【作者】 郭卫丽；

【导师】 巩振辉；

【作者基本信息】 西北农林科技大学 ， 蔬菜学， 2013， 博士

【摘要】 低温是限制植物生长发育和地理分布最重要的非生物胁迫因子之一。辣椒(Capsicum annuum L.)起源于中南美洲的热带和亚热带地区，对低温敏感，其最适合生长温度为21-27℃，低于12℃和高于30℃生长受到抑制。我们试图通过探索辣椒的防御机制，以期提高其耐寒性。本研究以抗寒辣椒品种P70为材料，0.57mMABA溶液喷施幼苗，72h后进行低温处理。一方面探索外源ABA对低温下辣椒幼苗抗氧化酶活性及其基因表达的影响；另一方面利用抑制消减杂交技术（SSH）分离ABA调节的低温抗性相关基因，并对其功能进行分析。主要内容为：（1）为了探索ABA调节的响应低温胁迫反应的生理生化机制，外源ABA预处理后进行低温10℃/6℃（昼/夜）处理。研究结果为：低温引起叶片坏死斑出现，MDA和H2O2含量增加；在遭受低温胁迫72h期间，辣椒MDHAR、DHAR、GR、POD、APX酶活和AsA、GSH含量增加；而在24h期间SOD和CAT活性下降，这些结果表明在CAT失活的情况下，低温启动AsA-GSH循环代谢。常温下ABA预处理引起以上提及的酶活显著增加和AsA、GSH含量的缓慢下降。与仅进行低温胁迫处理相比，在低温胁迫下ABA预处理的幼苗SOD和POD活性增加，而其他的抗氧化酶活性降低，MDA和H2O2含量也表现同步下降；而且相应的Mn-SOD和POD基因被诱导表达。总之，外源ABA能够显著提高SOD和POD酶活及其基因表达，进而增强辣椒对低温诱导氧化胁迫的抵抗性。（2）外源ABA可有效缓解低温对辣椒幼苗的伤害。然而，ABA调节的基础分子机理仍不清楚。为了从ABA预处理的遭受48h低温（6℃）的辣椒幼苗上分离差异基因，我们构建了抑制消减杂交文库（SSH）。总共获得235个高质量的ESTs，拼接成73个unigenes，含有18个contigs和55个singletons。其中37个unigenes（50.68%）与NCBI非辰余数据库中的已知功能基因高度同源；其他36个unigenes（49.32%）同源性低或同源基因功能未知。GO功能注解分析37个unigenes分为9类功能。另外采用qRT-PCR技术对18个基因的表达进行分析：ABA预处理低温胁迫的辣椒幼苗10个基因的表达量比清水处理低温胁迫的植株高2倍多；而ABA+低温处理的幼苗其他8个基因的表达量下调，表达量是遭受低温胁迫对照植株的1/3或更小。这些结果表明在响应低温胁迫反应时，ABA能够有效地正负调节辣椒基因表达。（3）根据SSH文库分离的差异基因，利用VIGS技术进行初步筛选，其中对5个ESTs克隆全长，分别命名为CaNAC2、CaMBF、CaF-box、CaMADS-box、CaDHN，含有完整的开放阅读框（ORF），GenBank登录号分别为JX402928、JX402927、JX402925、JX402926、JZ198814。辣椒CaNAC2开放阅读框为1230bp，属于NAC2亚家族成员。CaNAC2定位于细胞核且具有转录激活活性。CaNAC2主要在辣椒的根和幼嫩种子表达。盐胁迫和ABA处理诱导CaNAC2表达，表明其可能参与ABA介导的盐胁迫反应；而甘露醇和SA处理抑制CaNAC2表达。我们采用病毒诱导沉默技术（VIGS）分析CaNAC2对辣椒抗逆性的调控作用。结果表明：在叶面喷施ABA和清水后进行相同的低温处理下，CaNAC2沉默植株丙二醛含量高于基因未沉默植株（对照），表明CaNAC2沉默引起辣椒膜脂过氧化程度加剧。在ABA+低温处理下，CaNAC2沉默植株过氧化氢含量高于对照；然而，仅进行低温处理的CaNAC2沉默株过氧化氢含量很低，表明过氧化氢可能参与信号转导。与对照相比，高盐胁迫下CaNAC2沉默的离体叶片保持较绿，表明该基因的功能缺失可能与植株保绿有关；而甘露醇处理CaNAC2沉默的离体叶片表型基本没有变化。（4）辣椒CaMBF开放阅读框为420bp，分子量为15.3KD等电点为9.86。CaMBF表达在花和幼嫩种子较高，根中基本无表达。在高盐、渗透胁迫、重金属汞和SA处理下CaMBF表达均受到抑制，而低温诱导其表达。将带有35S的组成型CaMV启动子CaMBF超量表达载体导入拟南芥，结果表明：转基因拟南芥CaMBF受高盐、低温和ABA处理下调表达。转基因植株生长较大，莲座叶的叶长和叶宽分别比对照大70%和60%。将野生型和转基因植株进行低温4℃处理，转基因植株的耐寒性比野生型弱。并且拟南芥CaMBF的超量表达负调节一些逆境防御基因如RD29A、ERD15、KIN1、RD22、PDF1.2和PR2。（5）辣椒CaF-box编码638个氨基酸，属于F-box蛋白超家族的EBF分枝。CaF-box主要分布在茎和幼嫩种子中。ABA、SA和低温处理诱导CaF-box表达；甘露醇处理抑制CaF-box表达。利用VIGS技术诱导CaF-box沉默，与未发生CaF-box沉默植株相比，在低温胁迫下，CaF-box沉默植株丙二醛和电导率增加，表明CaF-box沉默引起辣椒的耐寒性减弱。在高盐和渗透胁迫下，CaF-box沉默离体叶片明显黄化，表明CaF-box沉默植株出现早衰现象。（6）辣椒CaMADS-box开放阅读框为726bp，属于SEP1/AGL2亚家族。CaMADS-box表达量在花中较高，根、叶和种子中几乎无表达。ABA处理对CaMADS-box表达无影响；SA处理12h CaMADS-box表达缓慢下降；而高盐和渗透胁迫CaMADS-box上调表达。辣椒CaDHN开放阅读框为660bp，属于SK2型脱水素。CaDHN主要分布于果实和花中。在低温、高盐、渗透胁迫和SA处理下CaDHN表达增加。更多还原

【Abstract】 Low temperature is one of the most important abiotic factors limiting the growth,development and geographical distribution of plants. Pepper plant (Capsicum annuum L.)originates from tropical regions and is very sensitive to low temperature. The optimal growthtemperature for pepper plants ranges from21°C to27°C, and growth is retarded below12°Cand above30°C. As part of this effort, we are interested in investigating of plant defensemechanisms, in order to improve plant resistance to environmental stresses. In this study,‘P70’ is a typical pepper cultivar that is fairly tolerant of low temperatures. The pepper variety(cv.‘P70’) seedlings were pretreated with0.57mM abscisic acid (ABA) for72h and thensubjected to chilling stress. We investigated the effect of exogenous ABA on total antioxidantactivity and related genes expression in pepper seedlings subjected to chilling stress.Meanwhile, suppression subtractive hybridization analyzed genes regulated by application ofexogenous ABA in pepper plant leaves under chilling stress. Additionally, functionalidentification of several genes in pepper seedlings subjected to abiotic stress was done. Themain results of the study are as follows:(1) To elucidate how physiological and biochemical mechanisms of chilling stress areregulated by ABA pretreatment, the pepper seedlings were pretreated with0.57mM ABA for72h and then subjected to chilling stress at10°C/6°C (day/night). Chilling stress causedsevere necrotic lesions on the leaves and increased malondialdehyde and H2O2levels.Activities of monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase(DHAR), glutathione reductase (GR), guaiacol peroxidase (POD), ascorbate peroxidase(APX), ascorbate (AsA), and glutathione (GSH) increased due to chilling stress during72h,while superoxide dismutase (SOD) and catalase (CAT) activities decreased during24h,suggesting that chilling stress activates the AsA-GSH cycle under CAT deactivation in pepperleaves. ABA pretreatment induced significant increases in the above-mentioned enzymeactivities and progressive decreases in AsA and GSH levels. On the other hand,ABA-pretreated seedlings under chilling stress increased SOD and POD activities andlowered concentrations of other antioxidants compared with untreated chilling-stressed plants.These seedlings showed concomitant decreases in foliage damage symptoms, and levels ofmalondialdehyde and H2O2. Induction of Mn-SOD and POD was observed in chilling-stressed plants treated with ABA. Overall, the results indicate that exogenous application of ABAincreases tolerance of plants to chilling-induced oxidative damage, mainly by enhancing SODand POD activities and related genes expression.(2) Application of exogenous ABA effectively alleviates the symptoms of chilling injury,such as wilting and formation of necrotic lesions on pepper leaves; however, the underlyingmolecular mechanism is not understood. The aim of this study was to identify genes that aredifferentially up-or downregulated in ABA-pretreated pepper seedlings incubated at6°C for48h, using a suppression subtractive hybridization (SSH) method. A total of235high-qualityESTs were isolated, clustered and assembled into a collection of73unigenes including18contigs and55singletons. A total of37unigenes (50.68%) showed similarities to genes withknown functions in the non-redundant database; the other36unigenes (49.32%) showed lowsimilarities or unknown functions. Gene ontology analysis revealed that the37unigenes couldbe classified into nine functional categories. The expression profiles of18selected genes wereanalyzed using quantitative RT-PCR; the expression levels of10of these genes were at leasttwo-fold higher in the ABA-pretreated seedlings under chilling stress than water-pretreated(control) plants under chilling stress. In contrast, the other8genes were downregulated inABA-pretreated seedlings under chilling stress, with expression levels that were one-third orless of the levels observed in control seedlings under chilling stress. These results suggest thatABA can positively and negatively regulate genes in pepper plants under chilling stress.(3) Based on differential genes identified by SSH technique, in accordance with thetobacco rattle virus (TRV)-based VIGS technique, five genes were isolated using ahomology-based candidate gene method and designated as CaNAC2, CaMBF, CaF-box,CaMADS-box, CaDHN, respectively. They contained complete open reading frames (ORF)and GenBank numbers were JX402928, JX402927, JX402925, JX402926, JZ198814,respectively.In this report, pepper CaNAC2belonging to NAC2subfamily, was predicted to encode aprotein of409amino acid residues. The deduced CaNAC2protein was localized in thenucleus of onion epidemical cells and proven to have transactivation and DNA-bindingactivities in yeast. The expression of CaNAC2was largely higher in roots and seeds than thatin other tissues. CaNAC2transcript in pepper leaves was induced by sodium chloride (NaCl)and ABA, indicating that this gene was involved in ABA-mediated salt signaling. However,the expression of CaNAC2was downregulated by mannitol and salicylic acid (SA). Heavymetal Hg treatment had no obvious effect on CaNAC2expression.To further examine the effect of loss-of-function of the CaNAC2gene on abiotic stresstolerance, we silenced the CaNAC2gene in pepper plants using a VIGS technique. Loss-of-function of CaNAC2in pepper plants led to increased susceptibility to chilling stress.Following chilling stress pretreated with ABA or water, the thiobarbituric acid reactivesubstances (TBARS) content of the CaNAC2-silenced leaves was significantly higher thanthat of the empty vector control plants. The combination of ABA with chilling stress led to anincreased level of H2O2in the CaNAC2-silenced leaves. However, H2O2content inCaNAC2-silenced seedlings subjected to chilling stress was very low, indicating that H2O2isinvolved in signaling transduction. Meanwhile, compared with the empty vector controlplants, the leaf discs of gene-silenced plants were more severely green under salinity stress.However, no phenotypic differences were observed between leaf discs from empty vectorcontrol and CaNAC2-silenced plants following treatment with300mM mannitol.(4) Pepper CaMBF contained an open reading frame (ORF) of420bp. The deducedCaMBF protein consisted of140amino acids with a calculated molecular weight of15.3kDaand an isoelectric point (pI) of9.86. The expression level of CaMBF in flowers and seeds washigher and was not easily detected in roots. CaMBF transcript in pepper seedlings wasupregulated by cold stress and downregulated in response to salinity, osmotic stress, heavymetal Hg and SA. Meanwhile, the expression level of CaMBF in CaMBF-overexpressingArabidopsis was downregulated in response to salinity, cold stress and ABA. Transgenicplants were larger than non-transgenic plants (control), especially the length and width ofrosette leaves were70%and60%larger than control. To study the tolerance ofCaMBF-expressing plants to cold stress, we subjected2-week-old wild-type and transgenicseedlings to4°C and observed visual damage symptoms. Transgenic plants were moresusceptibility than wild-type plants to cold stress. Furthermore, overexpression of CaMBF inArabidopsis negatively regulated some defense transcripts in response to cold stress such asRD22, ERD15, RD29A, PDF1.2and PR2.(5) Pepper CaF-box belonging to an ERF branch of F-box superfamily, encoded apolypeptide of638amino acids. CaF-box was mainly expressed in stems and seeds. ABA, SAand cold treatments induced CaF-box transcript; the expression of CaF-box was down-regulated in response to mannitol and heavy metal Hg treatments. To further examine theeffect of loss-of-function of the CaF-box gene on abiotic stress tolerance, we silenced theCaF-box gene in pepper plants using a VIGS technique. The measurement of TBARS andelectrolyte leakage levels revealed stronger lipid peroxidation and cell death in theCaF-box-silenced plants than in the empty vector control plants, suggesting thatloss-of-function of CaF-box significantly compromised cold stress tolerance in pepper plants.When treated with300mM NaCl and300mM mannitol, the leaf discs fromCaF-box-silenced leaves were more severely bleached, indicating that loss-of-function of CaF-box in pepper plants showed early senescence in response to salinity and osmotic stress.(6) Pepper CaMADS-box contained an ORF of726bp. The deduced CaMADS-boxprotein consisted of241amino acids and belonged to an SEP1/AGL2-like subfamily. Theexpression level of CaMADS-box in flowers was higher and was not easily detected in roots,leaves and seeds. ABA and heavy metal Hg treatments had no obvious effect onCaMADS-box expression; CaMADS-box transcript gradually decreased during12h of SAtreatment and upregulated by salinity and osmotic stress. Pepper CaDHN, belonging toSK2-type dehydrins, contained an ORF of660bp. Pepper CaDHN was expressed mainly inflowers and fruits. CaDHN transcript in pepper seedlings was differentially upregulated bycold, salinity, osmotic stress and SA and downregulated in response to heavy metal Hgtreatment.更多还原