Study On FT Function And Transcriptome Analysis Of Leaves During Floral Induction In Pears

FLOWERING LOCUS T（FT）is considered to be a mobile florigen gene,which is a key factor in floral induction control.Flower bud differentiation of fruit trees is a complex life process and one of the hot research areas for a long time.Production practice has proven that pears are more likely to form flower buds than other fruit trees.In this study,pear tree was used as a trail of materials to research the function of pear FT,revealing the role of pear FT in the floral induction and the factors regulating pear FT expressions.Meanwhile,a transcriptome analysis of leaves form short shoots with a higher flower bud formation rate and long shoots would help to explore a series of genes associated with floral induction.This provided a new insight for revealing the molecular mechanism of pear flower bud induction.This research can not only promote people’s understanding of the general laws of pear tree floral induction,but also provide guidance and reference for enriching theories of flower bud differentiation and revealing the internal mechanism of flower bud differentiation of other fruit trees.The main research results obtained are as follows:1.In this study,a total of 24 PEBP genes were identified,in which 10,5 and 9 were from Pyrus bretschneideri genome,Pyrus communis genome and Pyrus betuleafolia genome,and distributed on 8.5.and 7 chromosomes,respectively.These genes were divided into three subfamilies of TFL1-like,FT-like,and MFT-like.Each PEBP gene was consisted of four exons and three introns formation.The Ka/Ks ratios of 13 duplicated PEBP gene pairs suggested that the duplicated PEBP genes of pears had mainly undergone strong purifying selection.Expression patterns of PEBP gene from P.bretschneideri genome in leaves during different stages suggested that only gene12374 from PEBP genes of P.bretschneideri was relatively highly expressed in leaves during floral induction and no other PEBP genes were thought to be expressed in the leaves.2.According to the genomic data,primers were designed in the conserved region of the target gene and FT homologous genes of two varieties,’Xueqing’ and ’Xinli No.7’were cloned.Sequences of two FT homologous genes were exactly the same,which were named PbxFT.PbxFT was located on the nucleus,cytoplasm,and membrane of the cell,indicating that PbxFT protein could play a role in the nucleus,cytoplasm,and on cell membrane.Additionally,the phenotype of transgenic Arabidopsis suggested that PbxFT played a role in not only promoting flower bud differentiation,but also regulating the balance between vegetative and reproductive growth by inhibiting lignin synthesis in plants.3.Using ’Xueqing’ and ’Xinli No.7’ as materials,the 2667bp promoter fragment of the PbxFT was cloned from the two varieties.Two sequences were exactly the same.The cis-elements in the promoters of PbxFT were related to GA and IAA response,especially light response.The promoter activity was identified through the transient transformation of tobacco and ’Xinli No.7’ tissue culture seedlings mediated by Agrobacterium infection,and it was found that a 210 bp fragment upstream of the start codon ATG had promoter activity.The transient transformation test on ’Xinli No.7’ tissue culture seedlings was successfully realized,which laid the foundation for the subsequent study of other genes or promoter functions of pears in the original plant.4.Seven-year-old ’Xueqing’ trees were used as materials.The treatments of day-length reduction and the expression pattern of PbxFT in a day indicated that PbxFT expressions in leaves were regulated by day-length and circadian clock.Shortening the day-length can significantly reduce the expression of PbxFT and directly affect the flower formation rate.By spraying the growth regulator,the inhibitory effect of GA₃ on PbxFT gene expression was verified,and it was found that exogenous GA₃ may inhibit the’Xueqing’ flower buds formation by inhibiting PbxFT expression.Under natural conditions,the key environmental factor for pear flower induction may be day length,which is more critical comparing with endogenous GA.Exogenous GA can easily change this regulation mode.Pear trees have certain sensitivity to GA during the physiological differentiation of flower buds.5.For ’Xueqing’ cultivar,removing all leaves on a shoot resulted in no flower bud formation;axillary buds forming flower buds under or near retaining leaves on the shoots with removal of leaves except 3～5 basic or apical leaves.There were all flower buds except basic 3 buds,if it was without treatment on the current shoot.The results showed that the leaves of different positions of ’Xueqing’ had different functions during the development of current shoots.The apical leaves mainly provided the driving force for material transport.The middle leaves were the main source of floral induction signals,while the base leaf did not have these two functions.This suggested that in higher woody plants,the florigen might be synthesized in the leaves,and travelled to shoot meristem by driving force of the transpiration pull.6.The RNA-seq libraries of leaves from spurs with a higher flower bud formation rate and long shoots were constructed.After analysis of transcriptome data.456 differential genes were identified during the flower induction period,which were mainly enriched to be annotated to four pathways,including metabolism,genetic information processing,environmental information processing,cellular processes.Among them,many genes were significantly enriched in the flavonoid biosynthetic pathway（map00941）.Phenylpropanoid biosynthesis pathway（map00940）and plant hormone signal transduction pathway（map04075）.The expression patterns of candidate differential genes indicated that the significantly up-regulated expression of gene31333（IAA26）and the down-regulated expression of genes,such as gene22546（GAI）and gene22106（PAL1）.gene780（C4H）,gene22826（C4H）,gene4670（COMT）.gene6170（F5H）and gene37723（F5H）.were floral induction signals.