Molecular Colning And Expression Analysis Of MADS-box Genes From Garlic(Allium Sativum L.)

Garlic(Allium sativum L.)belongs to the genus Allium,family Alliaceae,is a diploid species(2n=2x=16)grows an annual or biennial vegetable.Native to West and Central Asia,Because the rich of nutrients and resistance to storage and other characteristics have been widely planted.To explain the Molecular mechanism of floral organ development of garlic.Conventional garlic varieties 'Yining Redskins' as test material in this study.We isolated and characterized six full length cDNA sequences of garlic MADS-box genes from conventional garlic varieties 'Yining Redskins' denoted as AsPI,AsAG,AsAGL6,AsSEP1,AsSEP3 and AsSVP by using RACE(rapid amplification of cDNA ends).In addition,investigation was also made to analyze sequence characteristics and phylogenetic relationships based on bioinformatics methods.We analyzed the expression pattern of these genes in different organs of onion according to semi-quantitative RT-PCR and the real-time fluorescence quantitative PCR.The results of this study laid a solid foundation for further interpreting the action mechanism of garlic MADS-box genes and molecular mechanism of floral development.1.In this study we used conventional garlic varieties 'Yining Redskins' as test materials,obtained six full length cDNA sequences contained open reading frames by the method of RACE and namedAsPI,AsAG,AsAGL6,AsSEP1,AsSEP3 and AsSVP.2.Phylogenetic analysis indicated that AsPI belong to the PI/GLO of the monocot B-function gene family respectively;AsAG belongs to the AG-like-lineage of the monocot C-function gene family;AsA GL6 belongs to the AGL6-like-lineage;AsSEP1 belongs to the LOFSEP lineage of the monocot E-function gene family.AsSEP3 gene belongs to the SEP3-like-lineage of the monocot E-function gene family.3.AsPI high expression abundance in flower bud,with the highest level in the flower bud of the first period.Almost no expression in vegetative organs such as roots,stems,leaves,garlic skin,with very low level in young garlic shoot;AsAG expressed the amount showed a trend of increase in bud three times,in the vegetative organs of false stem,leaves almost no expression,and very low level in the root,garlic skin,young garlic shoot;AsAGL6 high expression abundance in the third period of flower bud,almost no expression in vegetative organs;AsSEPl with the highest level in the young garlic shoot and flower bud,and AsSEP3 with the highest level expression quantity in the third period of all period of flower bud;But AsSVP high expression abundance in vegetative organs,very low level in floral organs,moderate levels of garlic skin and young garlic shoot,the expression pattern is corresponding to that AsSVP genes function in inhibiting flowering characteristics.4.Constructed overexpression vector for AsSVP gene of garlic.Transfection of Arabidopsis thaliana with agrobacterium infection method.The results showed that compared with wild-type svp mutant exhibit that delayed flowering time.Wild-type Arabidopsis thaliana grown for 28 days,there are 14 sitting lotus leaf and appear buds.Transgenic plants after 36 days of growth,there are 24 sitting lotus leaf and appear buds.In accordance with SVP gene has inhibitory effect on the flowering.