| Barley is one of the oldest cultivated crops and used as food, feed and industrial raw materials of beer, with strong heterosis. Therefore, using the cytoplasmic male-sterility(CMS) of barley to produce hybrids is an important objectives in barley breeding with the advantages of facilitate, seed purity, and low cost. The CMS formation mechanism is closely associated with mtDNA, but many researchers also believed that CMS may be related to the expression of nuclear genes which was regulated by cytoplasmic gene. For this reason, cloning mitochondrial gene in barley and analysing the differentially expressed mRNA will be the important method to further research the structurre of the mitochondrial genome in CMS barley. It also has an important theoretical significance for the mechanism of CMS.In this study, using one maintainer 6602B and seven cms lines 6602A,6605A,N-5A,N-9A,N-10A,N-11A,US-A as male parent and female parent respectively.The back cross was made by pollinating with 6602B for five years seven generations, then we abtained a set of isonuclear alloplasm material. We use etiolated barley of CMS line and its maintainer for our material to extract mtDNA. After analyzing the aligment of many other plant mitochondrial gene(orf256, orf300, orf25),3 pairs of primers(B256, B300, B25) were used for amplifing the gene of mtDNA in maintainer line and CMS lines by means of PCR technique. We obtained the expected products through 3 pairs of primers. Afterwards, we aligned DNA and protein sequences of orf256,orf300 and orf25 genes by DNAMAN and NCBI database for the molecular mechanism of barley CMS. In order to investigate the differential expression of nuclear gene related to CMS in barley, a DDRT-PCR technique was used to analysis and identify the fertility-related genes. The results showed that:1. By cloning and sequencing, we obtained the expected products gene and known that the size of mitochondrial gene fragments orf256, orf300 and orf25 were 937bp,933bp, and 622bp.2. Nucleotide sequences of eight mitochondrial gene fragments orf300 and orf25, had no changes between the CMS line and its maintainer line. However, the mitochondrial gene orf256 presented mutation of one nucleotide in CMS line although there was no difference of the size of full sequences between barley CMS line and maintainer line, one mutation nucleotide have not caused variation of coding amino acid. Compared with maintainer line, one nucleotide of G at position 318, in orf256 sequence of CMS line, was replaced with C. The same as in theory that plant mitochondrial genome largley including repeat sequence especially invert repeat sequence.3. The results of aligning DNA and protein sequences are as following:orf256 sequence showed more than 99% similarity ratios to the sequence of orf256 in Hordeum, and showed more than 94% similarity ratios to orf256 hypothesis protein; orf300 sequence showed more than 94% similarity ratios to the sequence of orf256 gene in other plant, and showed more than 94% similarity ratios to NADH dehydrogenase subunit 2L; orf25 sequence showed more than 87% similarity ratios to the sequence of orf25 in Nicotiana tobacum, and showed more than 100% similarity ratios to ATP synthase protein. These indicated that there were some highly conservative in mtDNA of different crops.4. Using DDRT-PCR techmique to analysing mRNA of maintainer line and CMS line with three anchor primers and 10 random primers. The result indicated that there are differentially specific expression fragment.5. We recovered the differential expression cDNA, then we aligned DNA sequence of B1, B2 by DNAMAN database. The result showed that we obtained two fragment(B1, B2) gene andt the size of fragments B1 and B2 were 475bp,462bp.6. By aligning DNA and protein sequences, we know that B1 sequence showed more than 72% similarity ratios to the sequence of Maize hypothy protein gene, and showed more than 72% similarity ratios to Maize hypothy protein; B2 sequence showed more than 95% similarity ratios toVitis unknow protein. |
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