Beet M14 Strain Flowering Specifically Expressed Gene Cdna Library Construction And Screening

The apomictic monosomic addition line of Beta corolliflora, designated as M14(VV+IC, 2n=18+1), was obtained from remote hybridization of B. vulgaris L. and B.corolliflora Zoss. in sugar beet. M14, which constituted of normal 18 B.vulgaris L. (VV,2n=18) chromosomes with the No.9 B. corolliflora Zoss. (CCCC, 2n=36). chromosome,was found having a chromosome transmission frequency of 96.5% by embryologyidentification and cytogenetic analysis. Thus sugar beet M14 was an invaluable materialfor cloning apomictic gene(s).Three suppression subtractive EST libraries and one differentially displayed ESTlibrary had been constructed in terms of three key developing phases of sugar beet M14.In this experiment, three ZAP differentially expressed cDNA libraries of M14 atflorescence were constructed with total RNA corresponding to the three EST libraries.Each titer of initial cDNA library was above 1.25×10~6 pfu/mL, while the length ofinserts was between 400bp and 3 000bp, which covered all the genetic information.Three EST were chosen at random from a suppression subtractive EST librariy forscreening corresponding cDNA libraryⅠ. Three positive single clones obtained byplaque in situ hybridization, which were preserved and sequenced, were designated asMe-c84 (797bp), Me-c86 (1182bp) and Me-c264 (sequencing) respectively. Then5'-RACE (rapid amplification of cDNA ends, RACE) was played to obtain 5' cDNAsequences of Me-c84, by which a specific DNA fragment was generated. The specificDNA fragment was collected from 1% agarose gel and subsequently sequenced. Thusfull length cDNA (1331bp), designated as M14-86, was obtained by assembling withMe-c86 using nucleotide sequence analysis soft. Then it was identified by RT-PCR.Then the sequences of Me-c84 and M14-86 were aligned with known sequences inNCBI non-redundant database. The results showed that Me-c84 shared 98% similarities with the 26S ribosomal RNA gene from Sarracenia purpurea, while M14-86 shared97% similarities with the 26S ribosomal RNA gene from Portulaca Grandiflora.