Studies On Polymorphisms Of EIN3 And ACS1G Associated With Sex Determination In Cucumber(Cucumis Sativus L.)

[Background]Cucumber(cucumis sativus L.)is classic materias in research of sex expression patterns in plant. Researches on genes determining on sex expression start very early. Sex expression not only is controlled by genetic factors, but also regulated by environment and chemicals. Long day, high temperature, GA introduce staminate flower. Short day, low temperature, ethylene introduce gynoecious flowers. Endogenesis ethylene, which is significant positive correlation with gynoecious flowers formation, is sex hormone for cucumber. Depth researches on ethylene biosynthesis and signal transductions promot researches on sex expression mechanism in molecular level. ACC synthase is the key emzyme for ethylene biosynthesis so that it plays key roles in a process of sex expression in higher plant. The EIN3 gene(ethylene insensitive3,EIN3) encodes a novel nuclear-localized protein with similarity to known eukaryotic transcriptional activators.EIN3 protein is bind with DNA and located in nucleus which combines with promoter's special sequence of ethylene responses genes and activate those genes and make them transcription Therefore, researches on sex expression mechanism is good trials from breakthrough point of key gene of ethylene biosynthesis and ethylene signal transduction in cucumber.[Objects]The article is established in researches on sex expressions at home and abroad, we hope to get cucumber, watermelon and winter squash EIN3 gene sequences, and submitted to Genebank, furthermore, by bioinformatics and homologous alignments lay the groundwork for evolutions and gene functions researches. By directly sequencing ,clone EIN3 gene in different sex type in cucumber and search for SNPs to find ideal genetic markers. Make a study of the differences between CsACSlG and CsACSl which lay foundations for researches on relationships between ACC gene and gynoecious; Locating the CsACSlG SCAR marker in a linkage map which is constructed by Beijing Vegetable Research Center, which create conditions for molecular maker assistant selection and linkage map integration and cucumber inbred improvement. [Methods]Based on analyses on cucumis melon, nicotiana tabacum and arabidopsis thaliana EIN3 gene mRNA conserved sequences,we aligned by DNAMAN 5.2.2 and find primers by Primer Premier 5.0 and Oligo 6 softwares, cloned and sequenced ,got CsElN3 gene sequence, then by DNAMAN 5.2.2 to analysis homology among CsElN3^ CIEIN3 and CmEIN3.By cloning and sequencing CsEIN3 gene in different sex type, searches SNPs by DNAMAN 5.2.2., and by Primer Premier 5.0 restrict enzyme digestion analyses to get RFLP makers. To verify if of the difference between CsACSl and CsACSlG in promoter region exist. Lastly using RIL as materias, locate CsACSlG SCAR marker in a linkage map which is constructed by Beijing Vegetable Research Center by joinmap3.0.[Results](1) Using cucumber, watermelon, pumpkin as materias, successfully cloned EIN3-\ike gene coding sequence named CsEINS^ CIEIN3 and CmEIN3 ,length of those sequences is 725bp,coding 241 amino acids. The three genes have successfully submitted to Genebank. Accession number are as follows: CsEIN3 is AY973275, CIEIN3 is DQ023225, CmEIN3 is DQ023223. By DNAMAN 5.2.2 to analysis homology among CsEIN3> CIE1N3 and CmEIN3,ihe results show that high homology among cucumber, watermelon and winter squash: CsEIN3-ClEIN3 is 99.3%, ClEim-CmEim is 99.6%, CsEIN3-CmEIN3 is 99.7%; Rate of point mutation is 0.6% among three plant, have one point mutation in about 145bp.By translated correctly amino acids sequences homologous alignment,the results show that CsEIN3-ClEIN3 is 98.8% , ClEIN3-CmEIN3 is 9.8%, CsEIN3-CmEIN3 is 100% , aminoacid sequence are high homology. In NCBI website blastn aligned CsEIN3 with plant EIN3 gene in Genebank found that CsEIN3 is highly homologous to cucumis melo >90%;Nicotiana tabacum, Lycopersicon esculentum, Arabidopsis thaliana >80%. Aminoacid seuquence are highly homologous to cucumis melon (Genebank accession number BAB64344,AAK67355)>90%.(2) Cloning and sequencing on CsEIN3 of different sex genotype cucumber,then Blastn found that 14 SNPs in coding region, have one average 51bp selection rating 1.96%,3 mutation in enzyme digested site,eight SNPs cause amino acid sequence variation by ORF blastp in NCBI website.(3) One set of stable RFLP- Bfm I enzyme digested reaction system was established. By digesting DNA from different parents, found there is a enzyme digest band only in 1983H.