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Molecular Cloning and Expression Analysis of Several Key Genes from Descurainia Sophia

Author: TangSanYuan
Tutor: GuanRongZhan
School: Nanjing Agricultural College
Course: Crop Genetics and Breeding
Keywords: Sophia Fatty acid desaturase Protease inhibitors Clone Expression
CLC: S451
Type: Master's thesis
Year: 2007
Downloads: 19
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Abstract


This study aims to develop and use with specific quality and resistance traits of wild species sophia excellent genes. The main results are as follows: Using the RACE-PCR method from sophia cloned two oleic acid dehydrogenase gene (DsFAD2 and DsFAD6), they are converted to oleic acid linoleic key enzyme. These two were full-length cDNA encoding amino acids 383 and 447. Sequence analysis showed that these two genes are three typical two required for the combination of catalytic iron ions histidine Boxes, indicating that both genes desaturase enzyme function. DsFAD2 FAD2 and other crops as no obvious N-terminal signal peptide, but found to have a C-terminal signal peptide is rich in aromatic amino acids, endoplasmic reticulum repair signals. DsFAD6 chloroplast found in the N-terminal signal peptide, so positioned in chloroplasts. RT-PCR showed: DsFAD2 and DsFAD6 was constitutively expressed; stress are affected by the injury-induced; under chilling stress DsFAD2 transcript levels almost unaffected, while DsFAD6 transcription is inhibited. Using the RACE-PCR method, cloned from sophia to three linoleic acid dehydrogenase gene (DsFAD3, DsFAD7 and DsFAD8), they are converted to linolenic acid linoleic key enzyme. RT-PCR showed: DsFAD3 and DsFAD7 constitutive expression of the two genes were tested, but in the stems, leaves and young pods expressed higher levels; DsFAD8 only in the stems, leaves and young pods and other photosynthetic tissues in the amount of expression . In injury stress, DsFAD3, DsFAD7 and DsFAD8 are subject to induction; under low temperature stress, DsFAD3 and DsFAD7 transcriptional level downwards and DsFAD8 transcript level. Using the RACE-PCR, cloned from sophia into a diacylglycerol acyltransferase gene (DsDGAT), the gene encoding protein catalyzed diacylglycerol acyl coenzyme A and fatty acids into triglycerides, fats in the organism organizations formation plays an important role. DsDGA face length cDNA encoding 524 amino acids. Encoded protein has an N-terminal signal peptide cytoplasmic membrane. RT-PCI wet shown: DsDGA tunnel was due to constitutive expression in stems and young pods expressed higher levels; in injury and chilling stress, are affected by induced expression. Right sophia pods cDNA library clones were sequenced to obtain a full-length gene, which protease inhibitor gene from Arabidopsis homology, so named as DsTI-2. The gene is 442bp, encoding 100 amino acids. Tissue expression analysis showed: DsTI-2 gene sophia roots, buds, flowers and pods of expression, but in the stems and leaves almost no expression. The injury induced by the stress of the gene. By Agrobacterium-mediated method, the DsTI-2 gene was introduced into wild-type tobacco were obtained with a kanamycin resistance tobacco. PCR and RT-PCR analysis confirmed the initial gene has been integrated into the genome of tobacco, and can express. Protease inhibitor activity testing showed that transgenic plants are trypsin and chymotrypsin inhibition, inhibition of untransformed plants is low. From leaf feeding insects indoors insect resistance identification further evidence of transgenic plants resistant to bollworm has some. Natural identification results show that transgenic plants is likely to also have some smoke planthopper resistance.

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CLC: > Agricultural Sciences > Plant Protection > Harmful plants and their removal > Weeds
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