Effects Of Cs-ACS1 Transgene On Sex Differentiation Of Cucumber

Gynoec and subgynoe breed is one of the main purpose in cucumber breeding. The appearing time, quantity and distribution of node is a major factor affecting cucumber yield. At present it is clear that the Sex differentiation of cucumber is controlled mainly by genetic factors. However, It is controlled by other factors, such as endogenous ethylene, which is one of the main factors. Many researchers believed that ACC synthase gene is colsely linked with the F locus which controls female flower of cucumber. Tereinto, Cs-ACS1 gene and F gene are highly homologous. Previous, someone mistakely that they thought Cs-ACS1 gene was F gene. Obviousely, Cs-ACS1 gene has a great relation with female differentiation of cucumber. In this paper, a fragment about 2333 bp was amplified from cucumber genomic by means of PCR, and clones to the pMD18-T vector. Next, to construct the sense and antisense plant expression vector, also, construct a plant expression vector with red fluorescent protein as reporter gene. In the end, transform these plant expression vectors into cucumber. Through observation, identification, investigation and analysis, we find Cs-ACS1 gene has a great relation with female gender Of cucumber. The main contents and findings of this paper are as follows:1.Cloning of Cs-ACS1A 2333 bp fragment was amplified from cucumber genomic by means of PCR, BLAST analysis showed that the cloned fragment shares 98%homology with Cs-ACS1, only one base was different from the recorded sequence, without causing any change in the amino acid sequence. The results showed that:The cloned gene was Cs-ACS1 gene.2.Construct the sense plant expression vector of Cs-ACS1 (pVCT2214) and transform it into cucumberInserted the Cs-ACS1 gene into pVCT2102, to construct the sense plant expression vector of Cs-ACS1 (pVCT2214). After enzyme digestion and PCR testing, we transform pVCT2214 into strong female lines of cucumber 0837 (breed by our laboratory) use method of AgroBacterium-medi-ated. A total of 12 transgenic cucumber was survival after transplanting. Through PCR detection, preliminarily proved the pVCT2214 have been transformed into the genome of cucumber; through observation of phenotype of the survival plants, found that 10 transgenic cucumber plants all blossom male flowers, And all the the terminal bud are male, indicating that exogenous Cs-ACS1 gene and Cs-ACS1 gene of strong female cucumber inhibit each other, restrict exogenous Cs-ACS1 gene expression, but also restrict the Cs-ACS1 gene of itself expression, which appears co-suppression.3.Construct the antisense plant expression vector of Cs-ACS1 (pVCT2215) and transform it into cucumberAfter sense plant expression vector (pVCT2214) was digested by restriction enzyme, we obtained antisense plant expression vector (pVCT2215). After testing, we transform pVCT2215 into strong female lines of cucumber 0837 by method of Agrobacterium-mediated. Two transgenic cucumber was obtained after transplanting. Through PCR detection, proved the pVCT2215 have preliminarily been transformed into the genome of cucumber; through observation of phenotype of the survival plants, found that two transgenic cucumber plants all blossom male flowers, all the the terminal bud are male, which appears cis-suppression.4. Expression of Cs-ACS1-REF gene in flower bud of transgenic cucumberInserted the Cs-ACS1 gene into vectors containing red fluorescent protein to construc plant expression vector pVCT2217. After testing, we transform Cs-ACS1-REF into strong female lines of cucumber 0837 by method of Agrobacterium-mediated, and a total of 6 transgenic cucumber was survival after transplanting, and all blossom male flowers. We extracted the genomic of transgenic cucumber for PCR, proved Cs-ACS1-REF has been transferred into the genome of cucumbe; when did whitened treatment for a week via fluorescence microscopy, we detected the flower buds of transgenic cucumer had expected red fluorescence while the transgenic cucumer and non-transgenic cucumer were been whitening process. All indicate that Cs-ACS1-REF gene mainly expressed in flower buds.