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Transformation of Three Cold Tolerance Candidate Genes from Chaling Wild Rice and Phenotypic Analysis of Transgenic Plants in Arabidopsis

Author: ChenShuZuo
Tutor: XuMengLiang
School: Hunan Normal University
Course: Botany
Keywords: Chaling wild rice Arabidopsis cold resistance related genes stresstolerance ABA
CLC: S511.9
Type: Master's thesis
Year: 2014
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Abstract


Chaling wild rice(Oryza rufipogon Griff.) is one of two northmost wild rice groups in the world. Previous studies have shown that it is very cold tolerant. Cloning its cold tolerance related gene(s) not only can provide cold tolerance gene resources for the cultivated rice transgenic molecular breeding, but also can improve our understanding of molecular mechanisms of cold tolerance in wild rice. So this study has important value.OrCr1,OrCr2,and OrCr3(Oryza rufipogon cold responsive gene1,2,3) are three genes highly induced by the cold stress, which are screened from Chaling wild rice using the GeneChip Rice Genome Array (Affymetrix) analysis and in silico analysis.To find out whether the three cold tolerance candidate genes has tolerance to cold or other functions, we got transgenic Arabidopsis plants containing OrCr1,2,3genes,respectively, through the method of Agrobacterium mediated transformation, and identified pure lines for transgenic plants.The stress tolerance of transgenic plants was evaluated. The results are as follows:1) The germination rates of OrCrl,OrCr2,and OrCr3overexpression transgenic lines are significantly higher than those of the wild type, and their seedling growth potential is much better than that of the wild type after cold treatment for20days at4℃.2) There is no significant difference between transgenic Arabidopsis plants containing OrCr1,2,3genes and wild type ones after heat treatment at43℃for3h at their vegetative stage.3) The germination rate and root growing of OrCr3overexpression transgenic Arabidopsis plants are higher and better than those of its wild type counterparter after salinity treatment (10days in150mM NaCl,1/2MS medium), however, there is no significant difference between transgenic Arabidopsis plants containing OrCr1,2genes and wild type plants. 4) The germination rate and root growing of Arabidopsis plants containing OrCr3genes are better than those of wild type after drought treatment (300mMol/L mannitol for10days), but the germination rate and root growing of transgenic Arabidopsis plants containing OrCr1,2genes are not significantly different from those of the wild type.5) The sensitivity of all the OrCr1,OrCr2, and OrCr3overexpression transgenic lines to abscisic acid (0.4to0.8μM) is increased. At the concentration of0.8μM abscisic acid, the transgenic Arabidopsis lines overexpressing OrCr1,2,3genes hardly germinate,but the wild type plants germinate normally. With the increase of ABA concentration for treatment, the root length of the transgenic plants get shorter compared with the wild type plants, and eventually without growth at0.8μM ABA, and the wild-type Arabidopsis root length is slightly lower in this ABA treatment condition than in normal conditions.The above results show that:ectopic expression of OrCrl,2,3gene in Arabidopsis thaliana can enhance the cold tolerance of transgenic plants and improve the exogenous ABA sensitivity, and ectopic expression of OrCr3genes can improve salt resistance of transgenic plants, but can not enhance the osmotic stress tolerance..

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