Formation Mechanism Of A Late-Ripening Bud Mutant Of Navel Orange (Citrus Sinensis Osbeck)

'Fengjiewancheng'(FJWC, Citrus sinensis Osbeck) is a novel late-ripening navel orange mutant of'Fengjie72-1'navel orang(FJ72-1). Academically, FJWC might constitute a useful experiment system with its parental line for the study of citrus ripening mechanism. To get the knowledge or information as much as possible about the occurrence mechanism of this mutant will play a great role in citrus fruit-mature-time breeding via gene engineering, and at last in maintaining consecutive and healthy development of citrus industry. In order to give clues as to what mechanisms are monitored in the mutant, difference of FJWC and FJ72-1 in coloration or pigmentation, sugar, acid and mRNA transcripts during fruit ripening were compared, and the occurrence mechanism of FJWC was discussed in the end. The main results were as follows:1. An efficient RNA isolation method was established, which was proved to be widely applicable in different citrus tissues at different developmental stages. The A₂₆₀/A₂₃₀ of RNA was over 2.0, and the A₂₆₀/A₂₈₀ ranged from 1.65-1.90, indicating low levels of polysaccharide and good purity of the RNA. The quality of the RNA was further proved by means of RT-PCR, DIG Northern blot analysis and cDNA-AFLP.2. Coloration analysis during fruit ripening indicating that FJWC's coloration was delayed about 30 days or so, which was because that the distinctive decrease of chlorophyll in the peel of FJWC occurred from the early of November to the late of November, later than that of its original cultivar which occurred from the middle of October to the early of November, and the obvious accumulation of carotenoid in the peel of the mutant began on Dec. 12th, while its original cultivar on Nov. 3rd. What's more, analysis of independent-samples T test showed that the chlorophyll content in the peel of FJWC from October to November was significantly higher, and the carotenoid content from December to January was significantly lower than that of the parental line, FJ72-1 respectively. Gene expression of some carotenoid biosynthetic genes and cholorophyllase gene using RT-PCR technique were showed that the time of some carotenoid biosynthetic gene expression reaching a maximum in the peel of the mutant were postponed for one month comparing to those of its original cultivar, while the gene expression of cholorophyllase in the peel of FJWC reached a maximum in January, and kept slightly lower level from October to December than that of FJ72-1.3. Analysis of sugar seasonal changes in the pulp and peel of both FJWC and FJ72-1 during fruit ripening discovered a major difference of sugar contents in the pulp between the two cultivars. Sugar contents of sucrose, glucose and fructose in FJWC pulp were significantly lower than those in FJ72-1 before the middle December, and became higher after then. In fruit peel, only sucrose contents in FJWC were significantly lower than those in FJ72-1 at the two early sampling dates during fruit ripening. Transcript analysis of sucrose metabolic gene showed that CitAI(citrus acid invertase gene) and CitSS1(citrus sucrose synthase genel) had different expression patterns in the pulp between the two cultivars during fruit ripening which were related with the sugar content difference of them.4. Acid contents of FJWC during fruit ripening were also changed comparing with FJ72-1. The citric acid contents in the pulp of FJWC were obviously higher than those in FJ72-1 pulp during the early ripening stages, while lower than the latter after the late November. In the middle of fruit ripening, malic acid contents in FJWC were significantly lower than those of FJ72-1. Transcript comparison of citric metabolic gene showed that the difference of citric acid between the two cultivars might be related with the high transcript level of citrate synthase gene and with the block of cytosolic aconitase gene in the pulp of FJWC during the early ripening stages.5. No reliable differences on DNA level between the two cultivars were discovered by SSR and AFLP analysis.6. Expression profiles of the two cultivars' fruit during fruit ripening were analyzed using cDNA-AFLP. After screening 120 pairs of selective primers which produced about 20000 clear transcript bands, only 144 clear transcript different fragments(TDFs) were found in 34 pairs of primers. However, only 129 TDFs were successfully recovered, sequenced and then blasted by blastx tool. The blastx results showed that most of the TDFs were related with signal transduction and transcription factors, senescence or maturation, stress reaction, ribosome protein and DNA replication.7. Using the sequence of TDFs, six genes related with fruit maturation were successfully cloned using RACE technique. There were named as CitPG(EF185420), CitCAD(EF185415), CitMads(EF185417), CitMAP(EF185418), CitNAC(EF185419) and CitEREBP(EF185416). RT-PCR indicated that they expressed differently in fruit peel or pulp between the two cultivars, which implicated that these gene might be related with the occurrence of late-ripening phenotypes.Taken together, many aspects of FJWC were changed compared with FJ72-1, the late-ripening phenotypes of FJWC is obviously not the results of some factor mutation or alteration, but is involved in delay of ripening progress. Furthermore, the alteration of transcript level might be one of the reasons of the late-ripening phenotypes.Fruit ripening is a comprehensive and complicated progress. Unlike climacteric fruit, non-climacteric fruit ripening, such as citrus, experienced a gradual and programmed progress. Hence, what made FJWC coordinate changes must located in the upstream stages of fruit ripening or development, that is, a factor having the function to trigger the onset of fruit ripening might be altered, which lead to the coordinate delay of FJWC fruit ripening progress.