| Ramie is one of the important natural fiber crops in China with a long history of cultivation and processing. It has a high biological yield that even can be harvested three times a year in the rain abundant areas. The adaptability, especially drought tolerance, of ramie is strong, so it can be planted at slopes or hilly without land competition with food crops. Thus, one of the important ways to meet the increasing natural fiber demand of human is to vigorously develop the bast fiber crop, especially ramie. Additionally, the cost of soil erosion control will be greatly reduced by cultivating ramie at sloping land in the south of China. Ramie has been one of the important crops which can conserve water and soil at sloping land in the south of China recent years. However, the starting point of research on ramie is low and the researchers are scarce, merely 421 EST sequences of ramie were registered in the Gen Bank(by the end of April 9, 2015), and the information of genome map for reference is lack. The development of molecular biology in ramie has been seriously hindered by the lack of biological information. In this study, a number of transcription factors associated with drought resistance were identified by transcriptome sequencing. The genetic transformation is needed to verify the functions of those transcription factors. There already has reported on transformation using seedlings developed from seeds, however, the heterozygosity of experimental material can easily lead to the separation of traits. Therefore, we established a genetic transformation system utilizing leaf midribs of ramie. The SNAC1 gene is from NAC family of rice responding to drought stress. We transformed it into ramie via the genetic transformation system we established. And the main results of this paper are as follows:1. In this study, ?Huazhu No. 5‘ was chosen as plant material. We established an efficient genetic transformation system which is mediated by Agrobacterium and using leaf midribs as explants in ramie for the first time. Successfully solved the problem of traits separation causing by seed-developed seedlings. Evaluated the sensitivity of leaf midrib regeneration to kanamycin, the optimal concentration ofkanamycin to leaf midrib regeneration during selecting cultivation is 40 mg L-1.2. Some factors that affect the transformation and regeneration of leaf midrib were optimized, including leaf age, Agrobacterium concentration, length of Agrobacterium solution infecting time, acetosyringone concentration in the co-cultivation medium and the co-cultivation period. The conditions that can harvest the highest efficiency of genetic transformation are as follows: the 40-day-old in vitro shoots were used as midrib explants, the Agrobacterium concentration at OD600 is 0.6, length of Agrobacterium solution infecting time is 10 min, the acetosyringone concentration in the co-cultivation medium is 50 mg L-1, the co-cultivation period is 3d. The average transformation efficiency is 23.25%. PCR using GUS and NPTII gene-speci?c primers, Southern blot and histochemical GUS staining analyses further con?rmed that the transgene was integrated into the ramie genome and expressed in the transgenic ramie. The establishment of regeneration and transformation system mediated by Agrobacterium in ramie can not only used in genes transformation for the purpose of traits improvement in ramie, but also as a common means to testing functions of genes by enhancing or inhibiting the expression of target genes.3. Transformed SNAC1 gene which is from rice NAC family(NAM,ATAT and CUC2)into ramie using established regeneration and transformation system of ramie leaf midribs. Harvested the transgenic plants by Agrobacterium-mediated transformation, carried out PCR and Southern blot to analysis the different transgenic families of SNAC1 gene. The analysis results showed that the SNAC1 transgenic lines 1, 3 and 18 are single copy and the transgenic line 20 is double copy, thus, randomly selected transgenic lines 3 and 20 to do subsequent analysis. Fluorescence quantitative expression analysis demonstrated that the target genes of transgenic line 3 and 20 were all overexpressed compared to the control.4. Stress resistance analysis was carried out at the seedling stage, the rapid-growth stage and the ?ber maturation stage in transgenic line 3 and 20, respectively. Used 10%(w/v) PEG6000 to simulate drought stress at seedling stage, while the control plants were incubated in 1/2 Hoagland solution. The result of stress resistanceanalysis against the seedling stage indicated that both the fresh weight and the dry weight of transgenic line 3 and 20 are higher than control under drought stress. The method of drought stress to ramie under rapid-growth stage is to remove excess water after saturated irrigation, then inhibit them with water and observe those phenotypes. The results of drought stress to rapid-growth stage showed that the photosynthesis rate, relative water content, proline content and POD(Peroxidase) activity of transgenic lines are all higher than control, but the malondialdehyde content is lower than control. The transgenic lines and control under ?ber maturation stage were also treatmented by drought stress. The fresh ground part weights, fresh stems weights, fresh bast weights and dry bast weights of transgenic lines under ?ber maturation stage are higher than control. In summary, the transgenic plants have the stronger drought resistance than wild-type plants.5. In this study, ?Huazhu No. 5‘ was treatmented by 15% PEG6000(w/v) which can simulate drought stress. Evaluated the relative water content, proline content, malondialdehyde content and POD(Peroxidase) activity of leaves and roots stressed under drought condition 0h, 12 h, 24 h, 48 h and 72 h, respectively. According to the variation analysis of those physiological indexes, leaves and roots that under drought stress 0h, 24 h and 72 h were selected for RNA extraction. Obtained c DNA collections and carried out the transcriptome analysis using Illumina paired-end sequencing techniques, and then generated 170 million of original sequence reads. A total of 16,798 genes were differentially expressed in leaves and roots under PEG stress 24h(L2 and R2)and 72h(L3 and R3), 9,281 genes were in leaves and 8,627 were in roots. Among those genes, 25 TFs(transcription factors) separately from AP2(3), MYB(6), NAC(9), zinc finger(5) and b ZIP(2) family were thought to be associated with drought stress. A further drought adaptability investigation in ramie can be carried as the foundation of those identified transcription factors. |
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