【作者】 王艳；

【导师】 王杰； 姜子德；

【作者基本信息】 华南农业大学 ， 食品科学， 2019， 硕士

【摘要】 随着市场需求和生产量的逐年增长,杏鲍菇贮运保鲜已成为限制其产业可持续发展的关键环节。本课题组前期研究发现,杏鲍菇在采后冷藏过程中会发生两次木质化引起的质地劣变,严重影响其商品价值。本论文对不同调控处理下杏鲍菇质地性状和主要生理指标进行分析,进一步探究杏鲍菇采后木质化的主要诱因,为控制杏鲍菇采后木质化劣变开发新策略提供理论指导;克隆、预测木质素生物合成关键酶基因PePAL2和Pe4CL3启动子序列,并结合转录组测序结果筛选参与调控PePAL2和Pe4CL3表达相关的转录因子,为阐明杏鲍菇采后木质化的分子机理奠定基础。主要研究结果如下:1.不同调控处理下杏鲍菇采后质地及主要生理变化杏鲍菇在四种不同调控处理条件下（A,1℃、0.05 mm自封袋、损伤;B,4℃、0.05 mm自封袋、损伤;C,4℃、0.07 mm自封袋、损伤;D,4℃、0.05 mm自封袋、无损伤）下贮藏21 d,测定其质地指标及主要生理变化。结果显示,所有处理组硬度、韧度均出现至少两次的显著增加峰值,其中无损伤处理D在贮藏0-3d硬度、韧度显著下降,其他损伤处理则显著上升至第一次峰值。贮藏前期,相比其他损伤处理组,无损伤处理D的CO₂/O₂浓度变化平缓,超氧阴离子、羟基自由基、H₂O₂累积量与抗氧化酶SOD、CAT活性变化一致,POD酶活相对最低,MDA、电导率增加最少,线粒体能量代谢酶SDH、CCO、Ca²⁺-ATPase和H⁺-ATPase活性变化最为平缓且相对较高,Pe4CL3基因表达最低,表明损伤是采后杏鲍菇第一次木质化的主要诱因。贮藏后期杏鲍菇相应生理变化则表明,以活性氧代谢失衡、细胞膜降解和线粒体功能紊乱为表征的细胞衰老是第二次木质化的主要诱因。2.PePAL2和Pe4CL3启动子的克隆及预测分析根据前期研究所得PePAL2和Pe4CL3的cDNA序列使用Hi-tail和I-PCR法分别获得PePAL2和Pe4CL3上游的5’端侧翼序列部分,长度分别为964 bp和1087 bp。使用3个转录因子数据库分别预测所获得的序列。结果发现,两个片段均具有数量不同的核心启动子元件,并存在低温、茉莉酸甲酯等多个响应元件,且PePAL2和Pe4CL3启动子上分别具有1个MYB转录因子识别位点（序列为CCGTTG）/1个参与干旱诱导的MYB结合位点（序列为CAACTG）和2个MYB结合位点（序列分别为TAACTG和CAACCA）,表明PePAL2和Pe4CL3基因的表达可能均受低温、缺氧等逆境胁迫和茉莉酸甲酯等植物激素刺激诱导或增强,且可能受MYB类转录因子的调控。3.参与PePAL2和Pe4CL3表达调控转录因子的筛选和表达特征分析基于不同调控处理下关键贮藏期杏鲍菇的RNA-seq结果,结合PePAL2和Pe4CL3启动子预测分析结果、贮藏期PePAL2和Pe4CL3的表达情况,筛选并分析参与PePAL2和Pe4CL3表达调控的转录因子基因。差异表达基因功能注释预筛选出6条可能参与杏鲍菇两次木质化的转录因子unigenes。unigenes表达模式（rpkm）显示预筛选unigenes中有5条候选转录因子unigenes的表达模式与PePAL2/Pe4CL3的表达趋势或杏鲍菇两次木质化劣变趋势一致。荧光定量PCR检测结果发现,只有PeMYB1/PeMYB2与Pe4CL3的表达显著相关（P<0.01,r=0.898/0.726）。表达特征分析发现,贮藏前期所有损伤处理组的PeMYB1和Pe4CL3表达相较于无损伤处理组均显著上调,整个贮藏期低温处理组PeMYB1相较于对照处理组显著上调,而PeMYB2在贮藏前期低温处理组相较于对照处理组表达显著上调。这表明PeMYB1可能受损伤和低温胁迫刺激分别在贮藏前期和整个贮藏期调控Pe4CL3或其他木质素合成相关基因表达增强,PeMYB2在贮藏前期可能响应低温胁迫参与第一次木质化相关基因的转录调控。更多还原

【Abstract】 The storage and preservation of Pleurotus eryngii has become a key to limit the sustainable development of its industry,as the increase of market demand and production year by year.Our previous study showed that an unexpected texture decay induced by lignification always occurred in Pleurotus eryngii stored at low temperature,which seriously affected its commodity value.In this study,the intrinsic active metabolites in Pleurotus eryngii under different stored conditions were analyzed,and the main inducers of postharvest lignification in Pleurotus eryngii were investigated,to provide theoretical guide for developing the new strategies of controlling the texture decay induced by lignification.The promoters of lignin biosynthesis key enzyme genes PePAL2 and Pe4CL3 were cloned,and combined with RNA-seq results,the transcription factors involved in the regulation of PePAL2 and Pe4CL3 were screened,to provide basis for elucidating the molecular mechanism of postharvest lignification in Pleurotus eryngii.The main results are as follows:1.Postharvest texture and main physiological changes of Pleurotus eryngii under different stored conditionsPleurotus eryngii was stored at four different conditions(A,1 °C,0.05 mm ziplock bags,wounding;B,4 °C,0.05 mm ziplock bags,wounding;C,4 °C,0.07 mm ziplock bags,wounding;D,4 °C,0.05 mm ziplock bags,no wounding)for 21 d,to determinate the texture indicators and major physiological changes.The results showed that the firmness and hardness of all treatment groups showed two significantly increased peaks at least.The hardness and firmness of the no wounding treatment D significantly decreased from 0 to third day,while that of other wounding treatments significantly increased to the first peak.During the previous storage,compared with other wounding groups,in no wounding treatment D,the change of CO2/O2 concentration was mild,the accumulation of superoxide anion,hydroxyl radical and H2O2 was consistent with the activities of antioxidant enzymes SOD and CAT,the activity of POD was relatively low,the increase of MDA and conductivity was the least,the changes of mitochondrial energy metabolism enzymes SDH,CCO,Ca2+-ATPase and H+-ATPase were the most gradual and activities were relatively high,and the expression level of Pe4CL3 gene was the lowest.These results indicated that the wounding was the main factor of inducing the first lignification in stored Pleurotus eryngii.The corresponding physiological changes of Pleurotus eryngii during the later storage showed that cell senescence,characterized by imbalance of reactive oxygen species metabolism,cell membrane degradation and mitochondrial dysfunction,was the main inducer of the second lignification.2.Cloning and predictive analysis of PePAL2 and Pe4CL3 promotersAccording to the cDNA sequences of PePAL2 and Pe4CL3 obtained in the previous study,the 5’ flanking sequences upstream of PePAL2 and Pe4CL3 were obtained by Hi-tail and I-PCR,respectively.The lengths were 964 bp and 1087 bp,respectively.The obtained sequences were predicted using three transcription factor databases.It was found that both fragments had a number of different core promoter elements and multiple response elements such as low temperature response elements,methyl jasmonate response elements.MYB transcription factor recognition site(sequence was CCGTTG)/ 1 MYB binding site involved in drought induction(sequence was CAACTG)and two MYB binding sites(TAACTG and CAACCA)were also both found in the PePAL2 and Pe4CL3 promoters,respectively.These results indicated that the expression of PePAL2 and Pe4CL3 genes may be affected by some stresses,such as low temperature,hypoxia,and some plant hormone such as methyl jasmonate,and their expression may also be regulated by MYB transcription factors.3.Screening and expression analysis of transcription factors involved in the regulation of PePAL2 and Pe4CL3 expressionBased on the RNA-seq results of Pleurotus eryngii under different treatment conditions,taken together the predicted results of PePAL2/Pe4CL3 promoters and the expression of PePAL2/Pe4CL3 during storage,the transcription factors involved in the regulation of PePAL2/Pe4CL3 expression were screened and analyzed.Six transcription factors unigenes,which may be involved in the two lignification of Pleurotus eryngii,was pre-screened according to the functional annotation of differentially expressed genes.The unigenes expression pattern(rpkm)showed that the expression patterns of five candidate transcription factors unigenes in pre-screened six unigenes were consistent with the expression trends of PePAL2/Pe4CL3 or the two lignification trends of Pleurotus eryngii.The results of real-time PCR confirmed that only PeMYB1/PeMYB2 was significantly correlated with the expression of Pe4CL3(P<0.01,r=0.898/0.726).The expression analysis showed that the expression of PeMYB1 and Pe4CL3 in all wounding treatment groups was significantly up-regulated compared with the non-wounding treatment groups in the previous storage,the expression of PeMYB1 in low-temperature treatment group was significantly up-regulated compared with the control treatment group during the whole storage,and the expression of PeMYB2 in low temperature treatment group was significantly up-regulated compared with the control treatment group during the previous storage.These results indicated that PeMYB1 may be stimulated by wounding and low temperature stress to enhance the expression Pe4CL3 or other lignin synthesis related genes during the previous and whole storage,respectively.And PeMYB2 may be involved in the transcriptional regulation of the first lignification-related genes in response to low temperature during the previous storage.更多还原