【作者】 张万萍；

【导师】 陈劲枫；

【作者基本信息】 南京农业大学 ， 蔬菜学， 2009， 博士

【摘要】 黄瓜(Cucumis sativus L.)是重要的设施蔬菜之一,也是典型的冷敏植物。在早春和秋冬季节,低温是产量和质量的限制因子,因此,黄瓜耐冷性的提高成为周年供应的一个关键环节,通过育种和栽培的综合手段克服黄瓜生产中的低温障碍是解决这个问题的主要手段,也是设施黄瓜的研究热点,而正确理解黄瓜的耐冷机制是耐冷育种和栽培的良好基础。为探明黄瓜耐低温胁迫的生理和分子生物学机制,本文以(8-15℃)亚适宜低温为胁迫因子,应用植物生理学、生物化学、分子生物学及功能基因分析的手段,研究低温影响黄瓜生长的机理,探明参与抗逆调控的重要植物生长类物质多胺(PAs)和水杨酸(SA)调节黄瓜耐冷性的生理生化作用机制,克隆分析了黄瓜耐冷相关基因CsLDC。取得了以下主要研究结果：1.黄瓜赖氨酸脱羧酶(CsLDC)的全长cDNA序列克隆和冷胁迫过程中CsLDC基因表达的初步研究高质量的RNA是研究基因表达以及后续下游试验的关键,常规的Trizol法对于种子和果实这类器官来说,很难提到理想的RNA。通过对几种方法的比较研究,对尿素-氯化锂法进行了改良,结果表明：对种子这类多糖、多酚以及蛋白质含量高的器官,改良尿素-氯化锂法能够获取高质量RNA,其28S、18S和5S电泳带型清晰完整,A260/A280值在2.0-2.1之间,用其进行其反转录的cDNA,不仅可以用作RT-PCR、也可以用做AFLP、RACE(快速扩增cDNA末端)以及Real time-PCR等后续研究。此外,该方法同样可以用于葫芦科其它作物如丝瓜、南瓜及甜瓜的种子,以及叶片RNA的提取。根据本课题组已报道的长度为132bp黄瓜耐冷相关基因(ccrtl32)片段序列(逯明辉,2005),运用RACE技术技术,克隆了长度为1069bp的全长cDNA序列。该序列的有效开放阅读框共含648个核苷酸编码一个长为215个氨基酸残基的蛋白质。经比对该全长cDNA及其编码的蛋白质序列,与几个植物的赖氨酸脱羧酶基因有较高的同源性,因此对该基因命名为黄瓜赖氨酸脱羧酶(Cucumis sativus Lysine Decarboxylase, CsLDC)。CsLDC含有高度保守的赖氨酸脱羧酶结构域以及一个高度保守的基序PGGXGTXXE,经预测,CsLDC蛋白的三级结构和已经报道的拟南芥赖氨酸脱羧酶晶体结构高度相似,与拟南芥赖氨酸脱羧酶进化关系较近。用RT-PCR和荧光实时定量PCR技术研究两个耐冷性不同的黄瓜品种在冷胁迫过程中基因表达的结果表明,低温胁迫下,耐冷品种长春密刺的种子及幼叶中CsLDC基因均能被强烈诱导表达,但在冷敏品种北京截头中表达量较低,提示该基因与黄瓜的耐冷性有关。2.外源多胺(PAs)调节抗氧化酶增强黄瓜耐冷性的研究低温处理后1d,耐冷品种长春密刺叶片中Put、Spd、Spm被诱导生成,Put含量达到最大,随后呈下降趋势,但Spd和Spm含量却持续上升,其中Spd含量始终高于Put和Spm含量。北京截头叶片中,Put含量在低温处理后1d增加,随后缓慢下降,而Spd含量一直呈下降趋势,Spm含量变化不大。外源Put和Spd处理均能提高两个品种内源三种多胺含量,但是长春密刺叶片中内源Put、Spd、Spm整体高于北京截头,外源Spd对提高内源多胺含量的影响大于外源Put,多胺抑制剂丙酮双脒腙(MGBG)却能抑制两个品种叶片中Spd、Sp(?)n的产生,但是对冷敏品种的影响大于对耐冷品种的影响。低温降低了两个品种可溶性蛋白的含量、抗氧化酶SOD、POD、CAT和抗坏血酸过氧化物酶(APX)活性,并影响细胞膜稳定性,但是对北京截头的影响大于长春密刺,这种影响可以通过外源Put和Spd而得到缓解,如减少了两个品种叶片由于冷害引起的电解质渗透和丙二醛含量,但是不同多胺种类所起作用因品种而异,多胺抑制剂MGBG预处理后可以抵消多胺(PAs)的作用。此外,组织化学染色和定量测定表明,应用外源Put和Spd可以消除或减少由低温胁迫引起的过氧化氢(H202)积累,而MGBG的作用刚好相反,这种情况在北京截头中尤为明显。值得注意的是,在低温胁迫下长春密刺叶片三种内源多胺含量均高于北京截头,低温处理前长春密刺和北京截头幼苗中SOD、POD和CAT活性没有明显差异,但低温处理整个过程前者APX始终高于后者。这些结果表明多胺(PAs)在黄瓜耐低温胁迫方面具有重要作用,而这种作用很有可能通过调节抗氧化系统而实现。3.水杨酸对低温胁迫黄瓜抗氧化系统和耐冷性的影响低温胁迫下两个品种叶片中内源SA含量明显高于种子,耐冷品种长春密刺的萌动种子和幼苗叶片内源SA含量均高于冷敏品种北京截头；长春密刺叶片中Pro和POD代谢水平高于冷敏品种北京截头,而MDA、电解质渗漏率和H202积累则低于北京截头,外源SA处理能显著提高低温胁迫条件下黄瓜叶片的POD活性及可溶性蛋白含量,降低MDA含量和电解质渗出率；相比北京截头来说,水杨酸对低温胁迫下长春密刺叶片中的H202积累具有更为明显的消除作用。结果提示品种间耐冷性不同导致了内源水杨酸代谢水平的差异,外源水杨酸可以提高黄瓜抵抗低温胁迫的能力,而且这种作用可能是通过调节体内的抗氧化系统来实现的。更多还原

【Abstract】 Cucumber (Cucumis sativus L.) is one of the typical chilling-sensitive plants and is also being considered as one of the important vegetables. However, low temperature in the early spring and late autumn or winter is the most directly limited factor of cucumber yield and quality. Therefore, it is becoming the focus of current research on cucumber through breeding and culture approaches, and understanding the cold-tolerant mechanism is the basis of breeding for cold-resistant variety. To uncover the chilling tolerant mechanism by which involves growth of cucumber in terms of both physiological and molecular aspects, this study employs a combination of approaches involving plant physiology, biochemistry, molecular biology and functional analysis of target gene to examine the influence of chilling stress on cucumber, while considering 15℃as the stress factor under sub-optimal temperature. Moreover, this sudy investigates the physiological and biochemical mechanisms underlying the chilling-tolerance mediated by important phytohormones in cucumber. Meanwhile, a chilling-tolerant gene of cucumber, CsLDC, was cloned and identified.Specifically, this study is focusing on and has made progress in the followings1. Cloning of a full-length cDNA of Lysine Decarboxylase (CsLDC) from Cucumis sativus and gene expression study of CsLDC in cucumber upon chilling stressHigh quality of RNA is the key for gene expression and downstream studies. It is very difficult to obtain high quality ofRNA from geminating cucumber (Cucumis sativus) seeds using commercial Trizol kit. We improved the Urea-LiCl method by comparing several different protocols. The results clearly showed that we succeeded in obtaining high quality of RNA with clear and intact bands of 28S,18S and 5S, and with an A260/A280 at around 2.0. This RNA can be used not only for cDNA synthesis, but also other downstream studies such as RT-PCR, AFLP, RACE and even Real-time PCR. Moreover, using this improved Urea-LiCl protocol we could get high quality of RNA from seeds of other Cucurbitaceae varieties, including squash, melon and towel guard, and other tissues besides seeds.A full-length cDNA sequence was cloned using RACE (Rapid Amplification of cDNA Ends) from cucumber (Cummis sativus) seeds, based on a 132bp fragment of cucumber cold related tolerance gene (ccrt132) reported previously by our group. This full-length cDNA contains an ORF (Open Reading Frame) of 648 nucleotides encoding a protein with 215 amino acid residues. Alignment analysis has shown that this cDNA sequence and its coded protein have high similarity of nucleotides and homology to reported plant Lysine Decarboxylase, respectively, thus it is named as Cucumis sativus Lysine Decarboxylase (CsLDC). CsLDC contains a highly conserved Lysine_decarboxy superfamily domain with a conserved motif PGGXGTXXE, and the crystal structure of CsLDC is predicted to be highly similar to that of reported Arabidopsis lysine decarboxylase x-ray A chain. The alignment of CsLDC with other plant LDCs suggested a closest relationship of CsLDC with Arabidopsis AtLDC. Moreover, the quantitative data of gene expression conduced by semi-quantitative RT-PCR and quantitative Real-Time PCR demonstrated that CsLDC is highly enhanced in cold-tolerant variety Changchun mici upon chilling stress, but not in cold-sensitive Beijing jietou, suggesting that CsLDC is highly associated with chilling tolerance of cucumber.2. Exogenous polyamines regulate chilling tolerance of cucumber (Cucumis sativus L.) through modulating antioxidative systemUpon chilling treatment, free spermidine (Spd), spermine (Spm) and putrescine (Put) were remarkably increased in the leaves of cv. Changchun mici 1 day after treatment. The induction of Put declined thereafter, whereas Spd and Spmlevels increased steadily. In the leaves of cv. Beijing jietou, Put content was increased only at 1 day after chilling, while Spd content decreased significantly upon chilling treatment and not much changes were detected for Spm. Exogenous PAs increased endogenous content of all three types of PAs, with higher levels in Changchun mici than that in Beijing jietou, and exougenous Spd showed much greater impact on endogenous PAs level than Put. The PAs biosynthetic inhibitor, methyglyoxal-bis-(guanylhydrazone) (MGBG), cancelled the effects of exogenous PAs on the endogenous PA levels, among which the effect of MGBG on chilling-sensitive variety was greater than on chilling-tolerant one. Chilling reduced soluble protein content, the activities of antioxidant enzymes, including superoxidase dismutase (SOD), peroxidase (POD), catalase (CAT) as well as ascorbate peroxidase (APX), and satiability of membrane; however, the reduction on Beijing jietou is much greater than Changchun mici, and the changes could be renovated by exogenous application of Put and Spd. It was also found that pretreatment with Put and Spd diminished the increased electrolyte leakage and malondialdehyde (MDA) content caused by chilling in the leaves of both cultivars. Hoever, the effects caused by PAs mostly depended on variety of cucumber, and pretreatment with MGBG could cancel the effect of PAs. Moreover, histochemical staining and quantitative measurements showed that exogenous application of Put and Spd eliminated but MGBG exaggerated the hydrogen peroxide (H2O2) accumulation caused by chilling stress, especially in leaves of Beijing jietou. Notably, Changchun mici was found to contain higher levels of all three types of endogenous free PAs compared to Beijing jietou. While no significant difference of SOD, POD and CAT activities was found between non-chilling stressed Changchun mici and Beijing jietou seedlings, the former exhibited higher APX activity than the latter. These results indicate that PAs play important roles in the tolerance of cucumber against chilling stress, which is most likely achieved by acting as oxidative machinery against chilling injury.3. Effects of salicylic acid on the antioxidant system and chilling tolerance of Cucumis sativusUpon chilling stress, the endogenous SA levels in the leaves of two different chilling-responsive varieties were obviously higher that that in geminating seeds, and Changchun mici contains higher SA levels in both leaf and germinating seed compared to Beijing jietou. The Proline content and peroxidase (POD) activity in Changchun mici are higher than Beijing jietou, while MDA levels, electrolyte leakage and H2O2 production in the former is much lower than that in the latter. Exogenous application of SA increases significantly POD activity and soluble protein content whereas it decreases MDA content and electrolyte leakage in the leaves under chilling stress. Compared to Beijing jietou, exogenous SA treatment could eliminate the accumulation of H2O2 in leaves of Changchun mici, much greater than in Beijing jietou, upon chilling stress. The data clearly demonstrate that SA production varies due to the difference of chilling responsiveness, and that exogenous SA can enhance the chilling tolerance ability, which might be achieved through modulating antioxidant system in cucumber.更多还原