Cloning And Expression Analysis Of Genes Related In The Glossy Navel Orange Mutant

The glossy navel orange mutant is a novel mutant occurred spontaneously from ’Newhall’ navel orange(Citrus sinensis [L.] Osbeck cv. Newhall). The most remarkable phenotypic trait of the mutant is the glossy surface of its fruit peels and leaves that is easily distinguishable from the Wild type. The mutants provide a valuable material for the study of the molecular mechanism that controling the wax biosynthesis and transportation pathway, In this research, the main biological characteristics, the genetic stability were studied, Moreover, two wax-related genes, named FATB and CUT1, were cloned from the Wild type ’Newhall’ and its glossy muntant. The expression patterns of these genes in Wild type and muntant at different fruit developmental stages were also detected in this research. The results of the research are as follows:1. Compared with the wild type, the glossy mutant has more glossy fruit suface, can be peeled off more easily, and was riped early. Scanning electron microscopy (SEM) revealed that the quantity of crystalline platelets were much less in the epicuticular wax layer of mutant fruits than that of wild type. Furthermore, the biological characteristics of mutant always remained stable under field conditions.2. The Trizol method and improved trizol method were respectively used to extract leaf and fruit peel total RNA from navel orange.The results of electrophoresis showed that the RNA bands were clear, high purity, and no obvious degradation, which can be applied directly to the following experiment. Two cDNAs represented the same gene, designated as CsFATB, were isolated from the wild-type navel orange and its glossy mutant (MT) with bright glossy fruit peels and leaves. The CsFATB cDNA is1407bp in length with a1254bp open reading frame (ORF), encoding a protein of418amino acids with a molecular weight of46.06kDa and a theoretical isoelectric point of7.76. Sequence alignment showed that The CsFATB protein share high identity with other plant FATBs. Semi-quantitative RT-PCR revealed that CsFATB expression levels were increased in WT but declined in MT fruit peels and leaves at later fruit developmental stages. Moreover, the CsFATB expression levels in fruit peels and leaves were higher in MT than in WT at the young fruit stages, but were lower in MT than in WT at the fruit matrue stage.3. The CsCUTl gene was successfully cloned by RT-PCR method from both wild type navel oranges and its glossy mutant. Sequence alignment showed that the43th,258th and548th base of CsCUT1sequence in the glossy mutant were different from the wild type ’Newhall’. The cDNA length of CsCUT1from wild type ’Newhall’ was1734bp with an open reading frame of1488bp, encoding a protein of496amino acids. However, the cDNA length of CsCUT1from was1733bp with an open reading frame of1287bp, encoding a protein of429amino acids. CsCUTl protein from wild type ’Newhall’shared79%-91%identity with other plants CUT1family proteins. Semi-quantitative RT-PCR expression analysis revealed that the CsCUT1expression in leaves and fruit of wild type ’Newhall’, and leaves of mutant were both up-regulated first, followed by down-regulated, but in fruits of mutant the CsCUT1expressionwas up-regulated with the maximum at November.