Dissertation
Dissertation > Agricultural Sciences > Crop > Economic crops > Fiber crops > Cotton

Cloning and Expression Analysis of Two 9-cis-expoxycarotenoid Dioxygenase (GhNCED1 and GhNCED2) Gene from Cotton under Drought-stress

Author LiuJiangNa
Tutor WeiYiNong
School Shihezi University
Course Crop Genetics and Breeding
Keywords cotton ABA NCED semi RT-PCR dehydration
CLC S562
Type Master's thesis
Year 2010
Downloads 42
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Object:Abscisic acid(ABA) is a hormone which plays an important role in the adaptation of plants to variety environmental stresses, drought, cold and salt stress all can make the accumulation of ABA level higher. Because many of these physiological progress are correlated with the endogenous ABA level, the regulation of ABA biosynthesis is the key element facilitating our understanding of these characteristics. Biochemical and genetic evidences demonstrated that oxidative cleavage of cis-epoxycamtenoids catalyzed by 9-cis- epoxycamtenoids dioxygenase(NCED) is the critical step in the regulation of abscisic acid (ABA) biosynthesis in higher Plants. The Environmental stresses induce the expression of NCED and thereby regulate endogenous ABA level. From now we did not find any report about NCED gene in cotton. In order to study the biologic function of 9-cis-epoxycarotenoid dioxygenase (NCED)gene under water-stress in cotton. Two NCED gene were cloned from cotton leaves (JIN13), namely GhNCED1 and ghNCED 2respectively. A semi RT-PCR system was established to detect the gene expression level in dehydrated different organs of cotton and the relation to the accumulation of ABA.Methods:Degenerated primers were synthesized based on the high homologous region among the NCED sequences of the NCBI has logged on, and used for cloning of two NCED genes from dehydrated leaves of cotton by using RT-PCR、RACE and nested PCR. A semi RT-PCR system was established to detect the gene expression of NCED in dehydrated cotton. Detect the change of ABA content in water-stressed cotton by HPLC.Results:1.Cloning of GhNCEDl and GhNCED 2 from cotton, Degenerated primers were synthesized based on the high homologous region among the NCED sequences of the NCBI has logged on, and used for cloning of two NCED genes from dehydrated leaves of cotton by using RT-PCR、RACE and nested PCR.. Two full length of NCED cDNA, namely GhNCEDl and GhNCED2 were cloned and seque NCED. The full length of GhNCEDl was 2130-bp long and contained 15bp 5’UTR,283bp 3’-UTR and an open reading frame (ORF) of 1827-bp which encodes a peptide of 609 amino acids and encodes a protein with a calculated molecular weight of 67.89 kDa and an isoelectric point of 5.94. The full length of ghNCED 2 was 2134-bp long and contained 13bp 5’UTR,327bp 3’-UTR and an open reading frame (ORF) of 1794-bp which encodes a peptide of 598 amino acids and encodes a protein with a calculated molecular weight of 66.72 kDa and an isoelectric point of 6.85.The protein sequences align closely at the C-terminus. The N-terminal regions of GhNCED and other NCED proteins are of low sequence identity. And we have found a 30-amino-acid chloroplast-targeting peptide predicted by Iposort algorithm is located at the N-terminus of the GhNCED2 protein.2. Analysis on the GhNCED1 and GhNCED2 expression and ABA biosynthesis in response to water stresses, Semi-quantification by duplex RT-PCR revealed that we can detect the expression of GhNCEDl in the cotton leaves, stem and roots, respectively. And we found that the gene did not exhibite a typical and significant response to water stress, but the expression level of the gene GhNCED2 in leaves was gradually increased with the water-stressed accumulation, and it reached the highest level at the time point of 6 hours after PEG6000 treatment then it was gradually decreased to lowest level after 24 hours water stressed. In different organs of cotton, the expression of GhNCED2 gene can be obviously induced at 6 hours after water stress treatment in stem and leaves, but the expression level of the gene in root was not obviously. So the result show that the expression of GhNCED2 exhibite a typical and significant response to water stress. We always found that the accumulation of ABA in cotton leaves is similar to the expression level of GhNCED2 under water stresses.Conclusion:Two NCED genes were cloned from dehydrated leaves of cotton by using RT-PCR、RACE and nested PCR., namely GhNCEDl and GhNCED2. A semi RT-PCR system was established to detect the gene expression of NCED in dehydrated cotton. We found that GhNCED2 exhibite a typical and significant response to water stress, and the expression level of GhNCED2 under water stresss is similar to accumulation of ABA in cotton. But GhNCEDl did not exhibit a relation to the accumulation of ABA.

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