Expression Analysis Of Genes Related To Aborted Flower Buds In Radish(Raphanus Sativus L.) And Cloning And Functional Analysis Of RsVPE1 Gene

Floral bud abortion（FBA）can result in reductions in yield of many crops,and reduce garden and greenhouse performance of many ornamental plants,even more important is which affect the reproduction and survival of plants.Plants exhibiting FBA are often eliminated during artificial breeding practices,even if they have good economic characteristics or specific traits,which results in some important materials difficult to save,affect the breeding potential and sustainability.We detected gene expression differences between aborted and normal buds of radish using cDNA-AFLP,and isolated radish FBA related gene RsVPE1,and uncovered its function by Agrobacterium tumefaciens mediated transformation of Arabidopsis thaliana.The main results are described as followings.1.We detected gene expression differences between aborted and normal buds of radish using cDNA amplified fragment length polymorphism（cDNA-AFLP）and real-time polymerase chain reaction（real-time PCR）.A total of 221 differentially expressed transcript derived fragments（TDFs）were detected by 256 cDNA-AFLP primer combinations,of which 114 were up-regulated and 107 were down-regulated in the aborted flower buds.A total of 54 TDFs were cloned and sequenced.A BLAST search revealed that all TDFs have homologous sequences and 29 of these corresponded to known genes,whose functions were mainly related to metabolism,stimulus response,transcriptional regulation,and transportation.Expressions of six TDFs with different functions were further analyzed by real-time PCR yielding expression profiling results consistent with the cDNA-AFLP analysis.2.TDF72,a transcript-derived fragment obtained by cDNA-AFLP,was up-regulated in the aborted buds and exhibited 89%sequence homology with the AtyVPE gen.Based on the sequence of TDF72,a VPE gene was isolated from flower buds of radishes with aborted flower buds or normal flower buds using RACE and RT-PCR.Since it was the first VPE gene discovered in radish,named RsVPE1.The full length gDNA and cDNA of RsVPE1 were 2,346 bp and 1,825 bp,respectively.The full-length gDNA of the RsVPE1 gene was comprised of 9 exons and 8 introns.The full-length cDNA of RsVPE1 contained a complete open reading frame（ORF）of 1470bp,which encoded a predicted protein containing 489 amino acid residues.Multiple amino acid sequence alignment revealed that the predicted amino acid sequence of RsVPE1 displayed homology with other proteins,including Arabidopsis thaliana gamma-VPE（NP₁95020.1,90%）,Arabidopsis thaliana alpha-VPE（NP₁80165.1,80%）,Vitis vinifera VPE（XP₀02276759.1,77%）,Ricinus communis VPE（XP₀02516472.1,77%）,Glycine max VPE（XP₀03525979.1,75%）,Medicago truncatula VPE（XP₀03603121.1,74%）,Cucumis sativus VPE（XP₀04142919.1,72%）,Sorghum bicolor VPE（XP₀02448237.1,63%）,Arabidopsis thaliana beta-VPE（NP₁76458.1,63%）;Zea mays beta-VPE（ACG47915,62%）and Oryza sativa VPE（NP₀010565054.1,59%）.Expression analysis demonstrated that RsVPE1 transcripts were produced in all tested radish tissues including leaves,flower stalks,normal flower buds,aborted flower buds and young siliques.However,its expression levels were various in different tissues.The highest expression level of RsVPE1 was detected in aborted flower buds,while the lowest level was discovered in flower stalks.Furthermore,the expression levels of Rs VPE1 gene in different organs of radish flower buds were analyzed.Interestingly,the levels of RsVPE1 transcripts were higher in the different organs of aborted flower buds than the corresponding organs of normal flower buds,with the highest expression levels in the sepals and the lowest expression levels in the pistils.3.The open reading frame（ORF）of RsVPE1 was cloned into the pCAMBIA2301-35S-Nos vector.The construct was transformed into Col-0 using Agrobacterium-mediated transformation method.Five homozygous transgenic lines were obtained by antibiotic resistance screening and PCR confirmation.Three independent homozygous transgenic lines（TL3,TL78 and TL83）were randomly selected for further analysis.The RsVPE1 gene expression level was checked by real-time PCR analysis（Figure 8a）.To examine the possible phenotype of homozygous transgenic lines,T3 progeny of the RsVPE1-overexpressed lines and the Columbia ecotype（Col-0）plants were grown in the greenhouse under normal conditions.Compared with Col-0 plants,no obvious morphological or developmental abnormalities were observed in transgenic plants when they were grown in same artificial soil at normal temperatures in the greenhouse.However,significant phenotypic changes,in the form of aborted flower buds,were observed in transgenic Arabidopsis plants in response to heat stress.There were 13.1 to 16.4 aborted flower buds in three transgenic lines.FBA began to display on the third day after heat stress.All aborted flower buds appeared on the eighth day after heat stress.Based on the flower bud ages and positions,we could speculate that FBA occurred in the flower buds of stage 9 to 12.Although the expression of RsVPE1 from the constitutive 35S-promoter should not be markedly affected by environmental conditions,RsVPE1 expression levels of three transgenic lines（TL3,TL78 and TL83）reached maximums at 24 h heat stress and were shown 2.1-fold,2.8-fold and 2.5-fold increase compared with their expression levels at 0 h,respectively.These results suggested that RsVPE1 gene and heat stress were important to the occurrences of FBA in transgenic Arabidopsis plants.4.A RNA interference（RNAi）expression vector,pRNAi-RsVPE1 was contructed.The construct was transformed into Col-0 using Agrobacterium-mediated transformation method.Nine homozygous transgenic lines were obtained by antibiotic resistance screening and PCR confirmation.Three independent homozygous transgenic lines were randomly selected for further analysis.Compared with Col-0 plants,there were significant phenotype changes in homozygous transgenic plants of RNAi expression of Rs VPE1.The homozygous transgenic plants can not yield seeds.The pistils and the pollens of homozygous transgenic plants viability were normal.However,the filaments of flower in stage 13 were slightly shorter,and the anther of flower in stage 13 and stage 14 did not dehisce,so that the pollens could not be released.Finally,they displayed functional male sterility.Meanwhile,the expression level of AtyVPE in transgenic Arabidopsis was decreased,which indicated that the lower expression level of AtγVPE resulted in filaments shorter and anthers indehiscent.In other words,it leads to the occurrence of functional male sterility.