The Relationship Between PIF Gene And The Biosynthesis Of Carotenoids In Citrus

Due to their healthy benefits and attraction to eyes, enrichment of carotenoids in fruit is among the important breeding goals in citrus. AtPIF1 is a transcription factor, which has been found to regulate genes that work in the carotenoids synthetic pathway, so discover the relationship between PIF gene and the synthesis of carotenoids in citrus has very important significance.Seike navel orange, Cara Cara navel orange, Washington navel orange and Newhall navel orange,’Guoqing 1’ Satsuma mandarin, Marsh grapefruit and Red Marsh grapefruit were used as experimental materials. The cDNA of their fruits and leaves were used to clone the CsPIF gene. Two complete cDNA sequence of CsPIFs were amplified from these materials. They nucleotide and putative amino acid sequence were analyzed. At the same time, Real-time PCR technology was used to analysis the copy number of these two genes and their tissue-specific expression. Furthermore, red light whether had effects on the expression of CsPIF gene and PSY gene, which encoded a rate-limiting enzyme in carotenoids biosynthsis pathway, or not was studied. In addition, over-expression was constructed of CsPIFL and CsPIFS. Main results were as follows:1.By using the information from public citrus genomic database, CsPIF gene was amplified from Seike、Cara Cara、Washington and Newhall navel orange,’Guoqing 1’ satsuma mandarin, Marsh and Red Marsh grapefruit by reverse transcript polymerase chain reaction(RT-PCR). There were two main highly homologous CsPIFs, CsPIFL and CsPIFS, in these citrus species.Their protein homology was up to 95%. There are 19 amino acid site differences between them and CsPIFL is 4 amino acids longer than CsPIFS. The two protein all have typical bHLH domain、APB motif and nuclear localization signal sequence. In addition, they were homologous to UNE(PIF8), which was a member of the PIFs family. Through Real-time PCR technology, copy number ratio of the two genes in different citrus speices were analysed preliminarily.2. By analysis the gene relative expression of total CsPIF in different tissue or organs, for example, leaves、juice sac in seven different citrus species, and flavedo of Marsh and Red Marsh grapefruit, the result showed that leaves had the highest gene relative expression of total CsPIF, followed by flavedos and juice sac had the lowest gene relative expression.3. Red light or white light were used to treat the callus and fruits at color break stage of’Guoqing 1’satsuma mandarin for lday or 2 days. In flavedo, experimental results showed that when treated with red light, the gene relative expression of CsPIF、CsPIFL and CsPIFS reduced, but on the contrary, the expression of PSY ascended. In callus, the results showed that when treated with red light, the gene relative expression of CsPIF、 CsPIFL、CsPIFS and PSY reduced. However, no matter in flavedos or callus, the average gene relative expression of CsPIFL was no more than half of the total CsPIF and 5 folds of CsPIFS.4. HPLC(High Performance Liquid Chromatography) was used to analysis the cotent of carotenoids in flavedo and callus of ’Guoqing 1’ satsuma mandarin which were treated with different quality of light. There were five main carotenoids, violaxanthin lutein、 a-carotene、β-carotene and phytoene, in flavedo and callus of ’Guoqing 1’ satsuma mandarin. In callus, phytoene was the main carotenoid. However, in flavedos, β-carotene was the main carotenoid and the cotent of phytoene was the lowest. Also experimental results showed that when treated with red light, phytoene and total carotenoids increased in flavedos, and when treated with red light for 1 day, the content of total carotenoids increased significantly. However, in callus, when treated with red light for 1 day, phytoene and total carotenoids increased, but decreased for 2 days.5.The over-expression vector of CsPIF were successfully constructed, it was useful to demonstrate the function of CsPIF in future.