Cloning And Expression Analyzing Partial CDNA Sequences Of FIE In Autonomous Endosperm Eulaliopsis Binata

Gametophyte Apomixis produces maternal genetic clone offspring without meiosisand fertilization, which will have good application in seed production andheterosis.Eulaliopsis binata, which belongs to the subtribe Apocopidinae in Gramineae, isa perennial grass. Recently, apomixis phenomenon was observed in E.binata, which wasapospory.We discovered autonomous endosperm type E.binata base on the embryologyand FCSS. Its polar nucleus or secondary nucleus proliferation produces tetraploidnon-fertilization endosperm. Whereas edible part of crops such as rice, wheat isendosperm, and which is direct related to the yield and quality of crops, thus the study ofautonomous endosperm type apomixes materials is valuable.RNA was extracted from inflorescence of Eulaliopsis Binata and used as templatefor cloning by RT-PCR with degenerate primers which were designed according to theFIE homologous sequences of Arabidopsis, rice and maize. As a result, one about 800bpfragment was obtained and then cloned to T-vector and sequenced. At the same time, westudied the spatio-temporal expression of EbFIE in apomixis E.binata by in situhybridization using cRNA probe which was made of rice's FIE cDNA clone.The main result:The result of sequencing showed the length of this fragment is 826bp, coding 275amino acid, named EbFIE1. Analysis with EbFIE1 cDNA sequence and deduced aminoacid sequence suggested that the fragment was similar to the reported FIE homologousgenes of maize, sorghum and rice. The partial cDNA is a part of FIE gene in EulaliopsisBinata.On this condition, the full-length cDNA of FIE will be obtained and we can studyfurther the homologue and function of wide type and mutative FIE gene.Nucleotide sequences of EbFIE1 similarity to maize FIE1, maize FIE2, and rice FIEand wheat FIE is 95%, 83%, 87%, 84%. Amino acid sequences of EbFIE1 similarity tomaize FIE1, FIE2, rice FIE, sorghum FIE,Arabidopsis FIE, catalpa FIE,eucalyptus FIE is95%, 76%, 88%, 94%, 72%, 80%, 84%.We found there was special spatio-temporal expression of EbFIE in apomixisE.binata by in situ hybridization using cRNA probe. The result showed FIE gene onlyexpressed in male gametophyte and female gametophyte. No hybridization signal in theother part of flower. From the MMC (microspore mother cell) stage to the MNM (mono-nucleus microspore) stage,FIE gene primarily expressed in microspore in stamen. megaspore At this time, only megaspore mother cell (apospory initiation cell) showedhybridization signal. At Embryo sac development stage, female gametophyte cell still hadFIE expression and the signal weakened in microspore. Mature embryo had intensitysignal, but there was none signal in endosperm. There was no FIE expression in sterileovary and anther. These result suggested that FIE gene expressed gradually weakening inmale gametophyte and enhanced in female gametophyte. The development of embryo andendosperm was regulated by FIE gene.Conclusion: In The research, patial EbFIE cDNA sequence was cloned byhomologous sequencing method through RT-PCR using the apomixes E.binata, thensequenced and analyzed in detail by Bioinformatics. It makes preparations for the obtainof full length FIE cDNA and the research about molecular mechanism of non-fertilizationendosperm initiation. The result of ISH indicate that the FIE gene has specialspatio-temporal expression in apomixis E.binata during the reproduction growth andcontrols the development of embryo and endosperm.