Genomic Analysis And Ultrastructure And Differential Expression Proteins Study Of Chloroplast In Wheat Albinism Line

Wheat albinism line is a natural mutant from the parent dwarf wheat Aibian 1. In the stage of every early spring , albinism line starts albinism from the interior leaf base, and the leaf blade albinism area enlarges from the bottom to the top with deercasing temperature. The albinism line could show enlarges from the interior leaf base and enlarging from bottom to the top in every early spring, and would return green with temperature increasing gradually, the albinism line returned to green from the heart leaf base, and its character performance restores normally, which have presented"returning white---green"character lasting approximately 30-40 days. The albinism line is different with the common leaf color mutant controlled by nuclear gene, whose physiological mutation is controlled by cytogene. The albinism line is the temperature sensitive pigment mutant which is induced by low temperature and can inherit stably. There are not any influences on the wheat quality and output. Albinism line has been followed with interest for its unique plasmatic inheritance, the special metabolism feature and the potential application value since it has been obtained in 1985. It has positive significances in the plant genetics and breeding for makeing use of the character of albinism and green and the ineffective tillering. Up to now, the genetics, physiological biochemistry, chloroplast growth and cytology of albinism line have been studied well. We have made the massive meaningful progresses. However, the molecular mechanism of the"returning white---green"in albinism line has not been studied in-depth. To some extent, it has restricted its application on the fundamental and applied research.In order to lay the foundation for showing the molecular mechanism of"returning white--green"of the albinism, through the electron microscope, the researcher has observed the changes of chloroplast ultrastructure according to the process of the"return white--green". Through the whole genome shot gun sequencing , we have finished the complete sequence determination and the bioinformatics analysis of the chloroplast genome in the albinism line, which have layed the foundation for study of the genetic mutation site of chloroplast genome in the albinism line. Using the two-dimensional electrophoresis and the mass spectrum appraisal technology, The differentially expressed proteins has been identified between the albinism line and its parent strain aibian 1. Using the RT-PCR and the RACE technology ,we have obtained the cDNA complete sequence whose expression corresponds to proteins in the albinism line. All of these are the foundation for the association study between the function and albinism.The major results were as follows:1. The transmission electron microscope observation indicated that: the chloroplast structure of the albinism line had the corresponding changes during the process of albinism and green. With the leaf albinism, the chloroplast metabolism process cannot carry on normally and along with the chloroplast structure ill-defined, thylakoid degradation, large chloroplast cavitate, plastid internal structure fuzzy and membrane degradation. After the leaf returned green, the chloroplast structure and a series of metabolism processed restore for the normal state as same as the comparison leaf blade chloroplast structure.2. Through the reciprocal crosses experiment , we have proved that the plasmatic inheritance character of the albinism line.3. For the first time, we have completed the nucleotide sequence albinism line wheat chloroplast genome. The results showed that the closed circular chloroplast DNA is 135898bp long, of which 12791 bp contained in the inverted repeats (IR). The IR disrupts the remaining DNA into 80003bp of large single copy (LSC) region and 12791bp of small single copy (SSC) region. The sequential analysis results also showe that: the whole length of the albinism line chloroplast DNA more 1354bp than chinese spring wheat, but less 565bp than barley.4. The whole chloroplast genome includs four ribosomal RNA (rRNA), thirty tRNA genes transfer 20 species of amino acids, 74 protein-encoding genes and 2 for unidentified open reading frame. Comparing with the chinese spring wheat chloroplast genome, we have identified three new gene in albinism line wheat chloroplast genome, called psbZ, ccsA and petN.5. The primers were designed based on the different section of chloroplast genome between the albinism line and chinese spring wheat. We have cloned 26 sections in the parent strain Aibian 1. The sequential analysis showed that there were only the individual single basic group difference among the clones sectors of the chloroplast genome in the parent strain Aibian 1 and albinism line.6. The two-dimensional electrophoresis analysis indicated: at the same period, there are quite differences from the expression of chloroplast proteins between the albinism line and its parent strain aibian 1. Through the MS, 11 proteins were identified. 9 of 11 proteins expression are down-regulated and 2 proteins expression are up-regulated in the albinism line. All of them are encoded by chloroplast gene or by nuclear gene and located in the chloroplast. They are the important enzymes and the proteins taking part in the chloroplast metabolism. There are important meaning to further study all the proteins and enzymes for showing the molecular mechanism of albinism line.7. According to the identified proteins, using the reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends(RACE) technology, for the first time, we have cloned six gene fragments which encode the corresponding proteins in the albinism line. Among which, three gene fragments are the first time report in wheat, which encodes the fructose-bisphosphate aldolase, HSP70(GenBank accession number: FJ830947) and cp31BHv (GenBank accession number: FJ830948).8.According to the gene fragments which encodes chloroplast fructose-bisphosphate aldolase, using RACE technology, for the first time, we have cloned the complete cDNA sequence in the albinism line which encodes the chloroplast fructose-bisphosphate aldolase. The length of the cDNA is 1512bp, called AlDPTA(GenBank accession number: FJ625793 ). In the albinism line wheat, the gene AlDPTA encodes 388 amino acid, including the R85, K175 and K257 which are the substrate special union position spot of fructose-bisphosphate aldolase. It takes part in the starch synthesis metabolism in chloroplast and locates the key position in the entire metabolism network.