| Lilium regale Wilson, belonging to the Liliaceae Lilium, is native to Minjiang river basin of Si Chuan province of China. It was an important parent material in breeding programs with good characteristics, such as drought resistance, salt tolerance, diseases resistance. Therefore, Isolation of stress-tolerant related gene and understanding patterns of expressed genes were crucial to provide insights into functional analysis of gene in lily and will lead to the increasing stress resistance of lily by using gene modification method on the basis of c DNA library of Lilium regale.In this study, full-length c DNA sequence of lily14-3-3 and lily WBC-ABC genes were cloned through a Lilium regale Wilson c DNA library by using the rapid amplification of c DNA ends(RACE) method after the lily ABC1 gene was cloned. Sequence analysis and functional prediction of 14-3-3 and WBC-ABC genes were based on Bioinformatics. Then, 3 genes expression in 5 kinds of abiotic stresses including salt, low temperature, high temperature, dark and drought is analysed by the real-time PCR. The main results are as follows:1. The expression of lily ABC1 gene was up-regulated by Na Cl, low temperature, high temperature and dark, but the expression patterns of them was different: after 1 hour of high salt and low temperature stress, the gene arrived at the peak of expression, then falled slowly; The time reaching the peak of expression under dark stress was also 1 h, but after 3h, but the expression levels continued to decrease after 3 h, which has been significantly lower than the control at 12 h; In the condition of high temperature, the gene reached the peak of expression at 6 h, and then decreased gradually; In contrast, lily ABC1 gene is not sensitive to the PEG-6000.On the other hand, the sensitivity of the gene for the various abiotic stress was also different: low temperature>dark>high temperature>salt>drought.2. The full-length c DNA sequence designated as lily14-3-3 from Lilium regale Wilson was 1111 bp in length, encoding a 264 amino acid proteins with a molecular mass of 29.57 k D. The p I value of this protein is 4.77. The phylogenetic tree revealved the the lily14-3-3 gene and GRF3 gene have a high similarity. Real time PCR analysis revealed that lily14-3-3 was responsive to the salt, low temperature, high temperature, salt, but the expression patterns of them is different: in the conditions of salt stress and low temperature stress, the lily14-3-3 expression showed a trend of increased firstly and then fell down. However, in the darkness and drought conditions, the trend is the opposite. In addition, lily14-3-3 gene responsive speed for different abiotic stresses are also different: At high temperatures, lily14-3-3 gene expression levels have a a significant difference to the control after treatment for 1 hour, while it is in the 6 hour of salt, low temperature and drought conditions.3. The full-length c DNA sequence designated as lily WBC-ABC from Lilium regale Wilson was 2000 bp in length, encoding a 557 amino acid proteins with a molecular mass of 62.48 k D. The p I value of this protein is 9.31. The protein consists of two parts located outside the membrane and the membrane composition respectively. Between 1-185 amino acids, the protein contains an AAA domain, belonging to the ATPase binding site. Real-time PCR results showed that, lily WBC-ABC gene reached the peak of expression at 12 hour, 3 hour, 12 hour respectively; In the salt condition, expression showed a overall upward trend except 12 hour, which having a slight decrease. However, lily WBC-ABC gene is not sensitive to the low temperature.Overall, athough lily ABC1 gene, lily14-3-3gene and lily WBC-ABC gene have different expression patterns under the abiotic stress conditions, all of them are able to response to the treatments, indicating that they play important roles in the resistant mechanisms of Lilium regale Wilison... |
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