of carotenoids and astaxanthin were not significantly different from that of the control group(P>0.05).[Conclusion]Heterologous co-expression of β-carotene ketolase(CrBKT)from C.reinhardtii andβ-carotene hydroxylase(HpBHY)from H.pluvialis can promote the biosynthesis and accumulation of astaxanthin and carotenoid in tobacco leaf tissue.The results of this study will provide a scientific basis for breeding of new tobacco germplasm rich in astaxanthin as well as the application of tobacco leaf bioreactor to efficiently produce astaxanthin.