Function Analysis Of MwLEA3 And Cloning Of MwRRT Of Mongolia Wheatgrass

Mongolia Wheatgrass (Agropyron mongolicum Keng), a perennial grass, belongs to Agropyron genus in Gramineae, not only has high feeding value, but also rich in resistant genes,such as drought-tolerant gene, cold- tolerant gene, salt-tolerant gene and so on. It could provide valuable resource for the improvement of resistance and breeding of forage and its closely related species including rice, wheat, maize and so on. In this paper, the function of the MwLEA3 gene of Mongolia Wheatgrass was verified on the basis of molecular biological identification and physiological and biochemical identification,and MwRRT retrotransposons was cloned from Mongolia Wheatgrass for the first time. The main results are as follows:1. The full length MwLEA3 gene of Mongolia Wheatgrass was cloned by RT-PCR amplification technique. DNAman analysis indicated its open read frame was 492bp, coded 163 amino acids.2. The fusion protein vector pA7-MwLEA3 for subcellular localization was constructed. The fusion protein vector and pA7-GFP vector plasmids were transformed into onion cells. The results showed that the fusion protein pA7-MwLEA3 was mainly distributed in the cell nucleus, while pA7-GFP were distributed in the whole cell. This result was demonstrating that the expressed protein encoded by MwLEA3 was a nuclear localized protein.3. The plant expression vector of pCAM-MwLEA3 was constructed and transformed into tobacco via Agrobacterium infestation. T₀ transformed tobaccos were tested by PCR,PCR-Southern,RT-PCR and drought stress. Molecular detecting results showed that MwLEA3 gene had been transformed into tobacco and expressed. The results of drought stress showed that the transgenic tabaccos had stronger resistance than wide-type ones.4. The results of kanamycin resistant study of 10 lines T₁ generation of transgenic tobacco plants showed that the separation law of T₁ generation of seven lines of transgenic tobacco was 3:1, showing coincidence with Mendelian genetic law. Other three lines did not show seperation ratio of 3:1.5. The ihpRNA expression vector was constructed with inverted repeats of MwLEA3 gene, which was promoted by 35S promoter. The voctor was transferred into Agrobacterium tumefaciens strain LBA4404 with freeze-thaw method. The callus of Mongolia Wheatgrass were infected with Agrobacterium tumfaciens containing the ihpRNA expression vector, and are now being cultured on differentiation medium.6. MwRRT retrotransposons was cloned from Mongolia Wheatgrass for the first time. It had a length of 1005bp, and consisted of a non-coding region from 1bp to 225bp and a coding region from 226bp to 1005bp that encoded 260 amino acids. Sequence comparison analysis with LTR class retrotransposons from wheat (Triticum aestivum), rice (Oryza sativa), barley (Hordeum vulgare) and sorghum (Sorghum bicolor) showed that it had about 70% idendity in nucleotide sequence and about 50% homology in amino acid sequence. We registered this gene in GenBank and the accession No. was GQ855806.1.