Study Of The Molecular Basis Of Fruit Trees Flowering Early

Nowadays, fruit crops variety improvement mainly depends on the conventional breedingtechniques, which has been limited due to several factors. Among them, long juvenile period,with the esseatial character of without the flowering ability of seedlings, is one of the severelimiting factors for improving fruit varieties and utilizing fruit germplasm.In order to improving varieties of perennial fruit trees, it's of great impol'tance to know howto making flowering early of seedlings and shorten juvenile period. Though there was a largeamount of research about the physio1ogical and biochemical basis of juvenile period and theeffects of environmental factors and cultUre techniques on seedlings flowering, little wasknown about the molecular mechanism and genetic modulation of fruit trees flowering, and fewrelated papers were open to the public. So, there is no doubt that the problem, shoYtening thejuvenile period of fruit crops is fOcus on making them flowering earlierRecently, studies on flowering related genes in model plants especially in ArabiodoPsis otherplants have made great progress, which provided useful references fOr simi1ar study in fruitcrops. Using Ify mutants as the materiaIs, about 80 sites re1ated with fiower were located, andkinds of flower inducing gene were cloned successfully. At the same time, the model of fiowerwas constructed. Some genes accelerate flowering earlief, others hinder it.The formation of normal flowers requires the expression of fiower-meristem-identity genessuch as LEAFy(LFy), which acts as genetic switches in the choice of floral versus short foot inArabiodoPsiS. In @ mutants, most flowers are replaced by shoots with subtending leaves. Wehave known that flower-meristem-identity genes are of high homology even among distantlyrelated plants species. Besides LFY in Arabmpsis, there are many other flower-meristem-identity genes been cloned, such as NFL(Nicotboa tabacum), BOFHperassica oleracea),CFL(Cucumis sativus L.) etc. HoweveF, no such a gene has been found and cloned in fruit treesso far.Using Citrus and Grape as the materials, this paper was focusing on studies of acceleratingflowering of fruit crops,and shortening juveniIe period further at the level of moIecular biologyby using two parallel technique rotltes as foIlowing: Cloning one of the key genes, the LFYlikegene from Citrus with long juvenile period, which is the crucial step of further studyingmolecular biology background and tl1e inner manipulative mechanism of citrus; TransferringLEAFYgene cloned from ArabmpiS into Citrtls and Grape in order to obtain transgene plantswith earIy flowering character.Mentioned above, it will step up the pace of shortening longjuveniIe period and improving its varieties. The results obtained are surnmarized beIow.Gene cloning route:Obtain the LEAFylike gene fragment ofcsLFro903 from Citrus by Uneven PCR. Onthe basis of two conserved fragments in C terminal, which is of high homology of aminoacid sequences among severa1 known LFY and LFY-like genes mentioned above, tWo 3'degenerate primer, Primer I and Primer II(the Nested Primer), were desigl1ed. UsingCitrus genomic DNA as template, run two rounds of Uneven PCR with differentannea1ing temperature using degenerate primer and l2 arbitrary l 0-rner primers as partner,respectively. After fractionating the specific PCR products on l.5% agarose geI, l .3kbfragment was purified. Being transferred into DH5 (,, the specific cIone was verified by thefollowing steps: conducting PCR with prilners of primer II and Sl299 arbitrary primer,and digesting with Hjnd lII and EcOR V In these reactions, pUCl9 plasmid and thepurified l.3kb PCR Product were used as CK (-) and CK (+), respectively. At Iast theLFKlike gene, named csLFrs903 temporarily,was obtained and sequenced. Thenucleotides tested from the terminaI containing the nested primeY, 568bp long, was partlyoverlapped with another fragment of the LFY-like gene obtained i...