Cloning,expression Of RAMOSA2 And Preliminary Transgenic Research In Wheat

Wheat (Triticum aestivum) as one of the important food crop in the world, its yield is widely concerned. Gramineous crops, such as wheat, rice, corn and so on. Their productions are directly influenced by panicle morphological structure. Therefore, the study of the molecular mechanism of ear is an important theoretical problem. At the same time it's significant to increase wheat crops yield and variety improvement. Multiple spikelet branchs and many grains are the characteristics of the spike brangched wheat, one of three factors for yield components significantly increase the number of grains per ear, which can increase the production performance of grains. Branches spike through ears branched formed on the main spike node quarter main spikelet bears the team more spikelet, thus increasing the number and spikelet number of grains per ear. To study the spike branch phenomenon is not only beneficial to clarify grain morphological development, but also can be used in the application of highly production wheat breeding.Previous studies have shown that the RAMOSA pathway is involved in the regulation of morphological development, and determined the differentiation of panicle meristem in maize. Are there some ramosa homologous genes in wheat? Would those genes mutation affect small panicle morphological structure, thus cause spike branches in wheat? To answer this question, I cloned ramosa2 gene from tetraploid Tritium turgidum L.(TtRa2)in this research. Analyzed the combining characteristics between recombinant protein(TtRA2)and DNA by electrophoretic mobility shift assay(EMSA). And we used gene gun genetic transformation to try to get ramos2 and ramosa3?transgenic plants,which decreased in expression level of these genes due to RNA interference(RNAi). The following results obtained in this paper:1. Cloned? the TtRa2 gene, which length is 774bp from tetraploid Tritium turgidum L.. TtRA2 contains LOB and RA2 domain, and LOB domain include three conservative blocks: C-block, GAS-block and Leu coiled zipper coil, which belongs to LBD proteinⅠclass family members. Homology analysis showed that TtRA2 has the significantly sequence similarity (97.3%, 94.9%, 81.3%, 80.1%, 77.0%, 75.2%)with six kinds of gramineae grasses (Triticum aestivum, Hordeum vulgare, Brachpodium distachyon, Oryza sativa, Zea mays, Sorghum bicolor). 2. Detected the expression level of TtRa2 by RT-PCR in five different tissues and organs: the roots at seedling stage, stem, leaves at seedling stage, young spike, seed in Tritium turgidum L.. The results showed that only can we detecte high expression of TtRa2 in young spike.3. Prokaryotic expression analysis shows that TtRA2 mainly exists in soluble fusion form, which accounts for 68.6% of the total protein. We use electrophoretic mobility shift assay (EMSA) test the specifical binding ability of the reorganization form TtRA2 and DNA sequences. The results showed that TtRA2 can bind the specific DNA probe, which contains"GCGGCG"core sequence. And the dissociation constant of the complex is 10 nmol·L-1.4. The results of tissue culture system optimization for mature embryo, immature embryo of Mianyang 19 results show that the following hormone ratio(differentiation medium: 1.0 mg/L ZT+1.0 mg/L IAA, rooting culture medium: 0.25 mg/L NAA) can effectively enhance the callus emergence rate and improve the root status of regeneration plant in wheat mature embryos and immature embryo.5. The sensitivity experiments for Basta of callus tissue determined the optimal selection pressure concentration of Basta(6 mg/L)in developmental stages.6.?Receptor materials are immature embryos and mature embryos of Mianyang 19. Used RNAi expression vector(pAHC-ra2 and pAHC-ra3)to transform receptor materials by gene gun genetic transformation. We obtained 475 plant resistance seedlings by transforming 5439 callus (immature embryo callus: 1819, mature embryo callus: 3620).