【作者】 张迎辉；

【导师】 郑郁善；

【作者基本信息】 福建农林大学 ， 园林植物与观赏园艺， 2014， 博士

【摘要】 福建山樱花(Prunus campanulata Maxim.)每年春节前后开花,花期长、花色艳丽,花型独特,观赏价值极高,而且适应性广、抗逆性强,为园林绿地中不可多得的优良乡土观赏植物。但从原产地向北引种到长江流域或以北地区后,受到冬季低温的影响,会引起开花稀少甚至遭受冻害。低温作为最常见的制约植物生长发育的环境因素之一,也是影响福建山樱花向更高纬度应用的主要限制因子。本研究对1年生福建山樱花幼苗进行低温胁迫处理,测定叶片的各项生理生化指标,找出胁迫的临界温度、临界时间,从生理角度探讨福建山樱花防御低温伤害的机制；在此基础上,以临界温度临界时间处理的福建山樱花叶片为实验材料,采用SMART法构建福建山樱花低温胁迫全长cDNA文库,对得到的部分EST片段进行测序和生物信息学分析,并在此基础上筛选受低温胁迫影响的基因；通过实时荧光定量PCR检测分析了不同温度梯度处理下各基因的表达变化,详细分析不同基因在不同时期的表达规律,将生理测定的结果与分子生物学的结果结合起来,以期进一步为福建山樱花耐寒品种选育奠定理论依据。具体研究结果如下：1、对福建山樱花的叶绿素含量与叶绿素荧光动力学参数进行测定,福建山樱花在6℃和1℃处理叶绿素含量均呈现出下降的趋势,温度越低下降趋势更明显,1℃处理胁迫第5d时,与对照差异达到极显著水平(P<0.01)。与对照相比,随温度下降,福建山樱花的初始荧光(F0)不断上升,叶绿素含量、PS Ⅱ最大光化学效率(Fv/Fm)、光化学猝灭系数(qP)不断下降；10℃处理的实际光化学效率(Yield)和光合电子传递速率(ETR)明显高于对照,而6℃和1℃处理则随胁迫时间不断下降；非光化学猝灭系数(qN)10℃和6℃处理呈上升趋势而1℃处理则不断下降。1℃处理的低温胁迫参数的变化最为显著,表明低温对福建山樱花幼苗产生了显著的光抑制,进一步表明福建山樱花只有在一定温度和时间范围内通过对PS Ⅱ活性和电子传递的调节来提高其耐冷性,保护光合机构免受伤害。2、不同温度梯度处理下福建山樱花各生理指标测定结果与对照相比,差异性显著；各温度下相对电导率和MDA含量随胁迫时间呈不断上升趋势,其余指标变化规律相似,随胁迫时间延长在10℃处理时呈缓慢上升趋势,在6℃和1℃处理下呈先升后降趋势,最高值基本都出现在1℃处理第3d。低温胁迫初期福建山樱花能通过自身调节机制适应胁迫,减轻低温引起的伤害,随着胁迫时间的延长,抗氧化酶和渗透调节物质对膜系统的保护作用及调节机制超过一定限度,就会造成代谢失调,引起不可逆的损伤。3、对福建山樱花幼苗叶片中的内源激素进行测定,10℃处理赤霉素(GA3)、生长素(IAA)、脱落酸(ABA)含量与对照相比无显著差异；6℃和1℃处理GA3、IAA含量显著低于对照,ABA含量、ABA/GA3比值显著高于对照。随胁迫时间延长,10℃处理三种激素无明显变化；6℃和1℃处理GA3、IAA含量随时间呈下降趋势,ABA含量、ABA/GA3比值基本呈先上升后下降的趋势。低温条件下,福建山樱花能提高ABA类生长抑制物质,降低GA、IAA等生长促进类物质,通过调节植物体内内源激素平衡来应对低温。4、应用因子分析法对所测的19个生理生化指标进行分析,福建山樱花各生理生化因子综合效率最高的温度与时间处理的结合点为1℃胁迫3d,超过这个结点,福建山樱花幼苗的生长就会受到抑制。5、以福建山樱花1℃3d处理的幼叶为材料,利用SMART法成功构建了福建山樱花叶片低温全长cDNA文库。经鉴定该文库滴度为1.2×106cfu/mL,插入片段的平均长度为1.5kb,蓝白斑检测重组率为94%,而菌液PCR检测得到的重组率达100%。利用该文库获得了834条有效表达序列标签(EST),对834条有效序列进行拼接和聚类,共得到667条单一序列,其中含有574个单基因和93个片段重叠群,文库冗余度为20.02%。对具有已知或推定功能蛋白的475条单一序列在blast2G0进行功能分类,发现涵盖的功能较齐全,且能反应特定时期细胞表达信息(低温胁迫),其中参与对刺激的反应的有84个。获得的ESTs编码的氨基酸序列与过氧化氢酶(CAT)、丙二烯氧化酶(OsAOC)、捕光叶绿素a/b结合蛋白(CAB)、热激蛋白(HSP)、脂肪氧合酶(LOX)、脱水响应相关蛋白(DRP)、锌指蛋白、钙依赖性蛋白激酶MYB转录因子等防御反应相关蛋白有较高同源性。将比对上具有已知或推定功能蛋白的475条unigenes在blast2GO进行功能分类,结果共获得1597个GO terms,这些GO terms归为3类：生物学过程、细胞组件、分子功能。其中生物学过程所占的GO terms最多,占46.93%,其次是细胞组件和分子功能,分别占38.80%和14.27%。6、在ESTs分析基础上,选取具有代表性的12个推定的与低温胁迫相关基因,应用qRT-PCR定量分析这些基因在各温度处理下表达的差异。其中8个基因受低温胁迫的诱导而上调表达,这些基因所编码的蛋白(酶)分别是脱水应答蛋白(DRP)、MYB转录因子(MYB)、热激蛋白(HSP)、DEAD-box解旋酶(DRH56)、谷胱甘肽过氧化物酶(GPX)、GA20氧化酶(GA20-ox)、捕光叶绿素a/b结合蛋白(CAB)和脂加氧酶(LOX)；受低温胁迫抑制表达的基因有3个,所编码的有丙二烯氧化物环化酶(AOC)、温度诱导脂质蛋白(TIL)、钙依赖性蛋白激酶(CDPK):编码过氧化氢酶的CAT基因只在特定温度下上调表达。福建山樱花低温胁迫下表达的基因参与了信号传导、活性氧清除、转录、胁迫和防御等多个过程,验证了低温对植物的光合作用、水分、离子转运、能量传递等过程有不同的影响,同时也说明植物的抗寒性是一种积累性的数量性状,是由多种特异的数量性抗寒基因调控的。更多还原

【Abstract】 The Prunus campanulata Maxim.(P. campanulata), which belongs to the Rosaceae family, blooms early, around the Chinese Spring Festival; its flowering period can be as long as50d, the bright colour of the particular campanulate flowers confers an extremely high ornamental value on P. campanulata, and its strong resistance and wide adaptability also contribute to it being an excellent native ornamental plant. This species originated in Fujian, Zhejiang, Guangdong, Guangxi, Taiwan in subtropical regions in which the threat of low-temperature damage is not present. However, this species will suffer frost damage if introduced from origination area to the Yangtze River basin and northern areas. Low temperature is one of the most important abiotic factors limiting the growth, development and distribution of plants. Low temperature is also one of the most important limiting factors affecting P. campanulata when it is planted at higher latitudes.After exposing the one-year-old P. campanulata seedling to low temperature stress, physiological and biochemical indexes were measured in leaves to detect the boundary temperature and time of stress. From the physiological and biochemical point of view, the mechanism of P. campanulata response to low temperature stress is clarified, based on physiological and biochemical tests, A full-length cDNA library is established using the young leaf of P. campanulata treated with1℃3d and sequencing a large number of ESTs. Cold-related genes were screened through ESTs sequencing and bioinformatic analysis and analyzed by real-time quantitative PCR. The main purpose of this study is to investigate the cold-resistance mechanisms and lay a foundation for resistance breeding of P. campanulata. The specific findings are as follows.1. The relative chlorophyll content and chlorophyll fluorescence parameters were measured in leaves to detect the influence of temperature on the photosynthetic activity. Compared to the control, P. campanulata exhibited a continuous increase in initial fluorescence (Fo) and decrease in chlorophyll content, maximum photochemical efficiency (Fv/Fm) and photochemical quenching coefficient (qP) with decreases in temperature. The actual photochemical quantum (yield) and electron transfer rate (ETR) in the15℃treatment were significantly higher than those in the control, while they declined under the6℃and1℃ treatment over time. The non-photochemical quenching coefficient (qN) increased under the15℃and6℃treatments and decreased under the1℃treatment. The most significant changes in the parameters occurred under the1℃treatment, indicating that P. campanulata exhibited low-temperature photoinhibition. The results indicated that P. campanulata could protect the photosynthetic apparatus through the increased dissipation of excess light only within a certain range of temperature and stress time.2. After Exposuring of P. campanulata seedlings to different temperature, electrical conductivity, the activities of superoxide dismutase (SOD), peroxidase(POD), catalase(CAT), the content of malondialdehyde (MDA), soluble protein, soluble sugar and free proline were measured in leaves to detect the influence of temperatures on the physiological and biochemical characteristics. The results showed that the content of eight indexes have a signifcant difference with control. Electrical conductivity and the content of MDA exhibited a upward trend under different temperature over time; other indexes sustained a slight increase under the10℃treatment and firstly rose then went down under the6℃and1℃treatment over treatment time. The highest values of SOD, soluble protein, soluble sugar and free proline occurred on day3of1℃treatment. At the beginning of the stress, P.campanulata could reduce the harm through self-regulation mechanism, however, it will cause metabolic disorders and irreversible damage if the protection of antioxidant enzymes and osmolytes protective on membrane systems and regulatory mechanisms exceed a certain limit.3. The content of GA3(Gibberellic acid), IAA (indoleacetic acid), ABA (Abseisic acid) were measured in leaves to detect the influence of temperatures on the endogenous hormones. Compared to the control, P. campanulata exhibited higher contents in GA3、IAA and no significant difference in ABA in the10℃treatment, while GA3、IAA content were lower and ABA、ABA/GA3were higher significantly than control under the6℃and1℃treatment. GA3、IAA content exhibited a continuous decrease and ABA、ABA/GA3firstly rose then went down over treatment time under the6℃and1℃treatment. Under low temperature, P. campanulata can adjust the balance of endogenous hormones in plants to cope with low temperatures.4.19physiological and biochemical indexes were analyzed by factor analysis, as a result, comprehensive maximum efficiency of P. campanulata was the treatment of1℃for3days according to composite score, the growth of P. campanulata seedlings would be inhibited over this node.5. Based on SMART(switching mechanism at5end of RNA transcript) technology, a full-length cDNA library was constructed form the tissue of young leaves of Prunus campanulata Maxim, treated with1℃3d. The results showed:the titer of the cDNA library was1.2×106cfu·mL-1, the average size of inserted cDNA fragment was1.3kb, the recombination rate was94%using blue-white screening, however, it was100%detecting by PCR. To evaluate EST information,834clones were single-pass sequenced and667valid sequences were generated, in which574singlets and93contigs were obtained after initial assembly with SeqMan program. Based on the results of Blast analysis,475unigenes were found to significantly match the proteins with known and putative function. With Blast2GO annotation,475unigenes were found to cover most of the biological functions and express a specific period information (low temperature stress). On the basis of similarity search and GO annotation,84unigenes were found responsive to biotic and abiotic stresses. Homology blast results show that the ESTs encoded amino acid sequence of catalase (CAT), allene oxide cyclase (OsAOC), heat shock protein (HSP), dehydration response proteins (DRP), lipoxygenase (LOX), zinc finger protein, calcium-dependent protein kinase, MYB transcription factor and other defence responses related proteins have a more high homology.475unigenes with known and putative function were distributed into functional categories with Blast2GO and generated1597GO terms (3categories)."Biological Process" was the major functional category, containing750GO terms (46.93). Second larger functional category was "Cellular Component", constituted by620GO terms (38.8%),227GO terms (14.27%) were classified into the "Molecular Function" category.6. Based on the ESTs analysis,12putative cold-related genes of P. campanulata were chosen to measure the expression pattern by qRT-PCR with gene specific primers designed with primer premier software (version5), the following is the results. Compared to the control, the expression levels of DRP, MYB, HSP, DRH56, GPX, GA20-ox, CAB, LOX were obviously induced by treatment temperature, while the transcript level of AOC, TIL, CDPK were lower in the treatment group than in the control group, the expression of CAT gene was induced only in6℃treatment. On one hand, the results indicated that cold related genes of P.campanulata involved in signal transduction, active oxygen scavenging process, transcription, stress and defense process, while plant photosynthesis, water, ion transport, energy transfer processes were verified to be effected by low temperature; on the other hand, these cold related genes may play different roles in low temperature stress.更多还原