衣藻β–胡萝卜素加酮酶基因在南瓜果实中的瞬时表达

Transient Expression of β-carotene Ketolase of Chlamydomonas reinhardtii in Pumpkin Fruit

为了研究β–胡萝卜素加酮酶基因在南瓜中合成酮基类胡萝卜素的功能,采用农杆菌介导瞬时表达技术,将含有衣藻β–胡萝卜素加酮酶基因CrBKT的pBI121-CMTPCRBKT载体和pBI121对照载体的农杆菌菌液注入授粉后2、5、10、15和25 d的南瓜果实中,3 d后观察发现,授粉后2和5 d的果实转化后果肉呈微红,其中授粉后5 d的颜色较深,而10 d以上的无红色色素积累。经高效液相色谱(HPLC)分析色素成分,发现积累的红色色素为酮基类胡萝卜素角黄素和虾青素。与对照相比,注入CrBKT基因的授粉后2和5 d的幼果中的类胡萝卜素含量显著增加,5 d的幼果约增加了1/3,且含有106.31μg·g^(-1)酮类胡萝卜素,其中角黄素占80.65%,虾青素占19.35%,而授粉后10 d以上的果实类胡萝卜素含量没有明显变化,也没有酮类胡萝卜素的积累。PCR扩增表明转化组织中表达了该基因。结果表明,CrBKT基因只能在授粉后5 d以内的幼果中表达,并将幼果中的类胡萝卜素转化为酮基类胡萝卜素;随着果实成熟,菌液在果肉组织中无法渗透而阻碍基因表达。

In this study,transient expression system was used to analyze the function of β-ketocarotase for biosynthesis of ketocarotenoid in pumpkin fruit. The Agrobacteria that contained pBI121-CMTPCRBKT vector with Cr BKT gene and the control pBI121 vector were individually injected into the pumpkin fruits at 2,5,10,15 and 25 days after pollination,respectively. It was found that the fruit pulp was light reddish in 2-and 5-day fruits with darker color in pulp of 5-day fruit while there was no red pigment accumulated in more than 10-day fruit. High performance liquid chromatography（HPLC）analysis of pigment composition revealed that the accumulated red pigments were canthaxanthin and astaxanthin. Compared with the control,the carotenoid content in the 2-and 5-day fruits injected with the Cr BKT gene were significantly increased,which increased by about 1/3 in 5-day young fruit and ketocarotenoid content was 106.31 μg · g^（-1）,of which canthaxanthin accounted for 80.65%,astaxanthin was 19.35%,while the carotenoid content of the fruits in more than 10-day had no obvious change and there was no accumulation of ketocarotenoid. PCR amplification indicated that Cr BKT was expressed in the transformed tissue. The result showed that the Cr BKT gene could be only expressed in less than 5-day fruit and it could convert carotenoid in young pumpkin into ketocarotenoid. However,the expression of exogenous gene was discouraged due to the bacteria could not osmotic into the flesh tissue with the fruit ripening.