Cloning And Molecular Characterization Of Myb Genes From Leymus (Poaceae: Triticeae)

Leymus Hochst. is an important perennial genus of Triticeae Dumortier (Poacear), which includes about 32 species and 19 subspecies. They are widely distributed in Eurasia and North America, South America is also a small amount of the distribution. The major morphological characteristics of Leymus species are:a rhizome, cross-pollination, glume from coniform to lanceolate, lemma awnless or with a short awn, spike 1 to rounds of the sections was born in cob, anther length. Leymus species normally grows on the beach, rivers and lakes sandy desert dunes, grasslands, meadows, hills and forest. Some Leymus species bear such desirable traits as disease and insect resistance, bigger spikes, more and bigger grains and efficient photosynthesis. Meanwhile, most Leymus species have a strong cold, drought, alkaline, disease resistance, insect resistance, resistance to the capacity of sand, and are excellent grass. Leymus is of significance to improvement of Triticeae cereal crops. To research the Leymus about stress genes is of great significance.In plant stress response system, transcription factors regulating the expression of functional genes is the key link of plant stress responses. As one of the largest family of plant transcription factors, Myb (v-Myb avian myeloblastosis viral oncogene homolog) transcription factor plays an important role in plant stress resistance. Plant Myb transcription factor is crucial in regulating secondary metabolism, cellular morphogenesis and responses to hormone and stress signals. At present, a large number of Myb gene sequences have been received from the common wheat, while the homologous genes been obtained from other Triticeae species is relatively small. This situation leads that the function of Triticeae Myb genes and the relationships among different number of Triticeae Myb gene family is unclear. Thus, study of Myb genes from wheat relative species is very necessary.In order to understand and use the Leymus Myb genes, this study clones and analyzes the molecular characters of Myb genes from five Leymus species. The main results are as follows.1. According to Panicum miliaceum L. family member of Actin gene as bait, one pair of degenerate primers (P1) were designed for cloning the Actin gene form Leymus multicaulis. With the primers P1 and P2 (the primers for P. miliaceum Actin gene) and using the total RNA from L. multicaulis leaves as template, the Actin gene fragments were amplified by RT-PCR method and cloned into pMD18-T vector. After the positive clones identified by PCR and sequenced, the sequences were compared with the Actin gene sequence registered in GenBank. The sequence alignment was revealed 81.8% to 96% indentation with some known nucleic acid in plants, and other Actin amino acid sequence homology of 82%. Sequence analysis showed that the clones were Actin genes and were named as:LmACT1 which length is 961 bp and encoding 312 amino acids; LmACT2 which length is 192 bp and encoding 42 amino acids. Respectively, the GenBank accession numbers of LmACT1 and LmACT2 were GU992386 and GU992387.2. According to the conservative of Myb gene R2R3 region, the degenerate primers and specific primers for full Myb gene were designed.19 Myb gene fragments were isolated from five Leymus species, among which, three gene fragments were nearly complete open reading frames encoding putative Leymus Myb genes, designated LrMyb3, LmMyb6 and LmMyb8 respectively. Amino acid sequence and phylogenetic analysis indicated that all the 19 putative Myb from Leymus belonged to plant typical R2R3-Myb members. Sequence compared with the Myb genes in Arabidopsis thaliana, the 19 Leymus Myb gene fragments of the R2R3 region amino acid showed three different types, they were W-x(19)W-x(19)W- x(12)F-x(18)W-x(18)W(13), W-x(19)W-x(19)W-x(12)M-x(18) W-x(18)W(4) and W-x(19)W-x(19)R-x(12)L-x(18)W-x(18)W (2).In wheat, the 70th amino acid of all the proteins encoded by Myb genes is phenylalanine (Phe-F). However, the 70th amino acid of the proteins encoded by Leymus Myb genes exist three different types, they were phenylalanine (Phe-F) (13), leucine (Leu-70) (2) and methionine (Met-M) (4). It indicated that the loci of Leymus Myb genes have changed, and there were diversity of the members of Myb genes in the same or different Leymus species.3. According to the similarity of nucleotide sequences, phylogenetic relationships of three full Myb genes (LrMyb3, HM071921; LmMyb6, GU011687; LmMyb8, GU011688) from Leymus were analyzed. The results showed that, LmMyb6, LmMyb8 and LrMyb3 clustered with ZmMyb39, HvMyb4 and TaMyb2 genes firstly, they have the higher sequence similarity and the closer relationships. LmMyb6, LmMyb8 and LrMyb3 were most high similarity with some drought-related Myb genes in plant. Therefore, we speculated the LmMyb6, LmMyb8 and LrMyb3 genes were related with drought in Leymus.4. Through the use of fluorescence quantitative 2-△△Ct method, LmMyb8 gene in roots, stems and leaves of fluorescence relative quantitative analysis showed that, LmMyb8 gene was expressed at high level in leaves and roots, while at low level in stems. It was not same as maize Myb genes' expression in roots, stems, but it was similar to TaMyb2, TaMyb3, TaMyb4 and TaMyb6 genes' expression in wheat.