Microsoft Word tb170239何茂兰-y研究报告

Size: px

Start display at page:

Download "Microsoft Word tb170239何茂兰-y研究报告"

诲吴
5 years ago
Views:

1 微生物学通报 Microbiology China Sep. 20, 2017, 44(9): DOI: /j.microbiol.china 研究报告金属胁迫下灵芝寡肽转运蛋白基因家族的转录表达 * 何茂兰沈柯宇秦澎向泉桔 ( ) 摘要 : 目的寡肽转运蛋白(Oligopeptide transporters,opts) 通过细胞质膜, 将细胞外环境中的物质转入细胞内, 从而参与各种生理活动植物中 OPTs 蛋白参与金属运输和分配, 从而促进植物对金属的利用和适应, 但真菌中 OPTs 是否参与这一过程的相关报道较少探究 OPTs 是否在灵芝对金属的适应过程中发挥作用方法以灵芝荣保 1 号为材料, 利用平板实验分析不同浓度的 Cu 2+ Fe 2+ 和 Se 4+ 对菌丝体生长和生物量的影响, 并采用实时荧光定量 PCR 分析不同金属离子胁迫下, 两个培养阶段 (15 d 和 30 d) 灵芝寡肽转运蛋白基因 (OPTs) 的转录表达水平结果 Cu 2+ Fe 2+ 和 Se 4+ 三个金属离子在实验使用的浓度范围内抑制灵芝菌丝体的生长量和生物量, 且在相同处理条件下的生长量与生物量的变化趋势相同荧光定量 PCR 结果显示, 除未检测到转录本的 3 个基因 (OPT7 OPT8 和 OPT9), 其余 10 个基因均表现出了差异性表达模式在培养前期 (15 d), 有 6 个寡肽转运蛋白基因 (OPT1 OPT3-6 和 OPT10) 的相对表达量在 Cu 2+ 的胁迫下发生上调, 而在 Fe 2+ 和 Se 4+ 的胁迫下, 所有的 OPTs 基因的表达量均被下调在培养后期 (30 d),se 4+ 胁迫下的 OPTs 基因的表达量仍旧被下调, 而 Cu 2+ 和 Fe 2+ 的胁迫下表达量均显著上调结论金属能够在不同的程度上影响灵芝的生长量和生物量, 同时寡肽转运蛋白基因对这些金属胁迫在转录水平上做出了响应, 表明寡肽转运蛋白基因可能在灵芝对金属的适应过程中发挥作用关键词 : 生长量, 生物量, 寡肽转运蛋白基因, 金属胁迫 Transcriptional analysis of oligopeptide transporter gene family under metal stress of Ganoderma lucidum HE Mao-Lan SHEN Ke-Yu QIN Peng XIANG Quan-Ju * (Department of Microbiology, College of Resources, Sichuan Agricultural University, Chengdu, Sichuan , China) Abstract: [Objective] Substrates in the environment can be transported into the cell by oligopeptide transporters (OPTs), and then contribute to various biological processes. OPTs proteins are involved in metal transports and distributions in plants, which can promote the plants to use and adapt various metals. However, if OPTs from fungi participates in this process is still unknown. To explore if OPTs Foundation item: Education Office of Sichuan Province (No. 15ZA0013) *Corresponding author: Tel: ; xiangquanju@163.com Received: March 23, 2017; Accepted: May 19, 2017; Published online ( May 31, 2017 基金项目 : (No. 15ZA0013) * 通讯作者 :Tel xiangquanju@163.com 收稿日期 : 接受日期 : 优先数字出版日期 (

2 : 2121 play a role in the adaptation process of Ganoderma lucidum to metals. [Methods] Mycelium growth and biomass of G. lucidum (No. 1 Rongbao) were tested in the presence of different concentrations of Cu 2+, Se 4+ and Fe 2+. The specific or differential expression profiles of OPTs were analyzed by real-time quantitative PCR (RT-PCR) from the samples cultured for 15 d and 30 d. [Results] Growth and biomass were inhibited under all metal concentrations of Cu 2+, Fe 2+ and Se 4+. The results of RT-PCR showed that, except three genes (OPT7, OPT8 and OPT9), whose transcripts were not detected, the other 10 OPT genes have differential expression profiles. At the early culture stage (15 d), the relative expression levels of 6 OPT genes (OPT1, OPT3-6 and OPT10) were up-regulated under Cu 2+ stress, but the expression levels of all OPTs gene were down-regulated under Fe 2+ and Se 4+ treatments. At the later culture stage (30 d), the relative expression levels of all OPTs gene were down-regulated under Se 4+ stress, but the expression levels were significantly up-regulated under Cu 2+ and Fe 2+ treatments. [Conclusion] These results indicate that these metals can affect the growth and biomass of G. lucidum, while the oligopeptide transporter genes responded to metal stress at transcriptional levels, suggesting that OPT genes may function in the metal adaption processes in G. lucidum. Keywords: Growth quantity, Biomass, Oligopeptide transporter gene, Metal stress ( ) [1-2] ( ) [3-4] (Oligopeptide transport protein OPT) OPT [5] OPT AtOPT [6] OPT BjGT1 [7] OsOPT7 [8] OPT OPT 8 OPT [9] 14 OPT [10] OPT [11] 13 OPT OPT [12] OPT 1 Cu 2+ Fe 2+ Se 4+ OPTs OPT

3 2122 微生物学通报 Microbiol. China 2017, Vol.44, No.9 1 材料与方法 1.1 菌种主要试剂和仪器 (Ganoderma lucidum) 1 Pb(NO 3 ) 2 CdCl 2 2½H 2 O CuSO 4 5H 2 O FeSO 4 7H 2 O Na 2 SeO 3 FastQuant cdna ( ) SYBR Green Master Mix DHP-9082 GI54DWS AIRTECH iq5 PCR Bio-Rad 1.2 金属离子标准液与培养基 CuSO 4 5H 2 O FeSO 4 7H 2 O Na 2 SeO 3 50 g/l CuSO 4 5H 2 O Pa 30 min FeSO 4 7H 2 O Na 2 SeO μm PDA PDB PDA Cu mg/kg Fe 2+ Se mg/kg OPT 3 Cu mg/kg Fe 2+ Se mg/kg 15 d 30 d 1.3 培养方法 ( D=1 cm) PDA 30 C 7 d 3 ( D=1 cm) PDB 30 C 15 d 30 d RNA 菌丝体生长量与生物量的测定 7 d 7 d 50 C 1.5 总 RNA 提取 cdna 合成和 qrt-pcr 15 d 30 d TRIzol RNA [13] 1% RNA FastQuant cdna cdna gdna (10 μl) Total RNA 2 μl 5 gdna buffer 2 μl RNase-Free Water 6 μl 42 C 3 min (10 μl) 10 Fast RT Buffer 2 μl RT Enzyme Mix 1 μl FQ-RT Primer Mix 2 μl RNase-FreeWater 5 μl gdna 42 C 15 min 95 C 3 min cdna cdna 20 C cdna SYBR Green Master Mix OPT RT-PCR RT-PCR Bio-Rad iq5 PCR PCR (20 μl) 2 Green Master Mix 10 μl 10 μmol/l Forward Reverse 0.2 μl 50 Rox Reference Dye1 0.4 μl ng cdna 2 μl ddh 2 O 20 μl 95 C 5 min 95 C 10 s 60 C 30 s 40 RPL4 [14] OPTs [12] C t Y /3 100 ΔC t OPTs C q [15-17]

4 : 数据分析方法 Excel SPSS 2 结果与分析 2.1 Fe 2+ Cu 2+ 和 Se 4+ 对灵芝菌丝生长的影响 PDA 7 d 1 Cu 2+ Se 4+ Se mg/kg Fe 2+ ( mg/kg) Fe 2+ ( mg/kg) 2.2 Fe 2+ Cu 2+ 和 Se 4+ 对灵芝菌丝体生物量的影响 2 Fe mg/kg Cu 2+ Se 4+ Se mg/kg Cu 2+ Se 4+ 和 Fe 2+ 三种金属胁迫下灵芝 OPTs 基因的转录表达 PCR OPT 3 OPT (OPT7 OPT8 OPT9) 10 ( 3) 15 d Cu 2+ 6 (OPT1 OPT3 OPT4 OPT5 OPT6 OPT10) OPT 图 1 ρ(fe 2+ ) ρ(cu 2+ ) 和 ρ(se 4+ ) 对灵芝菌丝生长的影响 Figure 1 Effects of ρ(fe 2+ ) ρ(cu 2+ ) and ρ(se 4+ ) on the growth of G. lucidum A Fe 2+ B Cu 2+ C Se 4+. Note: A: Effects of different concentrations of Fe 2+ on the growth of G. lucidum; B: Effects of different concentrations of Cu 2+ on the growth of G. lucidum; C: Effects of different concentrations of Se 4+ on the growth of G. lucidum.

5 2124 微生物学通报 Microbiol. China 2017, Vol.44, No.9 (OPT2 OPT11 OPT12 OPT13) OPT Se OPT Fe 2+ OPT4 OPT5 OPT d OPT Cu d OPT Se 4+ 4 (OPT1 OPT5 OPT6 OPT10) OPT (OPT2 OPT3 OPT4 OPT11 OPT12 OPT13) OPT Fe 2+ OPT OPT4 ( 14 ) OPT3 ( ) (15 d) OPT3 Fe 2+ (30 d) OPT3 Fe 2+ 3 讨论 [18-19] [20] 图 2 ρ(fe 2+ ) ρ(cu 2+ ) 和 ρ(se 4+ ) 对灵芝菌丝体生物量的影响 Figure 2 Effects of ρ(fe 2+ ) ρ(cu 2+ ) and ρ(se 4+ ) on the biomass of G. lucidum A Fe 2+ B Cu 2+ C Se 4+. Note: A: Effects of different concentrations of Fe 2+ on the biomass of G. lucidum; B: Effects of different concentrations of Cu 2+ on the biomass of G. lucidum; C: Effects of different concentrations of Se 4+ on the biomass of G. lucidum. [21] [22] 0.2% Fe 2+

6 : 2125 图 3 OPTs 在不同金属胁迫下不同培养阶段的相对表达量 Figure 3 Relative expression levels of OPTs under different metal treatments and culture stages A B C D E F G H I J 10 (OPT1 OPT2 OPT3 OPT4 OPT5 OPT6 OPT10 OPT11 OPT12 OPT13) (Fe 2+ Cu 2+ Se 4+ ) (15 d 30 d). Note: A, B, C, D, E, F, G, H, I, and J represent the transcriptional expression levels of OPTs in different metal ion (Fe 2+, Cu 2+ and Se 4+ ) stresses and two culture stages (15 d, 30 d).

7 2126 微生物学通报 Microbiol. China 2017, Vol.44, No.9 [23] S3 Cu mg/kg [21] Fe 2+ Cu 2+ Se 4+ [24-25] PCR (OPT7 OPT8 OPT9) 3 10 (15 d 30 d) (15 d) Cu 2+ Fe 2+ Se 4+ (30 d) Cu 2+ Fe 2+ OPT Se 4+ OPT Fe 2+ OPT2 9 OPT Cu 2+ OPT [6] [8] [26] OPT Fe 2+ OPT Cu 2+ OPT Cu 2+ (30 d) Cu 2+ OPT OPT 4 结论 3 Cu 2+ Fe 2+ OPT Fe 2+ OPT3 ( ) OPT Cu 2+ 参考文献 [1] Zang J, Luo JC, Huang KX. Studies on screening of the strains for production of Ganoderma lucidum polysaccharide by submerged fermentation and optimizating the culture conditions[j]. Modern Food Science and Technology, 2007, 23(9): (in Chinese),,. [J]., 2007, 23(9): [2] Ma XK, Ling WL, Fam H. Heavy metal ions affecting the removal of polycyclic aromatic hydrocarbons by fungi with heavy-metal resistance[j]. Applied Microbiology and Biotechnology, 2014, 98(23): [3] Zhu HL, Ban LT, Xu XP. Study progress on the tolerating and accumulating mechanism of edible mushrooms on heavy metals[j]. Journal of Anhui Agricultural Sciences, 2011, 39(13): (in Chinese),,. [J]., 2011, 39(13): [4] Radulescu C, Stihi C, Busuioc G, et al. Evaluation of essential elements and heavy metal levels in fruiting bodies of wild mushrooms and their substrate by EDXRF spectrometry and FAA spectrometry[j]. Romanian Biotechnological Letters, 2010, 15(4): [5] Wang ZS, Xiang QJ, Wang GJ, et al. Study of Phanerochaete chrysosporium oligopeptide transporter gene family[a]// Chinese Genetics Society Meeting[C]. Urumqi: Genetic Institute of Xinjiang Uygur Autonomous Region, 2011 (in Chinese),, 珺,. [A]// 2011 [C]. :, 2011 [6] Stacey MG, Patel A, McClain WE, et al. The arabidopsis AtOPT 3 protein functions in metal homeostasis and movement

8 : 2127 of iron to developing seeds[j]. Plant Physiology, 2007, 146(2): [7] Bogs J, Bourbouloux A, Cagnac O, et al. Functional characterization and expression analysis of a glutathione transporter, BjGT1, from Brassica juncea: evidence for regulation by heavy metal exposure[j]. Plant, Cell & Environment, 2003, 26(10): [8] Bashir K, Ishimaru Y, Itai RN, et al. Iron deficiency regulated OsOPT7 is essential for iron homeostasis in rice[j]. Plant Molecular Biology, 2015, 88(1/2): [9] Reuß O, Morschhäuser JM. A family of oligopeptide transporters is required for growth of Candida albicans on proteins[j]. Molecular Microbiology, 2006, 60(3): [10] Xiang QJ, Wang ZS, Zhang YZ, et al. An oligopeptide transporter gene family in Phanerochaete chrysosporium[j]. Gene, 2013, 522(2): [11] Tang QJ, Luo X, Liu YF, et al. Study on separation and purification low molecular weight of Ganoderma lucidum polysaccharide and in vitro immune activity[c]. International Symposium on Ganoderma Research, 2011 (in Chinese),,,. [C]. 2011, 2011 [12] Xiang QJ, Shen KY, Yu XM, et al. Analysis of the oligopeptide transporter gene family in Ganoderma lucidum: structure, phylogeny, and expression patterns[j]. Genome, 2017, 60(4): [13] Meng L, Feldman L. A rapid TRIzol-based two-step method for DNA-free RNA extraction from Arabidopsis siliques and dry seeds[j]. Biotechnology Journal, 2010, 5(2): [14] Xu J, Xu ZC, Zhu YJ, et al. Identification and evaluation of reference genes for qrt-pcr normalization in Ganoderma lucidum[j]. Current Microbiology, 2014, 68(1): [15] Chen AQ, He SE, Li FF, et al. Analyses of the sucrose synthase gene family in cotton: structure, phylogeny and expression patterns[j]. BMC Plant Biology, 2012, 12(1): 85 [16] Liu ZL, Palmquist DE, Ma MG, et al. Application of a master equation for quantitative mrna analysis using qrt-pcr[j]. Journal of Biotechnology, 2009, 143(1): [17] Chen AQ, Chen X, Wang HM, et al. Genome-wide investigation and expression analysis suggest diverse roles and genetic redundancy of Pht1 family genes in response to Pi deficiency in tomato[j]. BMC Plant Biology, 2014, 14(1): 61 [18] Tang LH. Study of mechanism with light-induced brown film formation in Lentinula edodes[d]. Shanghai: Doctoral Dissertation of Shanghai Jiao Tong University, 2014 (in Chinese). [D]. :, 2014 [19] Hu XG, Liu WM, Liu J, et al. The influence of Zn and La on the Grifola frondosa s mass production in liquid culture[j]. Science and Technology of Food Industry, 2005(1): (in Chinese),,,. Zn La [J]., 2005(1): [20] Xie BG, Liu JY. Effects of heavy metals of Pb, Cd, Hg and As on the growth of three edible fungis[j]. Edible Fungi of China, 2005, 24(2): (in Chinese),. [J]., 2005, 24(2): [21] Zhang XN. Studies on the effects of four heavy-metals bioaccumulation in Ganoderma lucidum[d]. Beijing: Peking Union Medical College, 2007 (in Chinese). [D]. :, 2007 [22] Cai AQ, Zhang PC. Effect of calcium, iron and manganese on mycelial biomass production by Ganoderma lucidum and ganoderma tsugae grown in submerged culture[j]. Acta Edulis Fungi, 2011, 18(4): (in Chinese),. [J]., 2011, 18(4): [23] Ye SQ, Wu H, Yu YG, et al. Research for different metal ion in Ganoderma lucidum polysaccharides fermentation[j]. Food Research and Development, 2011, 32(1): (in Chinese),,,. [J]., 2011, 32(1): [24] Hartmann T, Cairns TC, Olbermann P, et al. Oligopeptide transport and regulation of extracellular proteolysis are required for growth of Aspergillus fumigatus on complex substrates but not for virulence[j]. Molecular Microbiology, 2011, 82(4): [25] Hu YT, Ming F, Chen WW, et al. TcOPT3, a member of oligopeptide transporters from the hyperaccumulator Thlaspi caerulescens, is a novel Fe/Zn/Cd/Cu transporter[j]. PLoS One, 2012, 7(6): e38535 [26] Yen MR, Tseng YH, Saier MH Jr. Maize Yellow Stripe1, an iron-phytosiderophore uptake transporter, is a member of the oligopeptide transporter (OPT) family[j]. Microbiology, 2001, 147(11):

Microsoft Word - 793-797 tb20150504赵宏宇s-高校教改纵横.doc

Microsoft Word - 793-797 tb20150504赵宏宇s-高校教改纵横.doc 微生物学通报 Microbiology China tongbao@im.ac.cn Apr. 20, 2016, 43(4): 793 797 http://journals.im.ac.cn/wswxtbcn DOI: 10.13344/j.microbiol.china.150504 高校教改纵横生物工程专业发酵课程群建设探索 * 赵宏宇