The Function Analysis Of Flowering-regulating Factors AsFUL And AsSVP In Garlic(Allium Sativum L.)

Garlic(Allium sativum L.)is a perennial herb belongs to Allium genus of Alliaceae,is a widely cultivated vegetable in the world with high edible and economic value.However,flowers of garlic are abortion,so the existing cultivars of garlic must rely on asexual reproduction.This leads to the difficulty of conducting garlic genetics research and improve the economic traits through routine breeding.Based on the research of the group,the full length sequence of the MADS-box gene AsSVP,which was related to the flowering of garlic was obtained.In this study,'A cheng' garlic was used as the test material.We isolated and characterized another full length cDNA sequences of the MADS-box gene AsFUL 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 AsFUL and AsSVP genes in different organs of garlic according to semi-quantitative RT-PCR and the real-time fluorescence quantitative PCR.The AsFUL and AsSVP gene overexpression vectors were constructed and transformed into Arabidopsis thaliana by Agrobacterium.Then the positive transformation lines were obtained by antibiotic screening,PCR,RT-PCR and we observed the flowering time of the positive transformants.This research mainly focuses on explain the function of AsFUL and AsSVP genes in the development of garlic flowers,and provide molecular basis for realizing sexual reproduction of garlic.1.In this study we used 'A cheng' garlic as test materials,obtained one full length cDNA sequences contained open reading frames by the method of RACE and named AsFUL.Sequence characteristics analysis show that AsFUL encode deduced proteins with 241 amino acids residues,and the amino acid sequences have a typical MIKC-type structural domain and C-terminal of AsFUL protein have specific motifs of A-class MADS-box genes.Phylogenetic analysis indicated that AsFUL belongs to the FUL-like-lineage of the monocot A-function gene family.2.The semi-quantitative RT-PCR and real-time quantitative PCR analysis showed that the AsFUL gene was expressed in the floral organ and had low abundance in the stems and young leaves,but was not expressed in other vegetative organs.AsSVP gene was expressed in all organs,including high abundance expression in vegetative organs,such as roots,pseudostellites and young leaves,but had low abundance in flower organs.3.Constructed overexpression vector for AsFUL and AsSVP genes of garlic by using the Gateway technique,and then the recombinant expression vector pH7WG2D,1-AsFUL and pH7WG2D,1-AsSVP were transferred into Agrobacterium LBA4404.4.Transfection of Arabidopsis thaliana with agrobacterium infection method.The results of PCR and RT-PCR showed that the foreign gene was successfully introduced into Arabidopsis thaliana genomic DNA and expressed at the transcriptional level.The phenotypic observation of the transgenic lines showed that the flowering time of the AsFUL gene overexpression lines was earlier than that of the wild type plants for about 7 days,and the floral organs were mutated,such as the petals were changed to sepals and a small number of flowers appeared 5 flowers,indicating that AsFUL gene was a flowering promoting factor and also involved in the regulation of floral organ morphology.AsSVP gene overexpression was significantly delayed in flowering time,and the number of leaf and branch increased significantly.And the downstream flowering related genes FT,SOC1 and LFY were all down-regulated,indicating that the AsSVP gene was a flowering inhibitory factor and had the function of promoting branch development and leaf increase.