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Genetic Transformation and Function Analysis of Cinnamoyl CoA Reductase (GhCCR4) Gene in Cotton (Gossypium Hitstum)

Author: QinChao
Tutor: FanLing；QuYanYing
School: Xinjiang Agricultural University
Course: Crop Genetics and Breeding
Keywords: GhCCR4 Transient expression Genetic transformation Tissue-specific expression Gene function
CLC: S562
Type: Master's thesis
Year: 2009
Downloads: 86
Quote: 2
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Abstract

Cotton is one of the world's most important natural fiber crops, cotton occupies a very important position in China's national economy. In recent years, the cloning and cotton fiber secondary wall thickening development related genes, from the molecular level to improve cotton fiber quality has become a major research direction. Cinnamoyl coenzyme A reductase (Cinnamoyl-CoA Reductase, CCR) is a key enzyme in the phenylpropanoid metabolic pathway, this enzyme may has a potential role in the regulation of the phenylpropanoid metabolic pathways of carbon flow. Construction of to clear cotton CCR gene function and lignin content, we utilize the rate-limiting enzyme genes have been cloned from cotton lignin biosynthesis - cinnamon coenzyme A reductase gene (GhCCR4), its transient expression vector cotton ovules, the study of gene expression by particle bombardment transformation of cotton fiber development. Then dip method using Agrobacterium transformation of cotton, and analysis of the function of the GhCCR4. The experimental results are as follows: 1) Experimental constructed by the CaMV35S promoter driven pGUS-CCR4 fusion expression vector, using a gene gun bombardment method transformed cotton ovules, determine the optimum conditions of conversion of 0 DPA ovules: bombardment pressure of 1350 psi, the bombardment distance to 9 cm, and the bombardment for two times. GUS staining results show that GhCCR4 genes in cotton fiber elongation and secondary wall thickening of sustained expression. Different developmental stages of fiber length measurement results found in 8 d to turn GhCCR4 gene fiber length and control compared to no significant difference, but in the 27 d fiber length was significantly shorter than the control. The fiber transmission electron microscopy slicing observed secondary cell wall turn GhCCR4 gene compared with the control thickening by 17%. The above results indicated, GhCCR4 the expression of genes in fiber cells of the cotton fiber elongation play inhibition and enable a certain extent, cell wall secondary wall thickening. 2) comparison of cotton shoot tips Agrobacterium-mediated transformation of a number of factors the perfect shoot tips of Agrobacterium transformation system. Cotton Agrobacterium-mediated transformation process should be two days without vaccine shoot tips as Agrobacterium infection receptor, the best ratio of the concentration of the hormone for KT 0.5 mg / L, IAA, 0.1 mg / L, relatively better shoot tips growing, budding highest rate; the kanamycin optimum screening concentration of 50 mg / L; the cuttings rooting method to shorten the regenerated plants fertility cycle. Using the above method, filtering the resistance conversion rate reached 35% or more, and the conversion cycle is shortened to 2-3 months. 3) the use of Agrobacterium tumefaciens in resistant seedlings batch to kanamycin dispensing and GUS chemical detection, in order to avoid the emergence of false-positive plants, first, to obtain positive plants; report CaMV35S promoter and NPT Ⅱ gene The primers were designed, positive plants PCR amplification, PCR testing positive rate of about 30%. Use a combination of resistance screening and molecular detection methods, 12 transgenic cotton. The detection of transcription levels while using a semi-quantitative PCR method, and found that there are obvious differences between them and the control, the higher the efficiency of antisense inhibition, CCR gene expression is suppressed. 4) improved hot borate method of RNA extraction of different periods of cotton fiber. Cotton GhCCR4 gene in the tissue detected by the herein are expression, particularly expression in the roots, stems, described the organized specific expression of this gene. Great changes in the level of transcription of different developmental stages of cotton fiber, weak expression in the the fiber development early stage (0-10 DPA) expression levels 15,20 DPA 25 DPA decreased expression of this gene in cotton fiber stretch The long development of the late highly expressed. 5) GhCCR4 gene overexpression and RNA interference transgenic cotton seedling morphology observed, lignin and cellulose content detection method comparison and analysis, and found that there are some differences between them and the control on plant morphology, reduced lignin content this may cause the inhibition of gene expression of the exogenous gene. In this study, the candidate target genes from the molecular level to improve fiber quality, and provide a theoretical basis for the study of the molecular mechanism of regulation of cotton fiber development.

Genetic Transformation and Function Analysis of Cinnamoyl CoA Reductase (GhCCR4) Gene in Cotton (Gossypium Hitstum)

Abstract

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