| As one of the main fruit trees in the south of China,citrus plays an important role in the process of rural economic development and rural revitalization.The development of citrus industry is mainly the alternation of old varieties and new varieties,but because of the long juvenile period of citrus,the breeding speed of new varieties is slow,which is difficult to meet the development needs of citrus industry.Therefore,it is of great significance for the development of citrus industry to study the juvenile regulation mechanism of citrus.FD gene plays a key role in the flower formation process of model plants.In citrus,alternative splicing of CiFD gene can produce different transcripts(CiFDα and CiFDβ).In this study,the functions of different CiFD transcripts were analyzed and the citrus transformation materials of CiFDα and CiFDβ were created.Through the experimental study of low temperature treatment,the regulatory pathway of CiFD involved in low temperature induced citrus flowering was preliminarily analyzed.The main research results are as follows:1.In order to study the function of different CiFD transcripts in the flowering process of citrus,the DNA sequence of CiFD(CiFD-DNA)and the sequences of CiFDα and CiFDβ were transferred into tobacco.The results showed that the flowering time of transgenic tobacco was significantly earlier than that the control,and the number of leaves at flowering was also significantly less than that the control.In addition,the expression levels of flowering related genes Nt FT,Nt LFY and Nt AP1 in CiFDα and CiFDβ transgenic tobacco were significantly higher than that the control.These results indicated that CIFD-DNA,CiFDα and CiFDβ may play an important role in the flowering process of citrus.2.In order to confirm the function of CiFDα and CiFDβ,the overexpression genetic transformation of CiFDα and CiFDβ was carried out in Poncirus trifoliata.A total of nine CiFD-DNA transgene lines,fourteen CiFDα transgenic lines and ten CiFDβ transgenic lines were obtained by DNA and RNA identification.It is worth noting that some transgenic lines flower very early and present flower buds in the test tube.Quantitative results showed that the expression levels of flowering related genes(Ci FT,Ci AP1 and Ci LFY)in the transgenic lines were significantly higher than that the control.The transgenic lines were subsequently transplanted into soil,and no flowering has been observed so far.These results suggest that CiFD is a positive regulator of flowering,and both CiFDα and CiFDβ may be involved in the regulation of flowering in citrus.3.GUS staining and enzyme activity analysis confirmed that CiFD promoter activity was induced by low temperature.At the same time,the expression trend of CiFDα and CiFDβ was detected in adult sweet orange treated with low temperature.It was found that CiFDα could be induced by low temperature,and the expression trend of CiFDβ was significantly up-regulated,while the expression trend of CiFDβ did not change significantly.These results suggest that CiFDα may play an important role in the process of citrus flowering induced by low temperature.In order to elucidate the pathway of CiFD regulating citrus flowering,the interaction of CiFDα and CiFDβ with the downstream target gene Ci LFY promoter was verified by yeast single hybrid experiment.The results showed that CiFDβ could interact with Ci LFY promoter,but CiFDα could not interact with Ci LFY promoter.These studies indicated that CiFDα was involved in citrus flowering process by driving downstream target gene Ci LFY at low temperature. |
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