Cloning, Identification And Function Analysis Of Meiosis Nuclear Division 1 Homologous Gene (mnd1) In Undaria Pinnatifida

The suitable culture conditions for the conversion from vegetative growth to reproductive growth of the gametophytes in short time were screened in this investigation. The results showed that a large number of sperms and eggs can be released from the gametophytes about 10 days under the conditions that the light intensity was 60～70μmol/m2·s, the photoperiod was 14:10 h (L:D) and temperature was 18±1℃. Furthermore, DAPI staining was employed for gametophytes nuclei observation in the experiment and the clear chromosomes and weak background could be obtained under microscope using this method.The suppression subtractive hybridization cDNA library of up-regulated genes from U.pinnatifida gametophytes under the development induction was constructed.110 ESTs were obtained after sequencing the positive clones, and accounted for 91.67% of the total sequencing samples. Among these ESTs,106 fragments were non-redundant sequences. PCR analysis indicated that the length of the ESTs were ranged from 100 to 2000bp and the average length was 612bp. Based on the functional similarity with amino acids coded by homologous genes in algae and higher plants, the ESTs could be divided into several functional groups:cell growth and development (accounting for 16.70%), metabolism (accounting for 10.00%), photosynthesis and photo-induction (accounting for 3.60%), signal transduction (accounting for 4.50%) and other functions (accounting for 1.80%).70 sequences with unknown function or no comparative result accounted for 63.40% of the total ESTs. The similarity values distributed between 20% to 60% and the deduced amino acid sequences of the ESTs have higher similarity with the sequences of algae and higher plants. It was shown that the G and C usage in the third position of the codons have no significant difference in U. pinnatifida and the GC frequency of codon usage was 53.05%. The stop codon usage preferred to TGA for U. pinnatifida. The GC usage frequency in the third codon position of red algae was higher than that in brown and green algae.A homologous sequence of meiosis-related gene in U. pinnatifida was cloned with the full length cDNA of 868bp from the library by using of RACE method. The length of open reading frame is 609bp, encoding 202 amino acids that with conserved Mndl protein domain. 4 introns and 5 exons were found in the coding region of this gene. The results of the prediction for molecular weight, theoretical isoelectric point, signal peptide, transmembrane structure, phosphorylation sites, kinase phosphorylation sites, subcellular localization and secondary structure, as well as phylogenetic analysis indicated that the isolated sequence was MND1 gene of U. pinnati/ida (UpMND1). The amino acid sequence of UpMND1 gene has a high similarity to the genes of other brown algae, especially to Ectocarpus siliculosus with the similarity up to 72%. The UpMND1 gene sequence has been submitted to the Genebank with the accession number JF357961.The real-time quantitative PCR results demonstrated that the relative expression abundance of UpMNDl gene were different in all developmental stages and different tissues. It was expressed ubiquitously in many organs, such as rhizoid, stem, leave and sporophyll. However, the highest expression was detected in the sporophyll, which was a reproductive organ. The relative expression level increased significantly during the early period of gametophyte development and also in the period of sperms and eggs formation.In order to investigate biological functions of UpMND1 gene, the expression vectors of this gene were constructed and transformed into wild and mnd1 mutant plants of A. thaliana and mndl mutant of Saccharomyces cerevisiae by the method of Agrobacterium-mediated and PEG-mediated transformation, respectively. Here the phenotypes and chromosomes stained by DAPI during meiosis of wild type, overexpression type, complement expression type and mutant type of A. thaliana were observed to analyze the role of the UpMNDl gene. The growth curve of transgenic yeast was plotted and compared with the mutant. The data revealed that the overexpression of UpMNDl gene in A. thaliana in vivo resulted in the synapsis and recombination of chromosomes cannot be successfully completed and the chromosome behavior disordered in the process. As well as the pollen fertility decreased in this state. The chromosome behavior in male gametes of A. thaliana that the UpMND1 gene overexpressed was similar to the mutant plant. Similar to the wild plants, the synapsis and recombination of the chromosomes in Atmndl mutants that the UpMNDl gene complementary expressed could be successfully completed and the chromosome behavior returned to normal level. The pollen fertility also returned to normal level in these plants. It was indicated that the fuction of the UpMND1 gene has correlation with synapsis and recombination in the meiosis of U. pinnatifida. The growth curve of transgenic yeast mutant was not significantly different from the controls, showing that the UpMNDl gene was not concerned with the regulation of normal mitosis. However, this gene played regulatory function during meiosis and the formation of reproductive cells of U. pinnatifida. The evolution of MND1 gene function was relatively conservative both in the higher plants A.thaliana (flowering plants) and the algae U. pinnatifida (cryptogamic plants).A 638bp promoter sequence on upstream of UpMNDl gene was cloned by high thermal asymmetric interlaced PCR (hiTAIL-PCR) method. The results that analyzed using online programs showed that the promoter contained CAAT-Box, TATA-Box and other cis-acting elements, for example, three light-sensitive cis-acting elements GATA-motif, GA-motif and the G-Box, two auxin-sensitive cis-acting elements TGA-element and ABRE and a meristem specific activation component CCGTCC-Box. The expression levels of reporter gene in transient expression system of A.thaliana protoplast cells controlled by the promoter with different length of 5 'deletion sequences were different. The results suggested that the distinguishing cis-acting elements could enhance or suppress gene expression in the regulation. The cis-acting elements in -296～-166bp region of the promoter showed a promoting effect on the downstream gene expression, and the cis-acting elements in -621～-296bp region could inhibit the expression of downstream gene.