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Cloning And Expression Of Four Drought Induced Genes In The Wheat Cultivar 'Luohan No.2'

Posted on:2009-01-09Degree:MasterType:Thesis
Country:ChinaCandidate:J Y WangFull Text:PDF
GTID:2143360248956108Subject:Crop Cultivation and Farming System
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Based on the data obtained from the gene expression profiling under the drought-stress in the roots of wheat cultivar'Luo Han No. 2', four drought-induced genes were cloned by Reverse Transcription-Polymerase Chain Reaction (RT-PCR), and their expression patterns under the drought- and salt- stresses, as well as the ABA treatment, were studied; The sense and antisense expression vectors of one of the cloned genes were constructed and were used in the genetic transformation of wheat and rice plants by the particlebombardment, Agrobacterium-mediated and pollen tube mediated transformation methods, and some transgenic plantlets were obtained. The main results showed as follows:1 Four drought-stress correlate genes were cloned by Reverse Transcription-Polymerase Chain Reaction (RT-PCR). The cDNA of TaLEA4 includes a sequence of 764bp in length and a coding region 510bp,which contains 94bp in 5' UTR and 160bp in 3' UTR . The deduced amino acids sequence (170 aa) of this gene is about 17.53KD and the pI is 6.05. The genemic cDNA of TaLEA4 in Chinese Spring was studied and a 100bp intron was found in the coding region of the gene. The TaRAB12 includes a sequence of 1013bp in length and a coding region 726bp,and the deduced amino acids sequence (241 aa) of this gene is about 23.98KD. The TaRAB56 is a gene which including a sequence of 464bp in length and a coding region 363bp,and the deduced amino acids was composed by 120aa.The TaRAB910 gene included a sequence of 950bp in length and a coding region 549bp,which the deduced amino acids were composed by 182aa, and the further analysis showed that this gene can code a water-stress regulated membrane protein.2 Expression patterns of cloned genes under the drought-, NaCl- and ABA- stresses were studied via SQ-RT-PCR , then the results showed: in roots, the expression level of TaLEA4 was gradually increased with the water-stress accumulation, and it reached the highest level at the time point of 24 hours after PEG treatment, however, the expression level declined at the time point of 48 hours after PEG treatment; in leaves, the expression of the gene is strong at the time point of 0.5 hours after PEG treatment, but that is relatively weak at other time points detected; Undering the stress of ABA,the TaLEA4 have the Characteristics of differential expression . Expression patterns of the TaRAB56gene under the drought- and salt- stresses showed that: under the PEG mediated water-stress, the expression of TaRAB56 was increased gradually and then decreased in the leaves of LuohanNo. 2, and in root, the expression of the gene is relatively higher than that of control at time 6h of stress treatment. In Chinese Spring, the expression of the gene is greatly changed in the leaves and roots. Under the NaCl mediated salt-stress treatment, The expression of TaRAB56 was slightly stranger than that of control at the time point of 0.5 h in Luohan 2; while in Chinese Spring, the expression of the gene was not obviously changed during the salt-stress treatment in the leaves and roots tissues.The expression level of TaRAB910 under the ABA -stress in leave were significant differently comparing with it's expression in root, but the expression level of TaRAB910 in leaves were lower comparing with it's expression in roots when under NaCl stress.3 The sense and antisense plant expression vectors of TaLEA4 gene were constructed, and they were used in the transformation of rice and wheat plant via microprojectile bombardment, Agrobacterium-mediated and pollen tube pathway mediated transformation. The identification of transgenic plants showed that 2 transgenic wheat plants with the sense TaLEA4 gene and 6 transgenic wheat plants with antisense TaLEA4 gene were obtained via the transformation of microprojectile bombardment. By the method of pollen-tube-pathway, 223 transgenic wheat plants with antisense TaLEA4 gene were obtained, and among them, 185 plants were the wheat variety "ZhengMai9023" and 38 of them were the wheat variety "YuMai34"; From the 26 positive plants with sense TaLEA4 gene, 5 transgenic plants were the wheat varietiy "ZhengMai9023" and 21 were the wheat varietiy "YuMai34". The PCR identification of rice putative transgenic plants transformed by Agrobacterium-mediated methods showed that 4 positive transgenic rice plants with the sense TaLEA4 gene and 16 transgenic plants with anti-sence TaLEA4 gene were obtained.
Keywords/Search Tags:Wheat, Water-stress, Gene cloning, Expression pattern, Genetic transformation
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