Ddrt-pcr Technology To Study The The Am Fungi Amorpha Process Gene

Arbuscular mycorrhiza (AM) formed between plant roots and fungi is one of the most widespread symbiotic associations found in nature. The AM symbiosis is based on bidirectional nutrient exchange; AM fungus supports the plant with macronutrients such as phosphorus and nitrogen, whereas it receives photosynthetically fixed carbon from the plant. In order to clarity the molecular mechanism on arbuscular mycrrhizal formation, related genes that expressed in Amorpha fruticosa roots, colonized Glomus mosseae and non-mycorrhizal were profiled at different stage of formation respectively, using differential-display RT-PCR (DDRT-PCR). In this study, several differentially expressed genes and their potential role are elucidated and discussed. Considering their putative relationship in forming mycorrhizae, these sequences may be considered of interest for further studies and may provide some clues to determining how AM are established. This will open new avenues for mycorrhizal research to elucidate the mechanisms of woody plants mycorrhizal symbiosis. The endosymbioses formed between plants and microorganisms play an important role in argriculture and natural ecosystems.First, among the three extraction methods of total RNA (Biozol, SDS / phenol, and CTAB), when infection rates were 0%, 10%, 30%, 50%, 100% of Amorpha fruticosa roots inoculated AMF (Glomus mosseae), and measured by their purity and integrity through 1.1% agarose gel electrophoresis, the results showed that the infection in five different infection rate, both the total RNA extracted by Biozol were high purity and good quality in only 2-3h. Total RNA extracted byBiozol at infection rate 0% (pre) and 30% (late) was purified and reverse transcribed to cDNA.Second, there have been 60 primer combinations of 3 anchor primers and 20 arbitrary primers to amplify. Through separating these PCR products on 6% non-denaturing polyacrlamide gel electrophoresis, results indicate 20-50 bands in each lane. By comparison, 169 were isolated by differentially expressed fragments appearance, and 63 were at early stage, 106 were at late stage of establishment of arbuscular mycorrhiza. Initial screening of the cDNAs to discard false positives was performed by reverse Northern blot, and 12 fragments were isolated, and the positive rate at the early stage was 42%, while at the late stage there was 61% high false positive rate.Last but not the least, positive differential bands were cloned and sequenced, most of which was about 250-500bp. And they have been submitted to NCBI's EST database in GenBank, the sequence number is: GW317173, GW327914, GW327915, GW327873, GW327874, GW327875, GW327876, GW327877, GW327878, GW327879, GW327880, and GW327881. Meanwhile, 12 fragments were compared Blastx analysis. Five genes of 12 are arbuscular mycorrhiza-related genes obtained at Amorpha fruticosa-G.mosseae early-stage interaction; 7 genes were isolated at late stage of mycorrhizal development. There are three ESTs origin of the fungi, eight EST corresponded to plant genes. Putative functions were confirmed and sequence similarities were found by Blastx analysis. Root exudates and volatiles may promote or suppress spore germination, indicating the existence of spore'receptors'responsive to alteration in the chemical composition of the environment. Hence, induction of a subset of genes appeared to lead to elevated respiratory activity and an energy status necessary for extensive hyphal branching. DD-2 is similar with Lotus japonicas class III HD-Zip protein, as signals to response external environmental stress and important regulatory role in plants unique developmental process. DD-3, similar with the ATP-binding cassette sub-family E member, is a class of widespread transmembrane transport system that convey metabolites, ions, sugars, amino acids, lipids, steroids and drugs and more kinds of substrates through the cell membrane. DD-4, similar with Penicillium marneffei hypothetical protein, has a large number expression at the early stages. DD-5 relates with respiratory oxidase gene, required greatly expressed. At early stage of the mycorrhizal symbiosis, there has a great change in respiration and increase in respiration, energy consumption. DD-8, similarity with acetyltransferase GNAT family protein, the common modification of protein has important significance for the protein activity and structure. DD-7 and DD-9 have similarity with the genes of Flavobacterium johnsoniae and Ajellomyces capsulatus hypothetical protein, which possible play an important role at late stages of arbuscular mycorrhizal development to promote fungi formed arrbuscular structures in the plant cells. DD-10, Lotus japonicas genomic DNA, is very specific function at the establishment of arbuscular mycorrhizal symbiosis. DD-11, similar to Arabidopsis thaliana clone SINE9 transposon-insertion display band genomic sequence, found important in the evolution by U.S. scientists, have significance meaning for arbuscular mycorrhizal evolutionary. DD-12, similar with genes about photosynthesis, expressed at arbuscular mycorrhiza formation. All functions of these different fragments still remain to verify in further experiments, to confirm its real role in establishment of arbuscular mycorrhiza.