| Flowering is one of the most important growth stages of higher plants during their life cycles and it is regulated by genes and environment jointly.Generated mainly in leaf phloem,the protein coden by FT gene is then transported to shoot apical meristem.The feature of long-distance moving makes it play a crucial role in regulation of floral induction.Studying on the mechanism of regulating the expression of FT gene is of great significance in speeding up the process of genetic improvement of mulberry.In this study,we cloned the promoter of FT gene from mulberry genome by PCR,and its function was analyzed through 5,deleted mutation.The results lay a theoretical foundation for researching the mechanism of regulating the expression of FT gene, further exploring the flowering regulation mechanism and shortening juvenile phase in order to realize early fruit and high yield.The main contents and conclusions of this study are as follows:1. Induction of adventitious buds of mulberry leavesAdventitious bud induction of vitro leaves from Neo Ichinose was performed in the laboratory environment.We induced adventitious buds successfully with differentiation medium of MS+6-BA 5.0 mg/L+NAA 0.2 mg/L+1.0% sucrose+1.0% glucose+1.0%fructose+0.75% agar. This provided a reference for the establishment of the regeneration system of mulberry.2. Cloning and functional analysis of MaFT gene promoter in MulberryAccording to the whole genome sequences of Morus notabilis,the FT promoter with921 bp was gotten from Morus alba by PCR.We designated it as MaFTP and the NCBI accession number was KU550086. Even though MaFTP which did not have CpG island had92% similarity with upstream sequences of Morus notabilis FT homologous gene which was published in NCBI,there were still deletion and insertion of large fragments.The results of multiple sequences alignment showed that the similarity of FT promoter sequences from different species such as mulberry, poplar, apple, Arabidopsis and rice were extremely low. The above results indicated that FT promoter sequences were different in a number of species,and even in different varieties of the same species,they were not same completely.The analysis of promoter prediction tool showed that the sequences not only contained TATA-box, CAAT-box and other core components of eukaryotic promoters,but also had light,drought,circadian rhythm,abscisic acid, auxin,salicylic acid and some other responsive elements.Based on the distribution of cis-elements in MaFTP, primers were designed and 5,deleted fragments were amplified.We named them as MaFTP903、MaFTP762、MaFTP542、MaFTP289 、 MaFTP162 respectively.This five deleted fragments substituded 35 S CaMV promoter in expression vector pBE2113 and fused with GUS gene,and the new expression vectors were then transformed into the tobacco by Agrobacterium. GUS staining showed that except MaFTP903 whose activity was very low, other promoter fragments could drive the expression of GUS gene efficiently, and the expression levels were similar, suggesting that the core section of FT promoter was located in162 bp. Studying the activity of different deletion promoters and determining the core functional sites had guiding significance for further research of the transcriptional regulation mechanism of FT gene.3. Functional analysis of KdsA under drought stresspBE2113-KdsA-GFP expression vector was constructed and then transformed into the tobacco by Agrobacterium.The subcellular location of KdsA showed that KdsA protein was distributed in the whole cell and mainly in the cell membrane and nucleus.Under drought stress, proline content and the expression levels of P5 CS and DREB2 B in mulberry leaves which expressed KdsA transiently increased significantly.The results showed that the increase of KdsA gene expression could change the expression levels of drought-related genes and drought-related index.Obviously,KdsA played a certain role in drought stress. This experiment laid the foundation for the study of the function of KdsA gene in drought stress. |
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