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Agrobacterium Tumefaciens Mediated Transformation of PtLRP1Gene in Poncirus Trifoliata (L.) Raf. and Tobacco

Author: SunXiaoYan
Tutor: ZhangZhuoWen; MeiLi
School: Huazhong Agricultural University
Course: Forestry
Keywords: lateral root primordium Poncirus trifoliata (L.)Raf. Nicotiana tabacum L genetic transformation in situ hybridization
CLC: S188
Type: Master's thesis
Year: 2012
Downloads: 90
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Abstract


The absorbing abilities of roots are directly bound up with the root types of plants, while, root types are shaped by the soil nutrient concentration. The changes of root type are apparently caused by the changes of carbohydrate distribution, hormone transportation and allocation, and other physiological affecters in nutrient deficient conditions. But in the final analysis, it’s the results of gene expression. Former researches had showed that boron deficiency has great effect on root types, and the symptom is that the growth of lateral roots was severely limited of citrus. Recently, abundant researches and certain progress had done in Arabidopsis, rice and corn for molecular mechanism about the development of lateral roots. Few similar reports were found in woody plants. In this research, PtLRP1gene was inserted into tobacco and the aimed plant-Poncirus trifoliata (L.) Raf. To determine the function of PtLRP1and the molecular mechanism of lateral root growth response to boron deficient condition, transgenosis mediated by Agrobacterium tumefaciens and in-situ hybridization technique were done in this research.The main results are as follows:(1) The construction of pCAMBIA1302-LRP1expression vectorIn order to verify the function of PtLRP1gene, which effect on the development of lateral root, pCAMBIA1302-LRP1carrier was successfully constructed. The pCAMBIA1302carrier is a plant expression carrier common used, with a skeleton of pUC18, the CaM35S promoter in T-DNA area and hygromycin B&GFP report genes. There are BglⅡ and Spel two enzymes cut sites on the polyclonal sites, which at the ends of purpose gene on the PCAMBIA-1302carrier. The two enzymes cut sites in the purpose gene do not appeared through the test of PtLRP1gene sequences by primer5software, which means that the insert direction can be controlled accurately.(2) Obtain positive transgentic tobacco seedlingsIn this research, agrobacterium tumefaciens mediated dordrecht variations has been used to importing pCAMBIA1302-LRP1into tobacco plants. Induced buds and screening culture was done in the MS induced buds medium, which contains2.5mg/L hygromycin. Root training culture was dong when the seedlings growth to appropriate size and then plant in a sterile soil until blossom and yield seeds. PCR testing to confirm that PtLRPl gene had been transferred into tobacco plants. Transgenic tobacco seedlings and positive control seedlings have gotten a bright aim strip at1500bp by primer LRP1-F&LRP1-R, a bright aim strip at850bp by primer hyg Ⅱ-F&hygⅡR have gotten. Purification recovery and sequencing the aim strips which appeared in the testing. The results of PCR testing indicated that there were15positive tobacco seedlings with aim gene and21positive tobacco seedlings withempty carrier.(3) Obtained positive transgentic trifoliate orange seedingsAgrobacterium tumefaciens mediated epicotylar by genetic transformation method, transferred pCAMBIAl302-LRP1carrier in to Poncirus trifoliata. Induced buds and screening culture is in the MT induced buds medium which contains2.5mg/L hygromycin. After2weeks dark culture, the buds were then cultured under light, and subculture for each25days. When the length were above0.5cm, the resistant buds were cut off with part of stem segments and cultured on MT elongation culture medium, subculture for each25days. When buds growth to appropriate size, culture on the Rooting medium to root induction. Because of slow growth, the buds of Poncirus trifoliata were not enough for extraction DNA/RNA yet. The carrier pCAMBIA1302contains GFP report gene was used to identification of Seedling transformation of early Poncirus trifoliate seedlings by type fluorescent microscope. The results showed that green fluorescence appeared in the leaves and stems, especially in the leaves. But some of transferred plants have not appeared green fluorescence, whether the green fluorescence appeared plants were positive seedlings or not, further genetic identification is needed.(4) In situ hybridizationTo accurately understand the expression location of PtLRP1gene in the roots of Poncirus trifoliata, digaoxin was used to mark the PtLRP1gene-specific RNA probes in the in situ hybridization analysis of Poncirus trifoliata roots. Plant samples was been putted in FAA fixative, produced by vacuum, dehydrated, clearing, paraffin infiltration and finally embedded in paraffin wax dipped. Then sliced, the wax with material was pasted on a Poly-lysine slides, baking30min under30℃, then put it in oven for two days under37℃, baked for one day after microscopic examination with a microscope to pick out ten good films for in situ hybridization experiments. The entire processes of in situ hybridization experiment are finished on Clean Benches. Results showed that LRP1gene expression in the pericycle parts were clearly, which accordance to previous studies.

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