| Citrus is one of the most important fruits all over the world and the fruit quality is always the main goal for citrus breeding. Citrus breeding has been hindered seriously by several factors, such as long juvenility, micellar embryo, female/male abortion and high hetetozygosis. Genetic engineering opens a new avenue for citrus breeding and makes it possible for using a single gene to improve the fruit quality in citrus. In this study, the regeneration and transformation conditions of the precocious trifoliate orange have been built and optimized to establish a short juvenility genetic transformation system for gene functional verification related to citrus fruits. On the basis of the transformation system, a group of regenerated plantlets containing citrus carotenoids metabolic genes were obtained. Molecular identification and gene expression analysis were also conducted. The main results of this study are as follows:1. High efficient Agrobacterium-mediated transformation and regeneration sytems for precocious trifoliate orange (Poncirus trifoliata [L.] Raf.) and Cocktail (C. paradisi Macf.×C. reticulata Blanco.) were established. Some factors which affect regeneration and transformation were optimized, such as, the illumination of etiolated explants, the coculture conditions, the selection media and the mode of selection etc. Higher regeneration frequencies and transformation frequencies were achieved in all 4-day-illuminated precocious trifoliate orange stem segments and 5-day-illuminated Cocktail epicotyl segment compared to etiolated controls. The highest transformation frequency obtained in coculture medium, which is MT basal medium supplemented with 0.4 mg L-1 2, 4-D. However, increasing concentrations of 2, 4-D from 0.6 to 0.8 mg L-1 suppressed this effect. It was proper for precocious trifoliate orange to produce normal shoots on medium G (MT + 1 mg L-1 BA + 0.1 mg L-1 NAA). For precocious trifoliate orange, when over 1.5 mg L-1 BA was added to SRM, although more visible shoots were produced, most of these shoots were abnormal. Regeneration of precocious trifoliate orange was completely suppressed when hygromycin B was used as selection agent. In recovery shoots, 9 months after grafting, 36.4% of the plantlets flowered (8/22). For the rooting lines (8/25), the flower process was delayed for 12 months. 20 months after being grafted or transferred to greenhouse, 87.2% (41/47) of the transformants flowered. The results implies the in vitro regeneration plants could reserved the precocious traits of precocious trifoliate orange and the transformation system could be used for gene functional verification related to citrus fruits. 2. The anti-sense expression vector pCABCH containing BCH gene fragment was constructed and used to transform the embryonic callus of Valencia (C. sinensis Osb. cv. Valencia). 6 independent transgenic callus lines were obtained. The carotenoid contents were detected by HPLC. The result revealed that lutein andα-carotene content increased in most of transgenic callus lines. And the growth of Valencia transgenic calluses was greatly suppressed by 50-60 mg L-1 Hyg B.3. The anti-sense expression vector pBICI containing CRTISO gene fragment was constructed, and used to transform precocious trifoliate orange and Hongkong kumquat (Fortunella japonica [champ.] Swing.). 4 independent transgenic Hongkong kumquat lines and 23 independent transgenic precocious trifoliate orange lines were obtained respectively.4. The RNAi expression vector pLCYB containing LCYB gene fragment, pLCYE1 and pLCYE2 containing LCYE gene 5'-end and 3'-end fragment were constructed respectively, and the three vectors was used in precocious trifoliate orange and Cocktail tansformation experiments. Results of Real-time PCR revealed that the expression of PDS gene markedly increased in LCFE-suppressed transgenic lines. The expression of LCYB and ZEP decreased. HPLC analysis revealed that in LCYE-suppressed transgenic plants, the content ofα-carotene decreased andβ-carotene and Violaxanthin contents increased, there were no significant changes in respect of lutein content. Unexpectedly, both ofβ-carotene and Violaxanthin increases were inversely proportional to the expression of LCYB and ZEP. According to these findings, we hypothesized that PDS gene was activated by some mechanisms in LCYE-suppressed transgenic plants. And the feedback effects should be responsible for the improvement ofβ-carotene and Violaxanthin:ζ-carotene and lycopene contents upstream improved with the increased expression of PDS gene, and this led to the dramatical increase of metabolic products such asβ-carotene and Violaxanthin downstream. These synthesis genes LCYB and ZEP were suppressed by their metabolic products conversely.In LCYB suppressed transgenic precocious trifoliate orange lines, the expression of genes PSY, ZDS , CRTISO and ZEP presented a downward tendency. At the same time, the content ofβ-carotene and Violaxanthin downstream decreased in the transgenic leaves and there were no significant changes in respect ofα-carotene and lutein contents. The results showed positive correlation between the conten of LCYB expression and β-carotene: the content ofβ-carotene elevated with the increasing of LCYB expression. This process may lead to the accumulation of lycopene.
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