The Effect Of Sealing Materials On Immature Wheat Embryos Culture, And The Cloning And Analysis Of Alpha-tubulin Genes In Regeneration Organs

In this paper, the immature embryos of T. aestivum cv.'YA-1'were cultured in order to to investigate the effect of sealing materials on calli regenereation, and to explore the structure characters of a-tubulin genes in the flowers and roots directly regenerated from the calli. The major findings are as follows:1. The frequencies of both adventitious shoots and roots redifferentiated, as well as browning of YA-1 immature embryo calli had significant differences under the two kinds of sealing materials like silicone plugs and cotton plugs (P<0.01). The former had a lower adventitious shoot ratio of 19.21%, while that of the latter was up to 44.86%. The adventitious root ratio of the former was also significantly lower than that of the latter (19.69%< 39.90%). And its browning rate was outstandingly higher than the latter one (49.62%> 8.37%).2. The cDNA sequences of a-tubulin gene in chromosome 1A, which were amplified from flowers and adventitious root regenerated from immature embryo calli, and immature inflorescences of YA-1, were named F-1A, R-1A, and S-1A, respectively. All the open reading frames (ORF) of the three a-tubulin genes in chromosome 1A were 1353 bp. The identity of the three sequences was at 100%. The number of amino acids deduced from their nucleotides was 450, and their theoretical molecular weight was about 50 KD, and their isoelectric point was 4.71. At their a-tubulin amino acid sequence sites of 142～148, there was a tubulin signal fragment of GGGTGSG. A MREII domain was found at their N-terminal polypeptide while there was a tyrosine residue at their C-terminal. The identity of the cDNA sequences was at 99.41% between all the three tested sequences and the published a-tubulin gene DQ435673 which came from vegetative organs including leaves and shoots of T.aestivum'Quantum'in spite of 32 different base points being discovered.The cDNA sequences of a-tubulin genes in chromosome 3B, which were amplified from the former materials were named F-3B,R-3B,and S-3B respectively. All the open reading frames (ORF) of the three a-tubulin gene in chromosome 3B were 1356 bp. The number of amino acids deduced from their nucleotides was 451, and their theoretical molecular weight was about 50KD。But the isoelectric point of F-3B and S-3B were 4.65 while the one of R-3B was 4.70. At the sites of 142～148 of amino acid sequences, there was a tubulin signal fragment of GGGTGSG. A MRECI domain was found at the polypeptide N-terminus while there was a C-terminal tyrosine residue. The sequence identity was up to 100% between the two tested sequences of F-3B and S-3B, and the a-tubulin gene DQ435662 from shoot vegetative organs of T.aestivum'Quantum'reported. The sequence of R-3B cloned from the adventitious roots was different from the three ones of F-3B, S-3B and DQ435662 due to the substitution of A for G at the position of 529.The identity of a-tubulin gene nucleotides in 1A and 3B chromosomes was varied with the property of the tested materials. The identities between them were at 84.69% from both the regenerated flowers and normal immature inflorescences.And, the identity of the tested chromosomes was at 80.68% from the regenerated roots. However, all the identities of the deduced amino acid sequences between them from the three kinds of tested materials were at 91.8% and the differences occurred mainly at the sites after the 439. So, it was implied that there are not only homogeneous but also heterogeneous among bothα-tubulin genes and their amino acid sequences of wheat chromosomes 1A and 3B. And,α-tubulin genes and their encoding amino acid sequences from the two chromosomes 1A and 3B, were highly stability for different tissues or organs, indicating thatα-tubulin genes of wheat are highly conserved.3. F-1A from Triticum aestivum and the a-tubulin amino acid sequences of the seven published species like Zea mays, Oryza sativa, Glycine max, Grosspium hirsutum, Arabidopsis thaliana, Danio rerio, Homo sapiens were aligned, and the identity of 93% was obtained. Homology tree analysis showed that Triticum aestivum, Zea mays, Oryza sativa fell into the same group, revealing closest relationship of their amino acid sequences to a certain extent.