摘要
在一些可合成苦马豆素(SW)的真菌中,吡咯啉-5-羧酸还原酶(P5CR)可催化哌啶酸(PA)的生物合成。为了研究P5CR基因对真菌苦马豆素合成的影响,本试验克隆了小花棘豆内生真菌棘豆链格孢(Alternaria oxytropis)OW7.8的P5CR基因,并对其进行生物信息学预测;利用实时荧光定量PCR(qRT-PCR)技术分析不同培养时间(23 d、26 d、29 d、32 d)下处理组[培养基添加L-哌啶酸(L-PA)]和对照组菌丝体中P5CR基因表达水平;利用高效液相色谱-质谱联用(HPLC-MS)技术检测菌丝体中苦马豆素水平。预测结果显示,P5CR基因序列从ATG到TAG为1179 bp,开放阅读框(ORF)长954 bp,有1个内含子,编码317个氨基酸,分子量为33297.32 D,等电点为6.14,是稳定疏水性蛋白,无信号肽,可能有跨膜结构域,二级结构主要以随机卷曲为主。qRT-PCR结果显示,随培养时间延长,处理组P5CR基因表达水平先下降后上升,对照组P5CR基因表达水平逐渐下降。HPLC-MS结果显示,处理组菌丝体中苦马豆素水平均高于对照组(P<0.05)。结果表明,添加L-PA可促进真菌苦马豆素合成,P5CR基因对真菌合成苦马豆素有重要影响。
In some swainsonine(SW)producing fungi,pyrroline-5-carboxylate reductase(P5CR)catalyzes biosynthesis of pipecolic acid(PA).To study the effect of P5CR gene on swainsonine biosynthesis in fungi,the P5CR gene in endophytic fungus Alternaria oxytropis OW7.8 from Oxytropis glabra DC.was cloned and predicted by bioinformatics.Quantitative real-time PCR(qRT-PCR)was used to analyze the expression levels of P5CR gene in mycelia at different culture times(23 d,26 d,29 d,32 d)in treatment group[media supplemented with L-pipecolic acid(L-PA)]and control group.The swainsonine levels in mycelia were determined by high-performance liquid chromatography-mass spectrometry(HPLC-MS).The results showed that the length of P5CR gene sequence from ATG to TAG was 1179 bp;the length of the open reading frame(ORF)was 954 bp,encoding 317 amino acids,including one intron.The molecular weight of P5CR protein was 33297.32 D and the isoelectric point was 6.14,which was predicted as a stable hydrophobic protein with no signal peptide and a transmembrane domain structure,and its secondary structure was mainly random curls existing most likely in the cytoplasm.The qRT-PCR results showed that with the extension of culture time,the expression levels of P5CR gene in the treatment group decreased at first and then increased,while in the control group decreased gradually.The HPLC-MS results showed that the swainsonine levels in mycelia in the treatment group were higher than those in the control group(P<0.05).These results indicate that addition of L-PA promotes swainsonine synthesis in A.oxytropis OW7.8,and the P5CR gene plays an important role in the synthesis of swainsonine in this fungus.
作者
白璐
卢萍
王钰
珠丽
袁博
高峰
杜玲
BAI Lu;LU Ping;WANG Yu;ZHU Li;YUAN Bo;GAO Feng;DU Ling(College of Life Science and Technology,Inner Mongolia Normal University,Hohhot 010022,China)
出处
《中国兽医杂志》
CAS
北大核心
2023年第4期33-40,共8页
Chinese Journal of Veterinary Medicine
基金
国家自然科学基金项目(31960130,31460235,30860049)
内蒙古自然科学基金项目(2022MS03014)。
关键词
内生真菌
苦马豆素
吡咯啉-5-羧酸还原酶
生物信息学
高效液相色谱-质谱联用
endophytic fungi
swainsonine
pyrroline-5-carboxylate reductase
bioinformatics
high-performance liquid chromatography-mass spectrometry
