| Superficial scald is a physiological disorder of apple and pear fruits causing brown or black patches on fruit skin which appears during or after long time storage. Although superficial scald has been studied for nearly a hundred years, the etiology and biochemistry processes that lead to its development are not yet completely understood. The α-farnesene oxidation hypothesis proposed that superficial scald is caused by the oxidation of a volatile compound, α-farnesene, which is produced in fruit skin and converted to toxic conjugated trienols(CTols). Although there is a close relationship between CTols content and the superficial scald occurrence, direct evidence proving CTols as the causal agent is still lacking.α-farnesene synthase(AFS) is the final enzyme in the α-farnesene synthetic pathway inhibiting which will specially reduce α-farnesene synthesis without causing other unwanted changes. Hence, by silencing this enzyme the relation between α-farnesene and the disorder can be detected and proved. The long juvenile phase and transformation difficulties existed in fruit trees, however, hampered the studies from the molecular biology respect. Former studies in our lab selected ‘Fertility’ pear(Pyrus communis L.) as the transform material and developed the Agrobacterium-mediated transformation system in this cultivar. Based on this system both anti-sense and RNAi mediated α-farnesene silencing construct was successfully transformed to ‘Fertility’ pears, but further identification is still needed. Here we report the stability assessment of these transgenic lines from molecular levels and further analyzed their physiological characters. Relationship between the superficial scald natural resistance and AFS promoter elements in different cultivars was discussed. Finally we checked the feasibility of producing superficial scald-resistant non-transgenic fruits by combing RNA interference technology with graft methods. The main results are as follow:(1) Kanamycin resistance screen and Polymerase chain reaction(PCR) was used respectively to assess the transgenic lines in genomic level. All the transgenic lines whichwere propagated in vitro for five years or lived in the field for several years live normally in the selection medium or has the PCR product of predicted length, demonstrating that the foreign gene was stably integrated into the genome without losses through many years during in vitro cultivation or in variable wild environment.(2) Reverse transcription quantitative PCR(RT-qPCR) analysis was used to quantify the PcAFS expression level of transgenic lines, including those grown for one year and four years in the fields and scions grafted on rootstocks. An obvious decline in the transcription level of PcAFS gene was detected in the leaves of all the plants mentioned above, which demonstrated that the silencing effect was quite stable in the transgenic lines through many years regardless of graft procedure.(3) MeJA was used to increase the expression level of PcAFS gene in leaves. Nearly a hundred folds of transcriptional raise was detected, reaching the level comparable to fruits.The expression level was still kept much lower in the transgenic lines compared with wild type as expected, showing the silencing effect is quite stable regardless of the gene expressional level.(4) Further identifications in the product level proved finally that the vectors we constructed was very effective in reducing endogenous α-farnesene content in ‘Fertility’ pear.(5) Key enzymes and other terpene metabolites in terpene synthesis pathway were characterized. Neither changes in the expression level of these enzymes nor the contents of other terpene metabolites was detected. These results demonstrated that silencing of PcAFS gene, which eliminates α-farnesene production, has little influence on terpenoid synthetic network.(6) There seems to be no obvious difference in abiotic stress tolerance between wild type and transgenic lines because of the similar performance under oxidation and heat stress conditions.(7) The graft transmission of silencing effect was researched using graft procedures in‘Fertility’ pear, in light of the non-cell-autonomous RNAi properties extensively reported in the herbaceous plants. The feasibility of producing marker-free and environmental safetyapples with high superficial scald resistance was explored by grafting wild type scion to transgenic rootstocks. Leaves collected were used to detect this property. Results showed that the graft transmission of silencing effect seemed not to be very obvious at least in the early-stage graft scions.(8) Promoter sequences of α-farnesene synthase gene were obtained using TAIL-PCR technologies from ‘white winter pearmain’ and ‘Fuji’ apples, respectively. High similarities was found between the two promoter sequences, so as to the cis-acting elements, although the natural superficial scald resistance was significantly different between these two cultivars,which indicates that the cis-acting elements may not play decisive roles.(9) The promoter sequence of PcAFS gene from pears was also cloned. Surprisingly, this promoter shares little similarities with its counterpart from apples(48.31% similarity). Only a total length of about 350 base pairs upstream from the translation initiation site was highly homologous.(10) Predicated cis-acting elements from promoters of ‘Fuji’ apple and ‘Fertility’ pear were compared and their shared elements was selected and labeled on the ‘Fertility’ pears’ promoter. Based on their predicted function and locus, serious 5 ? truncated promoter fragments(named D1-D5) were amplified. These promoter sequence fragments were then inserted into the upstream of GUS reporter gene in pCAMBIA1391 and introduced into tobacco. GUS activity analysis on T1 generation showed that all the promoters can drive GUS gene expression, although the GUS activity was different in various promoter fragments.Surprisingly, the shortest promoter fragment which was shorted to only 226 bp still functions normally. |
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