【作者】 郑宝强；

【导师】 彭镇华；

【作者基本信息】 中国林业科学研究院 ， 园林植物与观赏园艺， 2009， 博士

【摘要】 本研究以卡特兰品种Brassolaeliocattleya Sung Ya Green‘Green World’为试材,观测和记录研究了卡特兰的生长发育规律以及北方温室温度和湿度的周年变化,研究了各年生器官中营养物质的周年变化,以及不同叶龄叶片相对于不同光强的叶绿素荧光特性。采用石蜡切片法观察了卡特兰花芽的形态发生和结构发育过程。在此基础上,在花芽分化期进行3种不同的温度处理,探讨了相应时期内的营养以及新叶中内源激素的动态变化;对处于不同花芽分化阶段的卡特兰进行不同的温度处理,研究了温度对开花性状和质量的影响;在花芽分化期喷洒和注射不同浓度的GA3、NAA、ABA等激素,探讨了激素对开花性状和质量的影响。最后对卡特兰花朵衰老过程中的某些生理生化变化进行了研究,并且构建了卡特兰ACO基因的反义表达载体pBI121ACC,为进一步应用反义技术培养花期长的卡特兰新品种奠定了基础。通过以上研究,得出以下主要研究结果:通过研究各年生器官中营养物质变化发现,卡特兰当年形成的假鳞茎储存较少的淀粉,营养物质主要以可溶性糖的成分存在,其它各年生假鳞茎和叶片中,营养物质以淀粉形态储存,花芽分化和开花消耗大量营养。在休眠期营养储存少,在新芽初生期各假鳞茎营养储存较多,所以卡特兰的换盆和分株繁殖宜在新芽初生期进行。对不同叶龄叶片的荧光特性研究表明,随着光照强度的增加和叶龄的增长,PSⅡ电子传递量子产率(Yield)、光化学淬灭(qP)、有效光化学量子产量(Fv’/Fm’)表现为逐渐降低,非光化学猝灭(qN)逐渐升高。新叶虽然在低光强下有着较高的qP、Yield、Fv’/Fm’,但当光强高于740μmol·m-2s-1时,光抑制现象严重,表观电子传递速率(ETR)降低,表明新叶对强光的适应能力较差,因此在栽培中,新叶生长期光照强度不宜超过740μmol·m-2s-1。一年生叶片ETR最高,光能利用效率最高,但光强超过1250μmol·m-2s-1时同样会导致光抑制,二、三年生叶片ETR相近,四年生ETR最低,光能利用率最低,最易受到光抑制。在北方温室温度条件下,卡特兰花芽分化从7月初花序原基分化开始,至9月下旬合蕊柱及花粉块形成历时约3个月。其过程可分为6个时期:未分化期、花序原基分化期、小花原基分化期、萼片原基分化期、花瓣原基分化期、合蕊柱及花粉块分化期。其中,小花原基分化期、合蕊柱及花粉块分化期历时长,分化较慢,其它时期历时短,分化较快。自萼片原基分化期开始,新生植株生长已基本停止。6月下旬,在卡特兰花芽未分化期开始不同的温度处理表明,25/20℃处理能显著的促进花芽分化,30/25℃处理花芽能正常分化,35/30℃处理抑制花芽分化;25/20℃和30/25℃处理条件下,叶面积增长速率差异不显著,在处理24 d时,25/20℃处理条件下花鞘增长速率快于30/25℃处理。35/30℃处理条件下,叶面积和花鞘增长速率明显落后于其它两个处理。在不同温度处理的开始时,随处理温度的升高,可溶性糖在新叶和新假鳞茎中下降幅度增大,在老叶和老假鳞茎中下降幅度变小,淀粉在各个器官中下降幅度也变小。处理18 d时,35/30℃处理条件下,新叶中可溶性糖含量明显高于其它两个处理,假鳞茎中可溶性糖含量则低于其它两个处理。在30/25℃处理条件下,可溶性糖和淀粉在各个器官中变化趋势基本一致。在25/20℃处理条件下,新叶的可溶性糖含量与其它各器官变化趋势相反。25/20℃、30/25℃处理条件下,新生叶片中GA3、ZR、ABA含量增加,IAA含量减少。35/30℃处理抑制新生叶片中GA3、ZR、ABA含量,促进IAA含量,而较低的GA3、ZR、ABA含量和较高水平的IAA含量不利于花芽分化;35/30℃处理使GA3/IAA、GA3/ZR的比值处于一个较为稳定的状态,而这种状态不利于花芽分化。保持较低的IAA/ZR与IAA/ABA水平,有利于花芽分化的继续进行。在6月下旬,卡特兰花芽未分化期开始25/20℃处理能够显著地促进开花,使盛花期提前56 d,但开花率低,开花以单花为主。在8月中旬萼片分化期开始25/20℃处理也能够显著地促进开花,使盛花期提前14 d,开花以双花为主,并且花朵显著增大。花芽未分化期开始35/30℃的高温处理能够抑制开花,萼片分化期开始35/30℃的高温处理能够延迟开花,花期延迟14 d。两次30/25℃处理与对照无差异。花蕾破鞘期进行10/6℃的低温处理能够延迟开花,使盛花期推迟36 d,在元旦开放。在花芽分化过程中喷施300 mg·kg-1和600 mg·kg-1的GA3能够使花柄和花葶的长度显著增加,使盛花期分别提前9.33 d和8.67 d,两个处理之间差异不显著。喷施不同浓度的NAA对卡特兰开花性状没有影响,当喷施NAA的浓度为200 mg·kg-1时使花期推迟6.67 d。注射GA3的浓度为60 mg·kg-1和120 mg·kg-1时,能够使盛花期显著提前13.34 d和22.34 d,使萼片、花瓣、花柄和花葶的长度显著增加;9月9日花鞘注射10 mg·kg-1的NAA能够使花期提前,能够使花朵显著增大。喷施和注射ABA对卡特兰的花期没有影响,注射ABA浓度为40 mg·kg-1时,开花率下降,花朵缩小。所以,在栽培中推荐使用注射的方法进行花期调控,不仅用量小,而且效果显著,注射60 mg·kg-1的GA3或10 mg·kg-1的NAA不仅能够使花期提前,而且使花朵增大,可以作为花期调控的重要手段。卡特兰花衰老过程中花瓣的可溶性蛋白质含量逐渐下降,细胞质膜透性、丙二醛(MDA)、超氧阴离子(O2·-)产生量随花瓣的衰老逐渐增加,超氧物歧化酶(SOD)和过氧化物酶(POD)活性逐渐降低。内源ZRs含量降低,内源IAA、ABA含量上升,乙烯释放量呈跃变型变化,GA3含量变化不明显。以花瓣为试材,首先提取总RNA,并根据其它兰花的ACC氧化酶基因保守序列设计一对特异性引物,然后通过RT-PCR法克隆得到1条967 bp的卡特兰ACO cDNA片断,共编码321个氨基酸残基。序列分析结果显示该克隆片断与已发表的其它兰花的ACO基因序列同源性均在85%以上,尤其与卡特兰原生种和其近亲属的同源性均在95%以上。将克隆的卡特兰ACO片段反向连接到植物表达载体pBI121中CaMV35S启动子的下游,构建了卡特兰ACO基因的反义表达载体pBI121ACC。本研究论文首次明确了卡特兰在北方温室环境条件下的生长发育规律、需肥规律、需光特性,首次明确了卡特兰花芽分化的规律和时期,为制定合理的栽培和管理措施提供了一定依据;通过研究不同温度处理对卡特兰花芽分化、营养物质、内源激素的影响,首次揭示了温度对营养物质、内源激素的调节作用,以及在此基础上对花芽分化的影响。首次成功的通过温度和激素处理对卡特兰花期进行了调控,能够使卡特兰在花卉的热销季节供应市场;通过克隆卡特兰ACO基因以及其反义表达载体的构建,为将来通过反义技术培养花期长的卡特兰新品种奠定了基础。更多还原

【Abstract】 The growth rhythm of Brassolaeliocattleya Sung Ya Green‘Green World’was observed and recorded in greenhouse in North China, and also the temperature and relative humidity was recorded. The changes of nutrient content in organs of different physiological ages and the chlorophyⅡfluorescence characteristics in leaves of different age were studied. The flower bud differentiation process was observed by the method of paraffin cut. The changes in nutrient content and endogenous hormones in new leaves were measured during the flower-bud differentiation phase of plants growing under 3 different temperatures. The effects of different temperature treatments, spraying or injecting different concentration of hormones (GA3,NAA,ABA) on Cattleya florescence and flower quality were studied. Physiological and biochemical changes in flower senescence were also studied. An antisense expression vector of the cattleya-ACO gene, which named pBI121ACC, was constructed. This provides the foundations of future transgenic research for prolonging the flowering period of transgenic Cattleya via antisense technology. The results are described as follows:Soluble sugar was the dominant carbon reserves with lower content of starch in new pseudobulbs, but starch was dominnant in older (2-4 years) pseudobulbs and leaves. Most of carbon nutrient was consumped during flower bud differentiation and florescence. There was lower nutrient storing during dormant period, but more during new bud sprouting. Therefore, new bud sprouting period appears to be the best time for repotting and division propagation.With increasing of light intensity and leaves age, there was a slower decrease in actual PSII efficiency(Yield), photochemical quenching (qP), and efficiency of energy conversion of open PSII (Fv’/Fm’), but a slower increase in nor-photochemical quenching (qN). The new leaves under lower light intensity had higher qP, Yield, Fv’/Fm’, but it is susceptible to high light intensity under which photoinhibition occurred and electron transport rate (ETR) decreased when the light intensity over 740μmol·m-2s-1. The one-year-old leaves had the highest ETR and the most efficiency of light energy, but they also exhibited photoinhibition when the light intensity over 1250μmol·m-2s-1. Two-and three-year-old leaves had similar ETR, and four-year-old leaves were most susceptible to photoinhibition with the lowest ETR.The flower bud differentiation process lasted for about three months from the start of inflorescence primordial differentiation in early July to column and pollinia formation at the end of September under the greenhouse climate condition in North China. The process could be divided into 6 phases: undifferentiation phase, inflorescence primordium differentiation phase, flower differentiation phase, sepal differentiation phase, petal differentiation phase, and column and pollinia differentiation phase. The phases of flower differentiation, column and pollinia differentiation were relatively longer. The new plant finished its growth when sepal differentiation phase began.The flower-bud differentiation was significantly accelerated under the treatment of 25/20℃, unaffected under treatment of 30/25℃, but inhibited under treatment of 35/30℃; The flower sheaths increased faster under 25/20℃than those under 30/25℃after 24 days of treatment. But no difference was observed in leaf area between them. The leaf area and flower sheaths increased slowest under the treatment of 35/30℃; When under a higher temperature in the primary stage of the experiment, the decrease of soluble sugar content in new leaves and new pseudobulbs was more prominent in comparison with old leaves and old pseudobulbs, while the starch content in all organs had a smaller decline. After 18 days of treatment, under the treatment of 35/30℃, the soluble sugar content was higher in the new leaves but lower in new pseudobulbs in comparison with other two treatments. The changes of soluble sugar and starch tended to consistently in all organs under the treatment of 30/25℃. The soluble sugar content in new leaves changed opposite to other organs under the treatment of 25/20℃.The contents of GA3, ZR, ABA were promoted while IAA was inhibited under treatment of 25/20℃and 30/25℃. The reverse trend was observed under the treatment of 35/30℃. The low content of GA3, ZR, ABA and high content of IAA were not good for flower-bud differentiation; The rate of GA3/IAA, GA3/ZR sustained at a stable level under the treatment of 35/30℃which was not good for flower-bud differentiation. It was required lower rate of IAA/ZR and IAA/ABA to keep the flower-bud differentiation continuing.When the different temperature experiment began at undifferentiation phase, it was significantly accelerated the blossom time of Cattleya under the treatment of 25/20℃, which 56 days earlier than that of control, but with lower flowering rate and single flower was dominate. It was not only accelerated the blossom time under the treatment of 25/20℃, but also significantly increased the size of flowers when the experiment began at sepal differentiation phase. The blossom day was 14 days earlier and double flower was dominated. The florescence was inhibited under the treatment of 35/30℃when began at undifferentiation phase, but postponed 14 days when began at sepal differentiation phase. There was no difference between the treatment of 30/25℃and the control. The blossom day would be postponed 36 days to the New Year’s Day after the anaphase low temperature treatment of 10/6 ℃on the period of flower buds out of sheathes.It was remarkably advanced the date of florescence and prolonged the length of pedicel and scape after spraying GA3 with 300 mg·kg-1 and 600 mg·kg-1. The blossom date was postponed 6.67 days by spraying 200 mg·kg-1 NAA, but there was no effect on flower quality by spraying different concentration of NAA. The blossom time was 13.34 days and 22.34 days earlier than that of the control by ejecting GA3 with 60 mg·kg-1 and 120 mg·kg-1, at the same time the length of sepal and petal as the same as pedicel and scape were significantly prolonged. The flower size was increased by ejecting NAA with 10 mg·kg-1. There was no influence on florescence date by using ABA, no matter spraying or ejecting, but the flowering rate and size of flowers were decreased when ejecting ABA with 40 mg·kg-1. So ejecting was the efficient way which was recommended to use to regulate the date of floresence. When ejecting 60 mg·kg-1 GA3 or 10 mg·kg-1 NAA, it was not only to advance the floresence date, but also to enlarge the flowers size.Soluble protein content of petals decreased gradually. Membrane permeability, MDA content and O2·- production rates gradually enhanced while SOD and POD activity reduced with the fading of petals. The contents of endogenous ZRs decreased, but IAA,ABA content increased during senescence, ethylene production rate was typical climacteric type, and GA3 content didn’t change obviously.Total RNA was extracted from flowers of cattleya, According to the conserved acid sequence for ACC oxidase in other orchids, we designed a pair of oligo nucleotide primers. Using RT-PCR method, a cDNA fragment about 967 base pair which encoded 321 predicted amino acid residues were amplified. The result of BLAST showed the sequence presented a very high match with the ACO genes from the other orchids, the homologue was higher than 85%, especially the original species and relatives which the homologue was higher than 95%. An antisense expression vector of the cattleya-ACO gene which named pBI121ACC was constructed, in which the antisense sequence was controlled by the CaMV 35S promoter.This research provides novel knowledge of growth-development, fertilizer and light requirement of Cattleya under the greenhouse climate condition in North China. It also gives new insights into the flower bud differentiation process, offering theory basics of developing proper cultivation strategies for flowering regulation. It was the first time to reveal the roles of different temperatures in nutrient substances and hormones, and consequently in flower bud differentiation. It was successfully, for the first time, to regulate flowering date for fluffing the demands during the flower fast-selling season by using different temperatures and hormones treatment. An antisense expression vector of the cattleya-ACO gene was constructed which would provide the foundations of the future transgenic research for prolonging the flowering period of the transgenic Cattleya via antisense technology.更多还原