Study On Tomato RAPD Clustering, CDNA Library Construction And Separating RGA From ToMV-resistance Tomato(Lycopersicum Esculentum Mill)

Tomato virus disease is a very serious disease in the world. ToMV is one of the most serious diseases in vegetable crops and it attacks many kinds of crops. It affects tomato yield and quality. The most important way to control ToMV is to use disease-resistance cultivator. Disease-resistance cultivators could be developed by the traditional breeding method or genetic engineering. As the development of the molecular biology and study of modern resistant gene engineering, the cloning and transformation of plant resistant genes would give the wide foreground for high efficiency breeding of tomato(Lycopersicum esculentum Mill).Twenty tomato materials were used in the study. The ToMV-resistance characters of tomato materials were studied by field and artificial inoculation methods. Tomato POD and PPO enzyme activities and POD, PPO isozyme were studied. Relationship between 20 tomato materials was judged by RAPD. cDNA library of ToMV-resistance tomato was constructed. Resistance gene analogues (RGA) were cloned from tomato genome DNA by degenerate primers designed according to the conserved sequences of disease-resistance genes in tobacco and Arabidopsis. The results were as following.1. Classification of tomato based on ToMV-resistanceThe disease-resistance to ToMV of 20 tomato materials collected from the markets of whole country and Sichuan Agricultural University were studied. According to the introduction of tomato materials, ToMV-resistance study in the field , ToMV-resistance per centage and index checkup after ToMV inoculation to tomato in greenhouse, using ToMV-resistance index as the main factor of ToMV-resistance grade, the ToMV-resistance of 20 tomato materials could be divided into fourgroups. The first group was high resistant (HR) tomato materials, including Huang Sheng Guo and America tomato; The second group was resistance (R) tomato materials, including Zhong-za No 9, W262-4, Oh-2-2-11 and Yellow-leaf tomato; The third group was disease-tolerance (T) tomato materials, including L-402, Saporous cherry tomato, California Red No 1, Early Hong-ba, Bao-da 903 and Chuan Hybrid No 10. The forth group was disease sensitive (S) tomato materials, Jin-ke Hong-mei, Big red tomato 903, California Red No 4, Improved America 903, Bao-dao Ju-xing, W262, Oh-2-2-6 and Jing Gang Big tomato No2 belong to this group.2. Study on optimum reaction condition of oxidation enzymeThe study on reaction condition of oxidation enzyme POD and PPO showed that the best pH of phosphor acid buffer for extraction of POD and PPO enzymes and for reaction was 6.0, the best PPO enzyme reaction time at 10°C was 10 min. There was the biggest OD at 398nm using catechol as the PPO base reaction substance.3. Relationship between oxidation enzyme activities and the ToMV resistance of tomato materialsThe study of POD, PPO and PAL oxidation enzyme activities in tomato materials showed that after inoculation of ToMV, POD, PPO and PAL oxidation enzyme activities of tomato materials were higher than those without inoculating ToMV. The increasing time of POD enzyme activity of most R tomato materials was higher than S ones, while the increasing time of PPO enzyme activity of most R tomato materials was lower than S tomato materials. There was no distinct correlation between the increasing time of PAL enzyme activity and the ToMV-resistance.4. Correlation between POD and PPO isozyme pattern and the ToMV-resistance of tomato materialsPOD and PPO isozyme patterns of tomato materials were studied. After inoculation of ToMV, the band color and band width of POD and PPO isozymes were different from the control (non-inoculation). There were 1 to 3 more POD isozyme bands in the ToMV-resistance materials and 1to 2 more PPO isozyme bands in the ToMV sensitive materials. 5. Construction of cDNA library of ToMV resistant tomatoThree tomato ToMV-resistance materials (W262-4. Yellow-leaf tomato and America tomato) selected by above methods were used in extracting RNA and mRNA, synthesizing cDNA and constructing cDNA library. The vector was Uni-ZAP Express vector X-ZAPII. The library host bacteria were E. coli XL 1-Blue. The results were as following.Extraction of RNA: Stratagene's RNA Isolation Kit was used in extraction of RNA. By this way, larger amount and integrity RNA was obtained and total RNA of each tomato materials was about 5.0mg. OD260=0.4741,OD280=0.2475, OD230=0.2175, OD260/280 of RNA was about 1.92. OD260/OD230=2.18. The 28 S rRNA and 18S rRNA bands were clear. They attested that the RNA quality extracted by the kit was high and it could meet the requirement of RNA for cDNA synthesis.Extraction of mRNA: According to the direction of Poly (A) Quik? mRNA Isolation Kit, mRNA was extracted. By the method, higher yield (about 20ug mRNA of each tomato materials) and high pure mRNA was gained. O.D260/280 of mRNA was about 1.98 and it reached the requirement for library construction.cDNA synthesizing and library construction: According to the direction of ZAP-cDNA Gigapack III Gold Cloning Kit, cDNA library was constructed using Yellow-leaf tomato. The length of cDNA synthesized by the kit was about 200 to 4000bp and they showed dispersion. Most of cDNA fragments were in 500 to 2000bp. Twenty-four phage plaques were picked randomLy for checkup of inserting cDNA fragment size. The results showed that the average size of cDNA was 700bp, smallest one was 200bp and the biggest one was 1 lOObp. The titer of primary cDNA library was 1.22xlO6(pfu/mL) and the titer of amplified library was 1.43xl09(pfu/mL). The recombinant rate of the library was 9836%. They all achieved the base need of the cDNA library.6. RAPD analysisDNA extracting technique: 7 kinds (A, B, C, D, E, F and G) of modified CTAB methods were used for extracting genome DNA. Higher quality DNA could get by G method (0.8g material+2mL extraction solution+ 0.2g PVP).The ratios of OThat and ODjjo were between!.8 to 2.0. It accorded with the need of RAPD analysis.RAPD analysis technique: The reaction system of tomato RAPD was built There was 25ul in total reaction system, including 2ul DNA (25ng.uL"'), 0.2umol.L'' primer, 200umol.L"' dNTPs, 1.25U Taq enzyme, 2.5ul 10xBuffer(Mg2+free) and 2ul MgCl2(25m mol.L*1).The cycling parameter of amplified procedure was as following: pre-denaturalization 5 min at 95°C, denaturalization 1 min at 94°C, anneal 1 min at 36°C and extending 1.5 min at 72°C for 40 cycles, finally extending 10 min at 72°C.The results of tomato RAPD analysis: 5 primers were screening out from 40 lObp random primers. Total 30 bands in 20 tomato materials were amplified by five lObp random primers. There were 28 polymorphic sites, which accounted for 93.3%. There were average 5.6 polymorphic bands amplified by each random primer.