草菇菌丝应答低温胁迫的差异蛋白质组学研究

Volvariella volvacea (Bull. ex Fr.) Sing, commonly known as the Chinese Straw Mushroom, is an important edible fungus in tropics and subtropics. Low temperature injury has been found in this mushroom, which results in autolysis of mycelia. This study will be conducted to examine differential proteomic response to low temperature stress in mycelia of two Volvariella volvacea strains. One strain is V23, which is sensitive to low temperature stress; the other strain is VH3, which is mutated from V23 and less sensitive to low temperature stress than V23. Mycelia of the two strains will be exposed to 0℃ for 0, 2, 4, 6, 8 and 10 h, respectively. Several experiments will be performed, including:.1. Differential physiological response to low temperature stress in V23 and VH3 mycelia. Physiological parameters, such as electrolyte leakage, malondialdehyde (MDA) and soluble protein content will be measured. This experiment will be useful for the selection of protein samples analyzed in experiment 2 and 3. .2. Differential proteomic expression between low temperature stressed-mycelia and controls grown under normal temperature. This experiment will aid to understand the mechanism of mycelia autolysis under low temperature stress. .3. Differential proteomic response to low temperature stress in V23 and VH3 mycelia. Important proteins associated with improving the ability to low temperature stress in Volvariella volvacea will be found and identified. .4. Verification of identified proteins with good traits for better adaptation to low temperature stress in Volvariella volvacea. .5. Differential gene expression between V23 and VH3 mycelia under low temperature stress. RT-PCR will be used to analyze these genes coding proteins verified by western blotting in experiment 4. .All these work will provide important information for the breeding of new strains of Volvariella volvacea with better performance under low temperature stress.

草菇起源于我国热带与亚热带地区，属高温菇种，低温保藏时其菌丝体会发生自溶死亡，而从蛋白质组学角度揭示该机理的研究，尚未见报道。本项目拟以对低温耐受性不同的草菇菌株V23（低温敏感型）和VH3（V23诱变株，耐低温型）为材料，分析：1、低温胁迫对草菇菌丝生理生化指标的影响，以确定进行蛋白质组研究的取样点；2、与各自未进行低温处理的对照相比，研究V23与VH3菌丝中蛋白质表达随低温处理时间延长的改变，以剖析低温胁迫引起草菇菌丝自溶的原因；3、在各个相同低温处理时间下，比较V23与VH3菌丝中蛋白质表达的差异，并对差异蛋白点进行质谱鉴定，结合耐低温生理指标的变化差异，筛选草菇菌丝中与耐低温胁迫相关的蛋白质；4、采用Western印迹，进行耐低温特异蛋白的验证；5、运用RT-PCR技术，分析这些耐低温特异蛋白的基因表达情况，以期获得提高草菇耐低温能力的潜在基因，进而为后续的草菇低温育种工作服务。

草菇味道鲜美，营养丰富，源产于中国热带亚热带地区，属于高温菇种，不耐低温冷藏。在常规低温冷藏条件下（0-5℃），其菌丝体会发生自溶死亡，子实体亦会变软、液化，这一特性严重制约了鲜草菇的流通及其菌种的低温保藏，影响到草菇产业的快速发展，成为一个迫切需要解决的科学问题。本项目选用对低温耐受性不同的草菇菌株V23（低温敏感型）与VH3（V23诱变株，耐低温型）为材料，首先分析了低温胁迫对草菇菌丝相对电导率、丙二醛含量、可溶性蛋白含量等生理生化指标的影响，发现低温胁迫引起了草菇可溶性蛋白含量的改变，提高了细胞膜相对电导率与丙二醛含量，但上述指标在VH3中的变化幅度比在V23中要小；随后开展了低温胁迫下V23与VH3菌丝中蛋白质表达差异分析，质谱鉴定结果显示这些低温响应蛋白的功能涉及到代谢、能量、细胞生长/分化、蛋白质合成与降解以及逆境防御等多个过程，其中抗氧化酶和热激蛋白是构成差异蛋白质的重要组成部分；最后对耐低温特异蛋白在转录水平上的表达情况及其功能进行了验证，推测VH3之所以较V23耐低温能力更强，可能是由于Mn-SOD、CAT、Hsp60、Hsp70所编码基因在转录水平上的相对高表达，可以为后续草菇耐低温菌株选育的特异分子标记建立提供参考依据。