Cloning And Transfermation Of The SDH Gene Related To The Cucumber Propamocarb’s Residual Property

Cucumber, widely loved by the people, is an important fresh vegetable. But as the cultivating technology constantly updated in recent years, the vegetable growth period becomes shortened. For the destruction of the ecological environment which leads to plant diseases inc reased year by year and insect resistance is becoming more and more strong, most of the vegetables need to spray pesticide for many times before enter the market. Howerver, the medicinal properties of pesticide and its durability have been more and more strong, and the pesticide residue in cucumber is growing severely, becoming the hot topic in the government, market and consumers. This problem not only affects people’s health, it also affects the export [1].Cucumber downy mildew is one of the major diseases of cucumber plants, it can happper in all kinds of regions and cultural facilities [2-8]. Downy mildew belongs to carbamate fungicide with systemic strong. Our country has nearly 80% of the farmers use frost mildew to treat cucumber downy mildew. Due to large-scale and large dose use, the propamocarb pesticide residues have a severel problem that can not allow to ignore for cucumber production.Succinate dehydrogenase（SDH） is one of the key enzymes in the process of putting organic matters into the synthesis of CO2, H2 O and ATP. It could generate fumaric acid in the process of catalyzing succinic acid in TCA. Fumaric acid as an antioxidant, has the function of capture and neutralize free radicals, so as to remove free radicals, reducing the dama ge to the organism. Another important feature of SDH is that it can produce ATP through electronic respiratory chain, providing energy for organisms and various life activities, so the activity of SDH has great influence on the whole living organisms in the process of metabolism. This study group early evaluate the difference expressed genes of cucumber fruit under downy mildew threat through applying Solexa techniques and methods of comparative genomics, selecting related genes SDH of cucumber with low propamocarb.This study used PCR clone to obtain SDH full-length gene, and named CsSDH. We built the subcellular localization expression vector, and observe under laser confocal microscope after inject tobacco leaf by the method of agrobacterium infiltration; By analyzing the D9320 and D0351 CsSDH genes in high and low defective agricultural pruducts after the treatment with propamocarb respectively by fluorescence quantitative RT-PCR method, and enzymelinked immunosorbent assay, we can know CsSDH gene expression characteristics and activity of succinate dehydrogenase in different time and different tissue. We construct PBI121-CsSDH plant expression vector, and obtain arabidopsis plants of CsSDH gene through the method of agrobacterium mediated, the main results were as follows:（1）We use the cucumber D0351 leaves as materials, then separate and clone the cucumber CsSDH gene, the whole length of this gene is 834 bp, encoding 277 amino acids, amino acid sequence contains two domain structure, having conservative domain structure at 48-152 aa is Fer₂, which structure domain belongs to Fer2 super family, playing an important role in the process of enzymatic reaction and electron transfer. While close to C-189-262 aa only is a Fer4₁7 domain structure, which belongs to iron sulfur protein superfamily（HCP-like superfamily）, playing an important role in nitrogen metabolism. By phylogenetic tree analysis, we found that cucumber CsSDH has the closest elation with melon Cm SDH, the homology is 96%, but thehomology is 85% compared with beans, the homology is 84% compared with soybean, the homology is 86% compared with chickpeas, the homology is 84% compared with thistles alfalfa, and has relative distance relationship with potato and tomato more, the homology is only 82%. The gene is a conservative gene. The CsSDH gene encoding protein is an unstable water protein without transmembrane structure, signal peptide and obvious hydrophobic area.（2）Under the threatening of propamocrab, the gene CsSDH in the cucumber variety with low pesticide residue D0351 reacts rapidly after the fruits suffering the threat from propamocrab. It responds with strong expression primarily in the early phage of deali ng with, and the activity of Succinate dehydrogenase remarkably increases and its quantity of gene expression in this cucumber variety obviously exceeds its quantity in the cucumber varieties with high pesticide residue D9320. The expression amounts of CsSDH in fruits and leaves are higher than that in stems, and the order of the expression amount in each tissue is stable as leaves>fruits>stems. In the cucumber varieties with high pesticide residue D9320, the quantity of gene expression presents as increasing expression at 0.5, 1, 3, 12 h, the difference between it and control group is not outstanding at 6, 9 h, and it reaches the top of increasing expression at 48 h. This is not corresponding with the expression pattern of this gene in D0351. With contrasti ng the expressions of the gene in cucumber varieties with high and low pesticide residue and analyzing them, the studying results find out the correlations between this gene and low pesticide residue, preliminarily decided as low-pesticide-residue related gene.（3）Construct CsSDH gene expression vector, named as PBI121-SDH; and translate the successfully constructed plant expression vectors into agrobacterium LBA4404 by freeze-thaw method.（ 4） Transmit the plant expression vectors into Arabidopsis, and obtain 28 translated PBI121-SDH positive plants through PCR examination. Harvest the seeds of T1 generation of the transgenic vegetables.（5）Have constructed CsSDH-e GFP fusion expression vectors, and translate them into agrobacterium. With agrobacterium infiltration method, observe it under laser scanning confocal microscope after injecting tobacco leaf. The study of subcellular localization presents the proteins of CsSDH gene coding mainly distributes on cell membrane.