RNA干扰茄病镰刀菌ALP基因及其基因沉默菌株的制备

摘要

目的 构建RNA干扰质粒载体抑制茄病镰刀菌碱性丝氨酸蛋白酶(ALP)基因,通过检测ALP的表达及酶活性变化筛选出基因沉默菌株.方法 构建2个ALP的干扰载体,转化茄病镰刀菌孢子,获得ALP基因沉默菌株△ALP1、△ALP2,通过RT-PCR检测△ALP1、△ALP2的ALP基因mRNA变化,并通过蛋白琼脂廓清率培养基检测ALP酶活性变化,对所得菌株的毒力进行判断.结果 RNA干扰后所获基因沉默菌株中,ALP的mRNA表达量显著低于标准茄病镰刀菌株(F =184.67,P＜0.01),其中△ALP2抑制效果较好、酶活性显著低于标准菌株及△ALPI(q=5.276、5.463,P＜0.01).结论 通过LiAc转化法对茄病镰刀菌ALP进行RNA干扰,可有效抑制茄病镰刀菌ALP的表达；ALP基因沉默茄病镰刀菌株的获取,为进行动物体内实验、深入研究ALP在茄病镰刀菌性角膜炎发病机制中的作用、探索新型治疗方法提供思路.%Objective To inhibit the expression of ALP gene in Fusarium solani by RNA interference using constructed plasmid vectors and to identifiy the gene-silenced strains by detecting the expression and enzymic activities of ALP. Methods Two kinds of vectors pBC-hygro were transformed into Fusarium solani by dsRNAi. The strains we got were named after △ ALP1, △ ALP2, respectively. The reduction of mRNA in Fusarium solani was detected by RT-PCR. Enzymic activities were analysized using special culture medium. The gene-silenced strains △ALP were screened out, and the strain virulence was assessed. Results The expression of mRNA of ALP in the gene-silenced Fusarium solani strains were significantly inhibited ( F = 184. 67, P < 0. 01) . Among the gene-silenced Fusarium solani strains, △ALP2 achieved better inhibitory effect on enzymatic activities than other strains ( q =5.276,5.463, P <0. 01) did. Conclusions RNA interference in this study can significantly downregulate the expression of ALP gene in Fusarium solani . The results provide us the gene-silenced Fusarium solani strains for promoting animal experiments in vivo and exploring the mechanism of the ALP in keratitis caused by Fusarium solani . Thus we might find a new therapeutic method for curing the disease.