【作者】 杨静；

【导师】 向长萍；

【作者基本信息】 华中农业大学 ， 蔬菜学， 2008， 博士

【摘要】 苦瓜（Momordica charantia L.）,是葫芦科苦瓜属的一年生蔓生植物,果实具有食用和药用价值。目前研究主要集中在药用功能方面,对苦瓜的生理和育种研究报道较少。苦瓜主要分布亚热带、热带地区,喜高温,低温限制了苦瓜的种植范围和种植时节。植株在生长季节遇到低温会生长发育不良甚至死亡。本试验旨在开展苦瓜低温生理和耐低温机制研究,为苦瓜设施栽培和耐低温育种提供理论依据和指导,同时在耐低温育种方面也作了一些工作。本文模拟早春非加热大棚环境,研究了不同夜间低温对2个苦瓜自交系的生理生化特性的影响,以了解苦瓜对不同夜间低温的耐受程度和耐受机理;接着利用甲基化敏感多态性扩增（MSAP）技术研究了低温处理诱导的苦瓜基因组DNA的CCGG位点的甲基化模式的变化,以探讨苦瓜低温适应的分子机理。本文还调查了外源水杨酸（SA）预处理对苦瓜耐低温性的影响,以期进一步研究苦瓜的低温生理,同时也为遭遇低温时苦瓜的栽培管理措施进行有益的探讨。最后,利用7个多年多代自交纯和的优良自交系进行配组杂交,用加性-显性模型对苦瓜的耐低温性进行了遗传分析,同时对各组合的耐低温性进行鉴定,结合其它农艺性状,筛选出优良的杂交组合。实验主要结果如下:1.12℃夜间低温处理7d对苦瓜幼苗叶片的各项指标影响较小,几乎不引起伤害。8℃夜间低温处理7d降低了苦瓜幼苗膜稳定性、光系统Ⅱ的潜在活性（Fv/Fo）、光化学淬灭系数（qP）和PSⅡ量子产额（ΦPSⅡ）,使剩余光能荧光耗散（E）比率上升,对苦瓜幼苗造成一定伤害,这种伤害在经过7d常温生长后可以得到部分或完全恢复。5℃夜间低温处理7d对苦瓜伤害最严重,不仅进一步降低了苦瓜幼苗膜稳定性、Fv/Fo、qP和ΦPSⅡ,还导致叶绿素含量、最小荧光（Fo）、最大荧光（Fm）和可变荧光（Fv）降低,叶绿素a/b比值升高,荧光耗散比率上升幅度增加,经过7d常温生长后的各指标的恢复程度在株系间有一定差别。Z-1-4的恢复程度比Y-106-5好,表现出较好的耐低温性。夜间低温处理下ΦPSⅡ主要受反应中心的组成而不是吸收的光能到达反应中心的效率的影响。夜间低温对光系统Ⅱ最大光化学效率（Fv/Fm）影响较小,相对来说,Fv/Fo受到夜间低温的影响较大。在研究夜间低温对植物的影响时,Fv/Fo是比Fv/Fm更敏感更适用的指标。2.苦瓜DNA的甲基化总体水平为22.92%-25.86%,内侧胞嘧啶的甲基化水平平均为19.63%,外侧胞嘧啶的半甲基化水平平均为4.66%。四个供试自交系的甲基化水平高低依次为:Y-106-5＞Y-81.3＞Z.1.4＞Y-70-1。低温处理诱导了苦瓜基因组DNA的CCGG位点发生了甲基化模式的改变。实验成功克隆到9个至少在2个自交系中发生甲基化模式变异的片段,测序结果经过BLASTn比对,Y1、Y2、Y5三个片段在NCBI数据库中找到部分同源序列。Y1序列与长喙田菁（Sesbania rostrata）的β-1,3-葡聚糖酶mRNA序列有一定相似性。片段Y2与GCN5相关N乙酰转移酶（GNAT）有一定相似性。片段Y5与黄瓜中编码叶绿体光系统1 A亚基的psaA基因高度相似（98%）。3.经0.5mmol/L水杨酸处理的幼苗在低温处理后具有较高的过氧化物酶（POD）、过氧化氢酶（CAT）与超氧化物歧化酶（SOD）活性和（POD+CAT）/SOD比值,表现出较强的抗氧化活性,光合色素含量较高,Fv/Fm和ΦPSⅡ受到的影响小,膜脂过氧化程度低,受到低温的伤害较小。高浓度SA和低浓度SA预处理对苦瓜耐低温性有不同的效应。4.遗传分析表明:低温处理后MDA含量、叶片电导伤害率（Eb）及离体叶片低温电导伤害率（Ea）的遗传以基因显性效应为主,还受到部分加性效应的作用,环境条件对这些性状的表现也有很大影响。各性状的广义遗传力较高,狭义遗传力相对较低,宜在较高世代进行选择。部分F₁代植株具有较低的MDA含量和电导伤害率值,苦瓜耐低温性表现出较强的超中优势和超亲优势,通过杂交可以得到耐低温性比较好的材料。低温胁迫条件下,自交系P1、P2、P3、P6对MDA含量、Eb、Ea这三个指标或其中两个指标都具有反向加性效应,耐低温性较好,可以作为耐低温育种的良好亲本加以利用。杂交种P1×P5、P1×P6、P1×P7、P2×P3、P2×P5、P3×P5、P4×P6对三个指标都具有负向显性效应预测值,耐低温性相对较好,有一定利用价值。本课题组利用上述材料对苦瓜其它部分农艺性状进行了配合力分析,结合耐低温性和其它性状可以进行苦瓜温室品种选育。本实验所选4个组合已经过鉴定并开始进行扩大试种,其中2个组合可以作为温室栽培品种。更多还原

【Abstract】 Bitter gourd （Momordica charantia L.） is a member of Cucurbit family. Its immature fruits are edible and have medicinal value. Present studies mainly focus on its medicinal functions, seldom on the aspects of physiology and breeding. Bitter gourd mainly distributes in tropic and subtropic areas. Low temperature limits the geographical locations and the planting season suitable for bitter gourd growth. Chilling temperature always accounts for significant losses in crop production. So this experiment aimed to study on low temperature physiology and low-temperature-resistance mechanism on this special vegetable crop. This work would help to shelter cultivation and low-temperature-resistancebreeding of bitter gourd.In this paper, in order to understand the low temperature tolerance and the mechanism of tolerance of bitter gourd, the effect of different low night temperature on the physiological and biochemical characteristics in two bitter gourd inbred lines was studied; then low-temperature-induced methylation pattern changes in CCGG sites of genomic DNA were analyzed by methylation-sensitive amplified polymorphism （MSAP） technique to explore the possible molecular mechanism in low temperature adaptation. The effects of exogenous salicylic acid （SA） application on the low-temperature tolerance of bitter gourd were investigated with a view to further study the physiological mechanism under low temperature and give some useful advice in cultivation and management when bitter gourd suffered hypothermia. Finally, based on half-diallel design, 21 F₁ hybrids were confected by 7 inbred lines of bitter gourd. Additive-dominant model was used to process genetic analysis of low-temperature tolerance. At the same time the character of low-temperature-tolerance of all F₁ materials was identified. In combination with other agronomic traits, excellent hybrid combinations were selected. The main research results were as follows:1. A 7-d 12℃low night temperature treatment had little impact on the physiological and biochemical indicators in bitter gourd leaves and caused little damage to bitter gourd seedlings. A 7-d 8℃low night temperature treatment reduced the stability of the membrane, PSⅡpotential activity （Fv/Fo） , the coefficient of photochemical quenching （qP） and PSⅡquantum yield （ΦPSⅡ） of the bitter gourd seedlings, and increased the part of light energy that dissipated as fluorescence （E） , resulting a certain injury. And the injury can be partially or fully restored after growing at room temperature for 7 days. A 7-d 5℃low night temperature treatment did the most serious harm to the bitter gourd seedlings. It further reduced the membrane stability, Fv/Fo, and qP andΦPSⅡ. It also led decreases in chlorophyll content, the minimal fluorescence （Fo）, the maximal fluorescence （Fm） and the variable fluorescence （Fv）. Increases in chlorophyll a/b ratio and E were observed. After being growed at room temperature for 7 days, all the indicators restored to a certain level. The extent of the restoration showed difference between lines. As for the two lines we tested there, Z-1-4 showed better low-temperature tolerance than Y-106-5.Under low night temperature,ΦPSⅡwas primarily droved by the composition state of the reaction center, rather than the efficiency of the absorbed light energy to reach the reaction center. The maximal photochemical efficiency of the PS II （Fv/Fm） was affected slightly and Fv/Fo was affected largely by the low night temperature. In the studies about effect of low night temperature on plants, Fv/Fo was a more sensitive and useful indicator than Fv/Fm.2. The total DNA methylation content in bitter gourd is about 22.92% -25.86%, the average internal cytosine methylation is 19.63 % and the average hemi-cytosine methylation is 4.66%. The order for the total methylation content in the four inbred lines is Y-106-5＞Y-81-3＞Z-1-4＞Y-70-1.Low temperature induced methylation pattern changes at the CCGG sites in genomic DNA. Nine fragments which were observed variation in at least two inbred lines were cloned and sequenced successfully. BLASTn search revealed that fragments Y1, Y2 and Y5 showed homology to certain sequences in NCBI database. Y1 matched withβ-1, 3-glucanase mRNA in Sesbania rostrata to a certain extent. Fragment Y2 showed a certain similarity with the GCN5-related N-acetyltransferase （GNAT）. Fragment Y5 exhibited high similarity （98%） with psaA which encoding chloroplast PS1A subunit in cucumber.3. 0.5mmol/L SA pretreated seedlings had high peroxidase （POD）, catalase （CAT） and Superoxide dismutase （SOD） activity and （POD + CAT）/ SOD ratio, showed strong antioxidant activity, maintained relative higher levels of photosynthetic pigments. Fv/Fm andΦPSⅡwere impacted slightly, and lipid peroxidation degree was low, indicating 0.5mmol/L can reduce the injurys caused by low temperature. Higher concentration of SA （1.0 and 1.5 mmol/L） and lower concentration of SA （0.5 mmol/L） had different effect on bitter gourd seedlings under low temperature stress.4. Genetic parameters indicated that the MDA content, electrolytic leakage of intact leaves （Eb） and electrolytic leakage of detached leaves （Ea） after low temperature stress were mainly dominant effect. All the traits showed some additive effect. Environmental conditions also have a significant impact on them. These indicators showed high broad heritability （H_B²） and relative low narrow heritability （H_N²）, so that the corresponding traits should be selected at a higher generation.Under low temperature stress, the inbred lines P1, P2, P3 and P6 showed negative additive effect for content of MDA, Ea and Eb. They displayed relative good low temperature tolerance and could be used as good breeding parents for low temperature tolerant breeding of bitter gourd. Hybrids P1×P5, P1×P6, P1×P7, P2×P3, P2×P5, P3×P5, P4×P6 showed negative dominant effect in all the three indicators. Combining low temperature tolerance and other agronomic traits, bitter gourd variety that suitable for greenhouse cultivation can be selected. The trial cultivation of 4 hybrids had already been started. Among them 2 hybrids could be used in greenhouse cultivation.更多还原