【作者】 杨芩；

【导师】 王永清；

【作者基本信息】 四川农业大学 ， 果树学， 2013， 博士

【摘要】 本研究利用延迟授粉、组织化学检测和荧光显微观察方法首次确定了枇杷有效授粉期,并创新性地从柱头可授性、花粉活力、花粉原位萌发、花粉管生长动力学特性与胚囊寿命,以及降雨和温度对这些要素的影响方面系统地研究了枇杷授粉受精的内外影响因子,探明了枇杷有效授粉期决定因子对坐果率和果实种子数的影响。同时为了进一步阐明花粉管动力学特性,首次建立和优化了枇杷AS-PCR反应体系,并在此基础上首次确定了‘大五星’等18个品种或株系的S基因型,以及克隆和鉴定了枇杷的8个新S基因。主要研究结果如下：1.有效授粉期的确定延迟授粉、组织化学检测和荧光显微观察表明,‘大五星’和‘龙泉五号’品种与‘川农一号’株系的柱头可授性可以持续至花后5-6d,‘大五星’和‘龙泉五号’在开花当天柱头就具有较强的可授性,并在花后1-3d具有强的可授性,而‘川农一号’在开花当天只有部分柱头具有可授性,同时柱头具有强可授性的持续时间相对较短。2.花粉管动力学特性花粉管动力学特性研究表明,‘大五星’枇杷自花和异花授粉后花粉在柱头上的萌发率几乎无差异,授粉后2h花粉萌发率约为35%,授粉后8h花粉萌发率达到最大值；在授粉后第4h约有12%的花柱可以在其上部观察到花粉管,授粉后第12h可以在部分花柱的基部观察到花粉管。异花授粉后生长穿过花柱的花粉管量显著多于自花授粉。自花授粉后花粉管生长穿过上部、中部和基部的花柱比率差异显著,这表明花柱上部和中部是‘大五星’枇杷自交不亲性表现的主要部位。3.花龄对花粉萌发与花粉管生长的影响花龄对自花授粉和异花授粉后花粉萌发和花粉管生长的影响研究结果表明,大蕾期和花后46d进行授粉可以在一定程度上减轻花柱中的自交不亲和现象,蕾期与延迟自花授粉能够获得较高坐果率也佐证了这一结果。4.有效授粉期决定因子对坐果率和种子数的影响相关性分析结果表明,有效授粉期与柱头可授性、花粉管生长穿过的花柱比率及胚囊寿命密切相关,‘大五星’和‘龙泉五号’较高的坐果率和较多的果实平均种子数归因于其柱头具有较强可授性的时期较长,花粉管生长穿过的花柱和出现花粉管的胚珠比率均较高,而‘川农一号’较低的坐果率和较少的果实平均种子数归因于其柱头具有较强可授性的时期较短,花粉管生长穿过的花柱和出现花粉管的胚珠比率均较低。连续三年的实验结果表明,所试3份枇杷材料的有效授粉期是由遗传因子所决定,‘大五星’和‘龙泉五号’为依赖于柱头可授性的基因型,‘川农一号’为依赖于柱头可授性与胚珠退化的基因型。5.降雨对柱头可授性和花粉活力的影响以‘大五星’为试材,研究了阴雨天气对枇杷柱头可授性和花粉活力的影响。结果表明：持续的阴雨天气可以使枇杷柱头可授性和花粉活力不同程度的降低,降低授粉效率,对枇杷生产有负面影响。柱头可授性检测表明,‘大五星’枇杷柱头可授期为开花当天至花后第5d,在此期任何一天只要天气晴朗,柱头随即恢复并具有较强的可授性,这一特性可以一定程度地减少阴雨天气带来的负面影响。6.降雨对枇杷授粉受精的影响田间模拟降雨试验结果表明,萌发花粉粒和坐果率在授粉前4、2h与授粉前(-Oh)或后立即(Oh)模拟降雨的处理和对照间有显著差异,特别是模拟降雨后马上授粉与授粉后立即模拟降雨的两个处理与对照间的差异更明显。然而萌发花粉粒和坐果率在授粉前8h及授粉后2、4、8h和12h模拟降雨的处理与对照间差异不显著。室内模拟试验结果表明,保留在柱头上的萌发花粉粒数在所有的处理中都有所减少,特别是在授粉前后Omin或30min将枝条浸入水中的处理,以及枝条依次经过浸入水中、摇动、授粉、浸入水中与摇动的处理显著少于对照。这些结果表明,花粉与柱头乳突细胞的识别,及花粉的水合是十分迅速且有力的,除了在授粉前后立即降雨外,其他的处理均不能把柱头上的花粉冲洗掉。7.温度对枇杷柱头可授性和花粉活力的影响离体花枝在不同温度条件下培养不同时间后散粉情况观察、花粉活力与柱头可授性检测结果表明,5℃低温可以推迟枇杷花期和散粉时间,直至培养第7d花粉活力仍可以维持在55%左右的较高水平,同时柱头可授性在温度回升后也能维持和恢复在较强水平,同时有超过90%的胚珠具有活力。然而高温则促进花开放和加快散粉,同时加快花粉活力和柱头可授性的衰退,在30℃条件下培养至第7天花粉活力仅为6.39%,同时在培养第3d,柱头可授性已明显衰退,在第5d已不具可授性。这表明如果在枇杷气球花期遇持续的低温天气转晴后,气球花仍能顺利完成授粉过程,不会明显影响枇杷生产。8.温度对枇杷花粉萌发的影响通过对离体花枝在不同温度条件下培养,授粉后不同时间的花粉萌发情况的研究表明,在一定的温度范围内随温度的升高花粉的萌发时间逐渐缩短,萌发率逐渐提高,在5℃条件下花粉的萌发率仅约为10%,在10、15和20℃时萌发率显著提高至75%以上,但是温度升高至25和30℃时,萌发率又分别显著下降至约65%和45%。9.温度对枇杷花粉管生长的影响温度对花粉管生长的影响研究结果表明,花粉管在花柱中生长的跟踪观察结果与花粉萌发结果一致,在一定的温度范围内,花粉管生长速度随温度的升高逐渐加快,在10、15和20℃条件下被花粉管生长穿过的花柱比率和有花粉管的胚珠比率显著高于其他温度。当温度为5和30℃时,在授粉后120h均未发现有花粉管穿过花柱生长至胚珠。10.温度对枇杷胚囊寿命的影响温度对胚珠退化影响的结果分析表明,在5℃条件下培养120h后退化胚珠的比率仍仅为6%左右,而当温度升高至25和30℃时仅培养12h后退化胚珠比率均高达85%以上。这些结果表明,高温可以加速枇杷胚珠的退化,随温度的升高,胚珠退化的速率逐渐加快,同时退化胚珠的比率也逐渐增加,而适度的低温则有助于保持胚珠的活力,表明枇杷胚珠具有一定的耐寒能力。11.枇杷AS-PCR体系建立与优化以‘大五星’为试材,通过正交实验设计对影响枇杷AS-PCR反应较大的Mg2+等5个因素的浓度进行筛选,并对扩增反应程序进行优化,运用正交设计直观分析法和DPS7.05统计软件对扩增结果进行方差分析。优化后的枇杷AS-PCR反应体系为：25μL反应体系中,含10×buffer2.5μL,Mg2+浓度2.0mmol/L,Taq酶1.5U,引物0.5μmol/L,模板DNA80ng,dNTPs0.4mmol/L。反应程序为94℃预变性1mim,94℃变性30s,54℃退火30s,72℃延伸1mim,35次循环,72℃延伸5mim,4℃保存。12.枇杷新S基因克隆与鉴定利用上述枇杷优化AS-PCR体系对‘大五星’等18个品种或株系的基因组DNA进行扩增,对获得的目的片段进行克隆测序及序列分析,共克隆12个基因,其中8个为枇杷的新S-RNases基因,分别命名为S11-RNase(ACCESSION:KC131137)、 S12-RNase(ACCESSION:KC131138)、S13-RNase(ACCESSION:KC131139)S15-RNase(ACCESSION:KC131135)、S16-RNase(ACCESSION:KC131136)、 S18-RNase(ACCESSION:KC131134)、S31-RNaae(ACCESSION:KC131133)和S41-RNase(ACCESSION:JX217035)。13.‘大五星’等18个枇杷品种(或株系)S基因型鉴定利用上述枇杷优化AS-PCR体系对‘大五星’等品种或株系的基因组DNA进行扩增,对获得的目的片段进行克隆测序及序列分析,确定了所试18个品种或株系的S-RNase基因型分别为：‘川农2号’S1-S41、‘黄密’S2-S5、‘贵妃’、‘有间’和‘早钟6号’S2-S6、‘香钟11号’S2-S15、‘龙泉5号’S2-S18、‘房光’S2-S31、‘冠玉’和‘大五星’S2-S41、‘早红’S5-S6、‘晚钟’、‘大白梨’和‘茂木’S6-S13、‘里间’S6-S15、‘软条白沙’S6-S31、‘川农1号’S11-S12和‘有永’S16-S41。14.枇杷S-RNase基因推导氨基酸序列同源性比较所克隆的12个枇杷S-RNase基因的推导氨基酸序列的同源性为53.3%—-97.8%,与蔷薇科梨属和苹果属S-RNase基因的同源性在48.6%—100%之间。其中枇杷S13-RNase基因与梨S3-RNase基因,及枇杷S2-RNase基因与梨S8-RNase基因的同源性都高达100%；枇杷S16-RNase基因与梨S1-RNase基因的同源性为96%；枇杷S18-RNase基因与苹果S1-RNase基因的同源性为93%；枇杷S16-RNase基因与苹果S6-RNase基因的同源性为93%。这些结果表明枇杷属、苹果属和梨属S-RNase基因在不同属间的同源性大于在同一属之内。此外,系统树构建结果表明,参试的33个S-RNase基因都是随机地分布在进化树的不同位置,并未形成属内的特异性群体。综合S-RNase同源性比较与系统树构建结果表明枇杷属植物的S-RNase在该物种分化前形成。更多还原

【Abstract】 In the present study, a series of experiments were carried out to determine the EPP by delayed and bud pollination, histochemical tests and fluorescence microscopic observation. And the primary factors influencing the pollination and fertilization were systematically researched, such as stigma receptivity, pollen viability, pollen germination in situ, pollen tube growth, ovule vitality, rainfall and temperature. And the effects of primary factors influencing the EPP on fruit and seed set was elucidated. Simultaneously, to further elucidate the pollen tube kinetics, an AS-PCR reaction system was established and optimized for the first time in loquat and the S-genotypes of18cultivars or lines were determined, and eight novel genes named S11, S12, S13, S15, S16, S18, S31and S41respectively were cloned from these cultivars or lines. The main results were as follows.1. Determination of EPPThe data of delayed and bud pollination, histochemical tests and fluorescence microscopic observation showed that the duration of stigma receptivity was long, lasting until five or six DAA. The stigmas had the moderate receptivity in ’Dawuxing’ and ’Longquan No.5’, then for3days after that, the stigmas had the intense receptivity, while in’Chuannong No.l’, only partial stigmas had a little receptivity at anthesis, and duration of intense receptivity was two to three DAA.2. Pollen tube kineticsThe results of pollen tube kinetics showed that there was no significant difference in pollen germination between self-pollination and cross-pollination. The rate of pollen germination was about35%in2HAP, about12%pollen tubes were observed in upper of styles in4HAP, and the pollen tubes were observed in bottom of styles in12HAP.And pollen tubes that reached upper styles, middle styles and bottom styles in cross-pollination were significantly more than those in self-pollination. Furthermore, there was significant difference in the rate of styles whose upper, middle and bottom were traversed by pollen tubes respectively. The aforementioned results showed that the upper and middle of styles were the main locations of self-incompability in loquat.3. Effects of fowler age on pollen germination and pollen tube growthThe data of the effects of flower age on pollen germination and pollen tube growth showed that the self-incompability was weak. Namely, the number of styles traversed by pollen tubes in self-pollination in big budding period or4-6DAA was more than other days. And the result was evidenced by the higher fruit set in self-pollination in big budding period or4-6DAA.4. Effects of determined factors of EPP on fruit set and seeds setThe results of correlation analysis showed that the EPP was closely related to stigma receptivity, rate of styles traversed by pollen tubes and ovule viability. The higher fruit set and more seeds per fruit in ’Dawuxing’ and ’longquan No.5’ were attributed to the longer period in which the styles had intense stigma receptivity, and more styles and ovules were traversed by pollen tubes, while the lack of seeds was attributed to the shorter period in which the styles had intense stigma receptivity, and lower rate of styles and ovules traversed by the pollen tubes in ’Chuannong No.1’. These differences in stigma receptivity and ovule longevity were not due to external factors, such as climatic conditions, for these experiments were performed simultaneously in the three cultivars. Consequently, the stigma receptivity and ovule degeneration seemed to determine the EPP in loquat. In conclusion, fruit and seed set of ’Chuannong No.1’ was genotype-dependent and can be limited either by stigma receptivity or ovule longevity, while that of ’Dawuxing’ and ’Longquan No.5’ may be limited by stigmatic receptivity.5. Effects of rain on stigma receptivity and pollen viabilityThe effects of rainfall on stigma receptivity and pollen viability were studied in ’Dawuxing’ loquat. The results showed that the stigma receptivity and pollen viability decreased with the rainfall lasting days, which should reduce pollination efficiency and had the negative effects on production of loquat. Moreover, the detection results of stigma receptivity showed that ’Dawuxing’ loquat had a longer flower receptivity period until5 days after anthesis, and the stigma receptivity could be recovered from negative effects of rainfall by a sunny day which could reduced the negative effects of rainfall on production to some degrees.6. Effects of rain on pollen-stigma adhesion and fertilizationThe results of field experiments showed that the number of germinated pollen grains and the percentage of fruit set differed significantly between the-4,-2,-0or+0treatments and the control in the field assays, especially in the case of the-0and+0h treatments in which the stigmas were washed immediately before or after hand-pollination. However, the-8,2,4,8,and12h treatments did not show significant differences from the control. On the other hand, the laboratory experiments showed that all washing treatments reduced the number of germinated pollen grains retained on the stigma, and immersion treatments at-30,-0,+0, or30min, and the additional ISPIS treatment showed significant differences from the control in the numbers of pollen grains retained on the stigmas. In conclusion, the results showed that the adhesion of pollen grains to the stigmas in loquat was rapid and strong, and the simulated rain was not able to wash-off pollen grains completely from the stigma surfaces, except in the-0and+0h treatments.7. Effects of temperature on stigma receptivity and pollen viabilityThe results in studying the effects of temperature on stigma receptivity and pollen viability showed that the blooming and pollen grain dispersion were delayed in the low temperature (5℃), the pollen viability could still reach a higher level (55%) up to the7d after cultivation, the ovule viability could exceed90%, and it was worth noting that the stigma receptivity was renewed and lasted a higher level with the rise of temperature. However, the blooming and pollen grain dispersion were promoted in the higher temperature (25-30℃), resulting in speeded pollen viability and stigma receptivity decrease. The pollen viability was only6.39%in the pollen grains cultivated at30℃at7DAA, simultaneously, the stigma receptivity decreased obviously in the flowers cultivated at30℃at3DAA, and there was no stigma receptivity at5DAA. These results showed that flowers at balloon stage might achieve pollination and fertilization after clearing up of weather after the flowers went through cold and wet weather, which was beneficial to the production in loquat. 8. Effects of temperature on pollen germinationThe difference in pollen germination of the flowers cultivated at different temperatures showed that the time of pollen germination was shortened gradually with the rise of temperature in some scope of temperature, and the rate of pollen germination was increased gradually. The rate of pollen germination was only10%at5℃, while the rate of pollen germination was improved obviously to more than75%at10,15and20℃. However, the rates of pollen germination were declined obviously to65%and45%respectively when the temperatures were raised to25and30℃.9. Effects of temperature on pollen tube growthThe effect of temperature on pollen tube growth was consistent with that on pollen germination. The speed of pollen tube growth was accelerated with the rise of temperature in some range of temperature. The rate of style or ovule traversed by pollen tubes in the flowers cultivated at10,15and20℃was significantly higher than those of flowers cultivated at5,25and30℃. And there were not styles or ovules traversed by pollen tubes in the flowers cultivated120HAP at5and30℃.10. Effects of temperature on ovule viabilityThe results of studying the effects of temperature on ovule viability showed that the rate of degenerated ovules was only about6%when the flowers were cultivated120HAP at5℃, while the rate of degenerated ovules was exceeded85%when the flowers were cultivated120HAP at25and30℃. The aforementioned results implied that the degeneration of ovules was accelerated gradually with the rise of temperature, while the low temperature was beneficial to maintaining the ovule viability, which indicated that the ovules might have the stronger ability of resisting the lower temperature.11. Establishment and optimization of AS-PCR reaction system for loquat’Dawuxing’,’Zaozhong6’and’ Longquan5’were used as materials. The concentrations of Mg2+, Taq polymerase, dNTPs, primer and template DNA were determined. Orthogonal design was used to optimized above components, and time and temperature of various procedures were also improved in AS-PCR in this study. The orthogonal design intuitive analysis method and DPS7.05software were adopted in the analysis of variance. The optimized PCR cocktail of25μL contained10×buffer2.5μL,2.0 mmol/LMg2+,1.5U Taq polymerase,0.5μmol/L primer,80ng template DNA, and0.4mmol/L dNTPs. The optimized reaction procedure was:94℃for1min, followed by35cycles at94℃for30s,54℃annealing for30s,72℃for1min, and terminated with a5min extension step at72℃.12. Identification and cloning novel S-RNases in loquatIn this investigation eight novel genes named S11, S12, S13, S15, S16, S18, S31and S41respectively were cloned from eighteen loquat cultivars using the optimization of AS-PCR reaction system for loquat. And they were deposited in GenBank under the accession numbers of KC131137, KC131138, KC131139, KC131135, KC131136, KC131134, KC131133and JX217035, respectively.13. Identification of S-genotypes of18cultivars in loquatPCR amplification from genomic DNAs of eighteen loquat cultivars was carried out using the aforementioned optimization of AS-PCR reaction system. Then the PCR products were extracted, cloned and sequenced. The amplified fragments were assigned to their respective S-RNases by Blast analysis. The S-genotypes of the eighteen tested self-incompatible cultivars were identified as follows:’Chuannong No.2’(S1-S41),’Huangmi’(S2-S5),’Zaozhong No.6’,’Youjian’ and ’Guifei’(S2-S6),’Longquan5’,’Xiangzhong11’,’Fangguang’,’Dawuxing’ and ’Guanyu’(S2-S41),’Zaohong’(S5-S6),’Dabaili’ and ’Wanzhong’ and ’Mogi’(S6-S13),’Lijian’(S6-S15),’Ruantiaobaisha’(S6-S31),’Chuannong1’(S11-S12) and ’Yoshi’(S16-S41).14. Alignment of the deduced amino acid sequences of S-RNasesThe deduced amino acid homology ranged from53.3to97.8%among these twelve S-RNases alleles in this study, and ranged from48.6to100%with S-RNases alleles from Pyrus and Malus. The deduced amino acid identities were100%between S13-RNase of Eriobotrya and S3-RNase of Pyrus, S2-RNase of Eriobotrya and S8-RNase of Pyrus,96%between S16-RNase of Eriobotrya and S1-RNase of Pyrus,93%between S18-RNase of Eriobotrya and S1-RNase of Malus, and S16-RNase of Eriobotrya and S6-RNase of Malus. The aforementioned results showed that the homology of S-RNases from different genera was higher than the S-RNases from same genus. Furthermore, the results of phylogenetic tree indicated that the thirty three S-RNases alleles from Eriobotrya, Pyrus and Malus were not assembled the specific group within genus, and were distributed randomly in the different locations of phylogenetic tree. Therefore, these results indicated that the S-RNases existence predated the speciation among Eriobotrya, Pyrus and Malus.更多还原