茄病镰刀菌ALP小干扰RNA载体的构建与表达

Construction and Expression of Alkaline Serine Protease Small Interfering RNA eukaryotic Vector in Fusarium solani

[目的]进一步研究碱性丝氨酸蛋白酶(ALP)在真菌性角膜炎发病机制中的作用,寻找有效的治疗措施。[方法]根据载体质粒pBC-hygro要求设计两对小干扰RNA,退火后连接于载体相应位点,经双酶切及测序鉴定后,分别转染标准茄病镰刀菌株,得到菌株ΔALP1和ΔALP2。采用蛋白琼脂廓清特殊培养基检测酶活性变化,Western-blot检测ALP的表达,并与标准茄病镰孢菌比较。[结果]检测ΔALP1菌株、ΔALP2菌株和对照菌株各6份,其CH值标准株为0.80±0.01,ΔALP1为0.80±0.03,ΔALP2为0.86±0.03,ΔALP1与标准株的差异无统计学意义(P>0.05),ΔALP2大于标准菌和ΔALP1株(P<0.01)。ALP蛋白条带的积分值(INT×mm2),标准株为1 240.29±289.77,ΔALP1为603.58±120.55,ΔALP2为401.84±117.94,ΔALP1与ΔALP2均低于标准株(P<0.01),ΔALP1高于ΔALP2(P<0.05)。[结论]成功地构建了ALP小干扰RNA载体及稳定转染菌株。

[Objective]To explore the role of ALP in the mechanism of Fusarium keratitis and find a novel therapeutic strategy. [Methods]Two pairs of hairpin siRNA template oligonucleotides for ALP based on pBC-hygro vector were designed using the manufacturer＇s web-based tool. These oligonucleotides were annealed and ligated into vector respectively. After confirmation by double enzyme digestion analysis and DNA sequencing, positive recombinant plasmids were transfected into Fusarium solani. The strains were named after △ALP1, △ALP2. Enzymic activities were analysized using special culture medium and gene-silenced strains △ALP were thus screened out. Thus the strain virulence was assessed. The reduction of protein of ALP was detected by Western-blotting. [Results] The CH were 0. 80 ±0.01,0.80±0. 03,0. 86±0. 03 respectively in the group of standard strain,△ALP1 ,△ALP2. The first two group had no differences（ P 〉0.05） ,but the group of △ALP2 was larger than the △ALPI（ P 〈0.01）. The protein strip integral value of the three groups was 1 240.29±289.77（INT ± mm2 ）, 603.58 ± 120. 55 （INT ×mm^2 ）, 401.84 ± 117.94 （INT ×mm^2 ） respectively. The groups of △ALP1 and △ALP2 were both less than the control（ P 〈0.01）. The group of △ALP1 was larger than the △ALP2 （ P 〈0.01）. [Conclusion]The ALP siRNA eukaryotic expression is successfully constructed and stably expressed in the Fusarium solani.