Full-length Gene Cloning And Expression Analysis In Citrus Of The Cold-regulated Gene Ptcor8

Origin of Citrus is China. It is the largest fruit crop in southern China. However, different degrees of low temperature damage to citrus production suffered serious losses. Research work on risstance to hardiness of citrus has both significant theoretical and practical importance on the world's citrus-producing countries, the resistance breeding research has also been one of the main contents of citrus breeding. The research group has screened and cloned a cold acclimation-related genes Ptcor8 (EU077497) from the Poncirus leaves. In this study, further study the open reading frame of exon and intron number and distribution; Researched Ptcor8 gene in different varieties of citrus in the dynamic expression; Studied the annual expression of Ptcor8 gene in trifoliate leaves and Ptcor8 gene expression of the relationship with trifoliate defoliate. The results are as follows:1. Get Poncirus, Fortunella, Lemon Ptcor8 Genomic DNA SequencesUsed Poncirus, Fortunella and Lemon DNA as the template for PCR amplifica-tion, by cloning and sequencing, Get the full gene sequence, Poncirus and Lemon full gene are 1834bp, Fortunella is 1832bp. This gene was composed of four exons and three introns. Their exons location and size are the same, there are 188bp,107bp, 72bp and 254bp, the total length is 621bp; their intron location are the same but size differences, Poncirus are 786bp,83bp and 344bp, the total length is 1213bp, Fortunella are 786bp,83bp and 342bp, the total length is 1211bp, Lemon are 789bp,83bp and 341bp, the total length is 1213bp. Using DNAMAN compared Poncirus cDNA sequence and Fortunella, Lemon, Poncirus Ptcor8 full-length gene ORF sequence, Poncirus cDNA sequences and full-length gene ORF sequence have 99.03% homology, there are three different amino acids; Fortunella and Poncirus ORF sequence have 98.71% homology, there are eight different amino acids; Lemon and Poncirus ORF sequence have 98.55% homology, there are eight different amino acids.2. Ptcor8 gene expression in different varieties of citrusIn the whole cold acclimation process in autumn, real time quantitative RT-PCR technology was acpuired to detect the Ptcor8 expression in different types of citrus. The results showed that during cooling when the temperature decreased to below 5℃and for 15 days, the expression of the gene significantly increased. The gene expression was the highest in Poncirus, followed by Grapefruit and Sugar orange. Lemon Ptcor8 gene expression continued to maintain a low or no expression.Ptcor8 gene expression analysis in the anniversary in trifoliate orange, the results showed that, the gene is low or no expression in April 3,2009 to August 29, a sudden and sustained low temperature cooling can make the gene expression increase, but the gene expression delayed the cooling.Semi-quantitative RT-PCR, and real-time quantitative RT-PCR technique can detected the expression of Ptcor8 gene in the deciduous and the non-deciduous Poncirus. What we can refer from this are that:the expression of Ptcor8 gene may not be directly related to the physiological defoliation of Poncirus, but closely related to low temperature and it's duration.