RNA干扰茄病镰刀菌ALP基因及其基因沉默菌株的制备

中国真菌学杂志　 2013, Vol. 8 　Issue (1): 10-13,19.

RNA干扰茄病镰刀菌ALP基因及其基因沉默菌株的制备

梁涛¹, 刘珂凤¹, 何宏², 赵桂秋¹, 王婷¹, 喻文倩¹

1. 青岛大学医学院附属医院眼科, 青岛 266003;
2. 青岛大学医学院附属医院检验科, 青岛 266003

收稿日期:2012-10-19 出版日期:2013-02-28 发布日期:2013-02-28
作者简介:梁涛,男(汉族),博士,副主任医师.E-mail:lt19722000@yahoo.com.cn

The study of inhibiting the expression of ALP in Fusarium solani by RNA interference and establishing the gene-silenced strains

LIANG Tao¹, LIU Ke-feng¹, HE Hong², ZHAO Gui-qiu¹, WANG Ting¹, YU Wen-qian¹

1. Department of Ophthalmology, Affiliated Hospital of Medical College of Qiingdao University, Qingdao 266003;
2. Department of clinical laboratory, Affiliated Hospital of Medical College of Qiingdao University, Qingdao 266003

Received:2012-10-19 Online:2013-02-28 Published:2013-02-28

摘要/Abstract

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ΔALP2抑制效果较好、酶活性显著低于标准菌株及△ALP1(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。

关键词: 茄病镰刀菌, 真菌性角膜炎, 丝氨酸蛋白酶, RNA干扰

Abstract: 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 ΔALP 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, ΔALP2 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.

Key words: Fusarium solani, keratitis/fungal, alkaline serine protease/serine protease, RNA interference

中图分类号:

R379.9

梁涛, 刘珂凤, 何宏, 赵桂秋, 王婷, 喻文倩. RNA干扰茄病镰刀菌ALP基因及其基因沉默菌株的制备[J]. 中国真菌学杂志, 2013, 8(1): 10-13,19.

LIANG Tao, LIU Ke-feng, HE Hong, ZHAO Gui-qiu, WANG Ting, YU Wen-qian. The study of inhibiting the expression of ALP in Fusarium solani by RNA interference and establishing the gene-silenced strains[J]. Chinese Journal of Mycology, 2013, 8(1): 10-13,19.

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