| Color is one of the most important sensory qualities of fruits. As a main category of pigments, carotenoids are of vital significance for many kinds of physiological activities in plants(Lu et al 2006), as well as the human health. Citrus is rich in carotenoids, and chromoplast is the major organ for the synthesis and storage of carotenoids in the flesh of citrus(Egea et al 2010), which has irreplaceable effect on the process of intracellular pigment metabolism.Recently, with the booming development of the functional identification for the gene Or in cauliflower and other species, researches on the mechanism about the differentiation of the chromoplasts in plants are becoming deeper and deeper(Li et al 2001). On the basis of the previous researches, with the flesh and the calli of several kinds of citrus which are mutants in color as the materials, this research, on the one hand, conducts relative expression analysis on the genes in plastid genome of citrus using the quantitative real-time PCR(q RT PCR); and on the other hand, we mainly identifies the possible function that the gene Or has during the differentiation of the chromoplasts in citrus by means of genetic transformation, and then explores the internal relation and mutual influence between the gene Or expression and carotenoids metabolism. The main results are as follows:1. As the number of intracellular chromoplasts increases from 0, the phenotype also gets colored in the juice sac of ripe Guanxi Honey pomelo(Citrus grandis [L.] Osbeck) flesh with three different colors(red、yellow and white) and the induced calli of Or transgenic、empty vector injected and wild-type Arabidopsis.2. In the flesh of ripe Guanxi Honey pomeloes with three different colors(red、yellow and white), the Or genes Cs7g18870 and Cs6g14310 have similar relative expression trend, in which they both get the highest level in the yellow one accumulating β-carotene, the second highest level in the red one accumulating lycopene which is a little bit higher than the level in the white one, whose disciplines are same to the trends of the content of total carotenoids in their juice sac. As for the “Anliu”(Citrus sinensis [L.] Osbeck) and “Honganliu” sweet oranges during the fruit developing periods, the relative expression of Or gene Cs7g18870 tends to be remarkably rising; the level of the “Anliu” sweet orange is much higher than that of the “Honganliu” in the late fruit developing periods(December); however, as the fruits develop, the relative expression of Or gene Cs6g14310 tends to increase in “Honganliu” sweet orange while forms a “U-shaped” trend in “Anliu” sweet orange, and for several periods, the relative expression level in the “Anliu” is much higher than the one in the same period of “Honganliu". The relative expression trends of this two Or genes are completely on the contrary of the trends of the content of total carotenoids in their juice sac. Accordingly, in the flesh of red and yellow mutants of Guanxi Honey pomelo, “Anliu” and “Honganliu” sweet orange, the relative expression levels of Or genes in red fruits(red mutant of Guanxi Honey pomelo and “Honganliu” sweet orange) are less than those in yellow ones(yellow mutant of Guanxi Honey pomelo and “Anliu” sweet orange); also, the two Or genes’ relative expression disciplines in Guanxi Honey pomelo are same to the trends of the content of total carotenoids in their juice sac; while these disciplines in sweet oranges are opposite to the trends of the content of total carotenoids in their juice sac.3. In the flesh of ripe Guanxi Honey pomelo mutants, most genes in the plastid genome have the highest expression level in the yellow one, the second highest level in the red one, while the weakest expression in the white one. Comparing the “Anliu” and “Honganliu” sweet orange horizontally, they have different relative expression patterns of genes in plastid genome; while comparing those genes according to the fruit developing periods, more than half of them have a rising relative expression value in the “Anliu” and “Honganliu” sweet orange, and is generally active in October. This trend seems more obvious in the “Honganliu" sweet orange. Additionally, those genes in the flesh of the “Honganliu” sweet orange present high level relative expression again in December.4. Two Or genes in citrus——Cs7g18870 and Cs6g14310 have been cloned, and their over-expression vectors——PH7WG2D-Cs7g18870 and PH7WG2D-Cs6g14310 have been successfully constructed through the method named Gateway. Then, these two vectors are transformed to the calli of wild-type and gene Crt B transformed Marsh grape fruit “Rm” and “Rm33”, and the calli of Valencia Orange “V”. Finally, the resistant calli of the above is successfully acquired, and it is found that the phenotypes of color in the resistant calli transformed with Or genes are similar to the empty vector control and the calli before transformation. After picking and successfully culturing, identifying 20 independent positive monophyletic Rm33 resistant calli transformed with gene Or Cs7g18870, it is found that although there are phenotypic differences in color among those 20 ones, they are varying in the range from creamy white of Rm to orange of Rm33. Similar to the instability of Rm33, those 20 resistant calli of Rm33 transformed with Or gene Cs7g18870 fails to present a different phenotype in color compared with Rm33. Accordingly, we may preliminarily infer that the Or gene Cs7g18870 is unable to change the color of Rm33 remarkably by itself alone. After analyzing the relative expression of related genes in the 20 independent monophyletic Rm33 resistant calli transformed with Or gene Cs7g18870, we find that the phenotypes in color of those resistant calli are most related to the relative expression level of gene Crt B, while with less obvious regular pattern to Or gene Cs7g18870, which is proportional to Crt B in relative expression in a broad sense among those colored resistant calli. Therefore, we guess that in those resistant calli, the phenotypes in color depend on the relative expression of gene Crt B. Although Or gene Cs7g18870 could not change the color of those calli, it might increase the relative expression level of gene Crt B in the colored resistant calli.5. By observing the No.20 independent monophyletic Rm33 resistant callus transformed with Or gene Cs7g18870 through the confocal laser scanning microscope, we reassure the successful injection of Or gene Cs7g18870 and the definite expression of relative protein from the angle of cytobiology. Moreover, we find it by accident that the fusion protein of GFP and Or is distributed not only around the periphery of cell compactly, but also covers some areas inside the cell with the shape “8” like a finger ring. According to previous studies, we infer that the expressed Or protein might be located in cytomembrane and nuclear membrane, or some other types of membrane structures. On the other side, by observing 20 independent monophyletic Rm33 resistant calli transformed with Or gene Cs7g18870 and empty vector control through transmission electron microscopy(TEM), it is found that all those resistant calli cells contain a large amount of amyloplasts. And generally there is no other forms of plastids, or just a little immature plastids without pigment accumulation in the creamy white resistant calli cells transformed with Or gene Cs7g18870. While there are some plastids which is not totally mature with pigment containing in plastoglobulus, and some chromoplast-like structure with obvious double membrane structure in most orange resistant calli cells transformed with Or gene Cs7g18870 and empty vector control. Both the chromoplast-like structure with a riper and clearer appearance of double membrane structure, and the crystalline pigmentation are found in the brighter orange resistant calli cells transformed with Or gene Cs7g18870. According to previous researches, the lycopene and β-carotene in the chromoplasts will be stored as crystalline form only if they reach a really high level(Schweiggert et al 2011), thus, we believe that the crystalline pigmentation found in the brighter orange resistant calli cells transformed with Or gene Cs7g18870 might suggest an increase of intracellular pigments accumulation at a certain degree, in other words, Or gene Cs7g18870 is futher proved to be potential to induce the differentiation of the chromoplast and to facilitate intracellular pigments accumulation. |
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