Study On Genes Related To Male Sterility In Dominant Genic Male Sterile Flax

Flax ( Linium usitatissimum L.) is one of the important crops in the northern region of China for oil and fiber. Flaxseed and flaxseed oil have health function and medicinal effects. Flaxseed linoleic acid can reduce the cholesterol level in the blood andα-linolenic acid(ALA), an essential fatty acid that generates eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), has a function of lowering blood lipids and reducing the formation of the thrombosis. Lignan in flaxseed plays a role in the prevention of cancer. In recent years, more and more wealth and medical function of flaxseed has been developed, demanding more quality varieties. But the expansion of genetic variation and the use of heterosis have been restricted, as flax is a crop of self-pollinaton. So Researching genes and promoters related to pollen and anther development in flax has become significantly important for creating genetically modified male sterile flax line and heterosis utilization in production. In this study, male sterility related gene MS2-F, homologous to Male Sterility 2 (MS2) in Arabidopsis thaliana was isolated by sequence homology cloning method, and its sequence and expression analysis were carried out. Structure and function of the deduced protein were also predicted. Two plant expressing vectors of RNA interference (RNAi) were constructed, using different fragments of MS2-F, and were used in genetic transformation of flax, resulting in Kanamycin resistant regenerated plants. The mRNA differential expression was compared by mRNA differential display technology between male sterile and fertile flower buds of dominant genomic male sterile flax, providing some valuable information for the revealing of the molecular mechanism of male sterility in flax. The results are as follows:1. A gene conserved fragment of 258 bp was amplified in flax flower buds according to the male sterility related genes in other plants including Arabidopsis thaliana by sequence homologous cloning method. Then, 5'and 3' ends of this gene were amplified based on the nucleotide sequence of the gene conserved fragment, by rapid amplification of cDNA ends technology. They were 975bp and 869bp respectively in size and a cDNA sequence of 1911 bp was obtained after splicing the sequences.2. A pair of gene specific primers was designed according to the cDNA sequence, and was used to amplify full length of cDNA and gDNA corresponding to the cDNA, The sequencing results proved that the complete cDNA and gDNA were 1709bp and 2696bp respectively. The results of sequence analysis revealed that the gene consisted of 8 introns and 9 exons.3. The complete cDNA contained an ORF of 1608 bp, which encoded a protein of 535 amino acids including two male sterile conserved domains: NAD-binding domain and male sterile C-terminal domain. MS2-F amino acid sequence identities to MS2Bnap in Brassica napus and MS2 in Arabidopsis thaliana were 59.65% and 59.16% respectively. The gene was exclusively expressed in flower buds and may act as an acyl CoA reductase during flax pollen development.In conclution, a male-sterility related gene, named MS2-F, was isolated in flax. The GenBank Accession Numbers of the cDNA and the gDNA are EU363493 and EU365361.4. Two RNAi vectors of MS2-F gene were constructed, one using a gene fragment that does not contain the male sterile conserved domains in the gene and the other using a gene fragment containing a piece of the male sterile conserved domain in the gene. By agrobacterium-mediated method, several kanamycin resistant regenerated plants were obtained. The construction of RNAi vectors and subsequent genetic transformation of flax were carried out in the hope of producing transformed flax in which the expression of MS2-F gene is interfered, and thus providing some useful clues for revealing the function of MS2-F gene during pollen development in flax.5. In the study of mRNA differential expression in male and fertile flower buds, seven positive differential fragments were confirmed by Reverse Northern Dot Blotting, and then sequence analysis of these fragments was carried out: 5 fragments were specific to male sterile flower buds: G-E07-100, G-E07-330, G-E07-830, G-S273-500, A-S267-300; the other 2 fragments were specific to male fertile flower buds: G-S274-250, G-S274-450.6. The positive fragments were sequenced and analyzed with BLAST. G-E07-100, homologous to GTP-binding protein of plant, may play an important role in signal transduction. G-E07-330 has a high identity with a known cDNA sequence that is specifically expressed in flax flower buds. G-E07-830, which is most homologous to the sequence of Beta-D-xylosidase, may influence carbon hydrate metabolism in flax anther and pollen, resulting in pollen abortion. G-S273 -500 is homologous to NAD+ ADP-ribosyltransferase. A-S267-300, homologous to a secreted protein, may play roles in such process as signal transduction, Morphogenesis and Apoptosis in male sterility flax and finally lead to male sterility. G-S274-250 and G-S274-450 had no significant similarity with any known genes in plants, so they may be newly found genes related to fertility in flax.7. G-E07-100, G-E07-330, G-E07-830 were selected as candidate genes. Expression analysis by Northern hybridization confirmed that they were exclusively expressed in the male sterile young flower buds of flax.