导　　师： 李唯

学科专业： 090102

授予学位： 博士

作　　者： ;

机构地区： 甘肃农业大学

摘　　要： 桃（Prunus persica）原产于我国，属蔷薇科多年生落叶乔木果树，在全球南北纬45°间范围均有分布。桃果实鲜丽、清香多汁、酸甜适口、味美质细、极宜鲜食，其果肉中的生物活性组成成分有抗氧化、预防糖尿病、癌症等作用。桃从发育、成熟到软化经历了一系列复杂的过程，不仅包括生长，还包含色泽、质地、风味、香气及内含物等变化。桃是典型的呼吸跃变型水果，其果实成熟后会产生大量乙烯，诱发果实“呼吸”迅速升高，导致果实贮藏物质迅速水解而软化腐烂。这一过程涉及许多基因表达和多种水解酶活性变化，是由多种因素共同作用而导致的综合性结果。RNA干扰技术的创立与应用为有效缓解桃果实采后迅速软化和快速实现桃种质创新提供了新的重要途径，可望通过该技术的应用，封闭或降低果实成熟过程中果肉细胞壁原果胶分解的多聚半乳糖醛酸酶（PG）和催化乙烯合成前提物1-氨基丙烷-1-羧酸（ACC）氧化脱羧的氧化酶（ACO）活性，阻滞桃果实采后迅速软化和腐烂，大幅度延长果实的有效贮藏时间和降低贮运成本，增加桃的生产附加值。 为此本研究以兰州地区果实成熟后极易软化的“白凤桃”为实验材料，测定和分析其成熟过程不同阶段，果实中活性物质和抗氧化能力的变化，以及与PG和ACO基因表达之间的关系，并以白凤桃基因组DNA为模板，克隆了PG和ACO基因，构建其RNA干扰植物表达载体，通过根癌农杆菌介导法建立桃胚和番茄的遗传转化体系，获得了卡那霉素（km）抗性植株。主要研究结果如下： 1.随着桃果实的逐渐成熟，果实硬度、可滴定酸度逐渐下降，可溶性固形物含量、果肉成熟指数、花青素和维生素C的含量则逐渐增加；总酚含量基本呈下降趋势，而总黄酮含量先升而后降。在果实成熟软化过程中，果肉芳香挥发物的总量显著增加，其中有7类主要的芳香挥发物变化明显：反式-2-己烯醛和顺式-3-己烯醇含量逐渐减少，γ-十二内酯、γ-癸内酯、δ-癸内酯含量则显著增加，而γ-己内酯和γ-辛内酯含量显著增加后略有回落，这些变化使得桃果实可食性增强。 2．采用DPPH法和FRAP法进行桃果实抗氧化能力分析，发现果肉总抗氧化能力随着果实成熟整体呈上升趋势，达到完熟期开始下降。皮尔逊相关分析表明，果实抗氧化能力增强促进了芳香挥发物总量、γ-十二内酯、γ-己内酯、γ-辛内酯、γ-癸内酯、δ-癸内酯、花青素和维生素C的代谢，呈显著正相关性。其中DPPH法测得抗氧化能力与γ-己内酯相关系数达到0.973，而FRAP法得到与维生素C的相关系数达到0.981。 3.桃果实成熟期间，随着果实硬度和可滴定酸度降低，可溶性固形物和果实成熟指数上升，ACO基因表达量不断增加，而且与芳香挥发物总量，γ-十二内酯，γ-癸内酯，δ-癸内酯，γ-己内酯，γ-辛内酯，花青素和维生素C呈正相关，而与反式-2-己烯醛，顺式-3-己烯醇和总酚类物质则呈显著负相关。PG基因的表达在桃果实在进入着色期后就显著增加，接近完熟期时下降并保持在一个相对稳定的水平。其表达量的变化与可滴定酸度和反式-2-己烯醛、顺式-3-己烯醇、总酚类物质呈负相关（P<0.05和P <0.01），与γ-己内酯、类黄酮和花青素呈正相关（P <0.01）。果实成熟期抗氧化能力的降低与PG基因表达量的降低密切相关。而ACO基因的表达与桃抗氧化能力的关系却明显不同，不论桃“着色期”中ACO表达量降低或升高，果实的类黄酮含量和抗氧化能力均呈缓慢降低的态势，表明ACO的表达与果实成熟期抗氧化能力的变化无关。 4.利用CTAB法提取了供试材料果实基因组DNA，以DNA为模板进行PG、ACO基因的克隆，并进行表达载体的构建，得到了PG和ACO的RNAi植物表达载体，同时进行PCR、酶切鉴定和测序，结果表明，成功构建了pCAMBIA2300-PG和pCAMBIA2300-ACO植物表达载体。 5.优化番茄再生体系，用携有ACO基因hpRNA片段和35S启动子的表达载体pCAMBIA2300，转化番茄。番茄暗预培养3d后，经OD600为0.6的根癌农杆菌菌液浸染8min后进行共培养（MS+IAA0.2mg//L+6-BA1.0mg//L+AS200μmol//L），3d后，用Cef进行洗涤脱菌，脱菌培养3d后，转去分化培养基MS+IAA0.2mg//L+6-BA1.0mg//L+Cef300mg//L+Km30mg//L诱导产生不定芽，将抗km的不定芽在生根培养基上继续选择，获得完整植株。经PCR和点杂交检测初步证明，ACO基因已导入番茄中。 6.以白凤桃果实种胚为外植体，进行桃胚直接诱导不定芽再生体系的优化：种胚开花75d后能在MS+NAA0.05mg//L+6-BA0.5mg//L上直接诱导分化成苗，分化率可达91.5/%。用携带有PG基因hpRNA片段和35S启动子的表达载体pCAMBIA2300，转化白粉桃幼胚。当幼胚预培养3d后，经OD600值为0.4的根癌农杆菌菌液浸染10min后共培养65h，转移去分化培养基MS+NAA0.5mg//L+6-BA1.0mg//L+Cef250mg//L+km50mg//L上诱导产生不定芽，将抗km的不定芽生根培养基上继续选择，获得完整植株。经PCR检测和Southern杂交检测初步证明，PG基因已导入桃中。 Prunus persica, a perennial deciduous fruit tree of Rosaceae, is native to China anddistributes in a wide range between45°N and45°S globally. The prunus persica fruit isflorid, fragrant and juicy, palatably sour and sweet, delicious and of fine quality, thus it isvery suitable for eating when it is fresh. The bioactive components in its pulp have manyfunctions, including antioxidant capacity and diabetes and cancer prevention, etc. Prunuspersica experiences a series of complex processes, from growth, ripening to softening,including growth and other changes, such as color, quality, flavor, fragrance and inclusions,etc. Prunus persica is a typical respiratory climacteric fruit and its fruit will generate alarge amount of ethylene after ripening, which induces the “breath” of the fruit to risequickly, causing the fruit softening and bletting due to the rapid hydrolysis of storagematters in the fruit. This process involves many gene expressions and activity changes ofmultiple hydrolases and it is a comprehensive result caused by combined action of manyfactors. The establishment and application of RNA interference offers a new importantway for effectively remitting rapid softening of prunus persica fruit after picking andquickly realizing germplasm innovation. It is hopeful to close or reduce thepolygalacturonic acid /(PG/) decomposed by protopectin in the pulp cell wall and catalyzethe ACO activity of1-amino propane-1-carboxylic acid /(ACC/) oxidative decarboxylationby applying this technology to retard the rapid softening and decay after picking. As aresult, it can significantly prolong the effective storage time of the fruit, reduce storageand transportation costs and increase the production value added of the prunus persica. In this study,“prunus persica”, a fruit growing in Lanzhou Area and softening easilyafter ripening, is chosen as the experimental material to determine and analyze differentstages in its ripening process, changes of active substances and oxidation resistance of thefruit, and the relations with PG and ACO genes expression. Also, the DNA in the prunuspersica genome is used as the template to clone PG and ACO genes and establish the plantexpression vector of RNA interference. Furthermore, kanamycin /(km/) resistant plant isobtained by building the genetic transformation system of prunus persica embryo andtomato through agrobacterium tumefaciens mediated method. And main study results are as follows: 1. As the prunus persica fruit gradually become ripe, fruit firmness and TAdecline, while TSS content, RI, and the content of anthocyanins and Vitamin C increasesgradually; basically, total phenolics content is on a declining curve, while total flavonoidcontent rises before dropping. In the process of ripening and softening, total aromavolatiles of pulp increase significantly and seven main aroma volatiles change obviously:the content of trans-2-hexenal and cis-3-hexenol decreases gradually, while the content ofγ-dodecalactone, γ-decalactone, and δ-decalactone increases significantly, and the contentof γ-hexalactone and γ-octalactone, however, significantly rises and then drops slightly.Such changes strengthen the edibility of the prunus persica fruit. 2. It is discovered that total antioxidant capacity of the pulp is on the rise wholly withthe ripening of the fruit and starts to decline when the fruit enters into the full ripeningstage by analyzing the antioxidant capacity of prunus persica fruit with DPPH and FRAPmethod. The analysis of Pearson’s Correlation Coefficients suggests that the fruit’sstrengthened antioxidant capacity facilitates the metabolism of total aroma volatiles,γ-dodecalactone, γ-hexalactone, γ-octalactone, γ-decalactone, δ-decalactone, anthocyaninsand Vitamin C in significant positive correlation. The coefficient of association betweenthe antioxidant capacity and γ-hexalactone reaches0.973as determined by DPPH method,while the coefficient of association between the antioxidant capacity and Vitamin C is upto0.981as determined by FRAP method. 3. During the ripening of prunus persica fruit, as the fruit firmness and TA drop, TSSand RI rise, ACO gene expression quantity is increasing and in positive correlation withtotal aroma volatiles, γ-dodecalactone, γ-decalactone, δ-decalactone, γ-hexalactone,γ-octalactone, anthocyanins and Vitamin C, while it is in significantly negative correlationwith trans-2-hexenal, cis-3-hexenol and total phenolics substances. PG gene expressionincreases significantly as the prunus persica fruit enters into color period, then it willdecline and maintain a relatively stable level when the fruit approaches full ripening stage.The change of its expression quantity is in negative correlation with TA, trans-2-hexenal,cis-3-hexenol and total phenolics substances, but in positive correlation withγ-hexalactone, flavonoids and anthocyanins. The above results suggest that there is correlation between the increase of ACO and PG gene expression and major bioactivecomponents of prunus persica fruit. 4. Genome DNA is extracted from the fruit of experimental material by using theCTAB method. DNA is adopted as the template for cloning PG and ACO genes andestablishing expression vectors to obtain the RNAi plant expression vectors of PG andACO along with identification and sequencing of PCR and enzyme digestion at the sametime. The result shows that pCAMBIA2300-PG and pCAMBIA2300-ACO plantexpression vectors are successfully established. 5. Tomato regeneration system is optimized and the tomato is transformed by usingthe expression vector of pCAMBIA2300carrying hpRNA fragment and35S promoter ofACO genes. After3days of dark preculture, the tomato is dipped in the agrobacteriumtumefaciens liquid with OD600of0.6for8minutes before its co-culture in the medium ofMS+IAA0.2mg//L+6-BA1.0mg//L+AS200μmol//L. Three days later, Cef is used forwashing and bacteria elimination of the tomato, which is cultured without bacteria foranother three days. Then it is transferred to the differential medium of MS+IAA0.2mg//L+6-BA1.0mg//L+Cef300mg//L+Km30mg//L to generate adventitious buds underinduction and those km resistant adventitious buds will continued to be cultured in therooting medium to get a complete plant. Through the preliminary evidence by PCR testand dot blotting detection, ACO genes have been imported to the tomato. 6. The seed embryo of prunus persica fruit is used as the explant to optimize theadventitious bud regeneration system under the direct induction of prunus persica embryo:The seed embryo can directly induce differentiation for sprouting on the medium ofMS+NAA0.05mg//L+6-BA0.5mg//L75days after it blooms and the differentiation rate isup to91.5/%. The young embryo of prunus persica is transformed by using the expressionvector of pCAMBIA2300carrying hpRNA fragment and35S promoter of PG genes. After3days of preculture, the young embryo is dipped in the agrobacterium tumefaciens liquidwith OD600of0.4for10minutes before its co-culture for65hours. Then it is transferredto the differential medium of MS+NAA0.5mg//L+6-BA1.0mg//L+Cef250mg//L+km50mg//L to generate adventitious buds under induction and those km resistant adventitiousbuds will continued to be cultured in the rooting medium to get a complete plant. Through the preliminary evidence by PCR test and Southern hybridization detection, PG geneshave been imported to the prunus persica.

关 键 词： 桃 果实品质 抗氧化能力 基因表达 遗传转化

分 类 号： [S662.1]

领　　域： [农业科学] [农业科学]