| 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. |
PDF Full Text Request