Mechanism Of Cotton Disease Resistance And Yield Increase Induced By Protein Elicitor From Fungi And Transformation Into Cotton

Protein elicitor is a new group of protein with the function of inducing plant resistanceand promoting plant growth, which were isolated from Botrytis, Alternaria, Aspergillus,Pyricularia, Penicillium, Rhizoctonia solani, Trichoderma, Fusarium etc. Although proteinelicitors with various origins are slightly different in the physical & chemical proprieties, theyare similar or close in biological activity. PEAT in this paper is a new protein elicitor isolatedfrom Alternaria tenuissima (Chinese patent (CN 1344727A) in 2002), with the nucleotide andamino acid sequences different from those of harpins and Cryptogea. At present, research onbiological activity, mechanism and trangenic plant related to PEAT was rarely reported. Inthis paper, by using cotton as object, our research focus on the influence of protein elicitor oncotton growth, the mechanism of disease resistance and yield increase, and transgenic plant.The results are as follows:1. Either rapid paper germination method or sand germination method was adopted,PEAT could promote cotton germination, enhance germination rate and dramaticallyaccelerate roots growth. The effect was remarkable when seeds were soaked for 6 h at theconcentration of 3μg·mL^-1.2. By analyzing the main agronomic and economical character, the number of plant bellwas significantly promoted by PEAT. The plant height, weight of single bell, lint-index,seed-index and lint-percent were increased in different degrees, whereas the weight of singlebell hull was decreased. The protein elicitor promoted cotton plant to be precocious andincreased cotton yield. By the treatment of soaking seeds, the cotton unginned-yield andlint-yield reached 3800.55kg/hm² and 1596.155kg/hm², respectively, which were 15.12%and 17.19% higher than those of control. As to the fiber quality, many parameters weremarkedly improved by applying the protein activator at the right moment with the appropriateconcentration. The treatment of soaking seeds plus spraying at bud period had the topcomprehensive value, which was 6.1% higher than that of control.3.By analyzing the major photosynthesis indexes , we found PEAT could enhance thechlorophyll content, photosynthesis speed, blowhole leading, transpiration speed, especiallyat late growth stage. Thus, leaves' aging and decline of photosynthetic capability weredelayed, photosynthetic period was prolonged, photosynthetic efficiency was maintained at ahigh level.4. PEAT induced the changes of POD, NR, C/N, MDA content and SOD activity in the four stages. There was an increase of POD, SOD, NR, and C/N, and a decrease of MDAcontent. Therefore it launched a series of metabolic reaction, activated growth and defensesystem ofptant, so as to defense pests, promote growth and enhance yield of crops.5. We constructed the expression library of the seedling cotton and then screened thelibrary by SSH technology. Clones were selected and their functions were analysed .The resultdemonstrated that the genes related to several pathways of the plant, such as resistance, cellsignaling, transcription, energy metabolism, cell protection et al. The insertion fragments ofSSH library were ranging from 200bp-1kb. 150 clones were randomly picked for PCR andsequencing. After matching the result in GenBank dbEST, 104 sequences were obtained. Ofthem, 78 have 48.54%-100% similarity with known genes, which is 75% of the total EST. 6have their homology with the sequences in GenBank with unknown function. 10 have nosimilarity with the sequences in GenBank, which might be new genes. The function of all the104 EST was determined by classifying them in WEGO and gene ontology. Most of the ESTwere related to physiological process and cell process, with the number of 48 and 45,respectively. 44 of them related to cell component, 30 related to catalytic activity, 28 relatedto binding.6. A system of 2-DE that is suitable for cotton was established. At first, TCA-acetoneprecipitation method was adopted to prepare protein sample. Protein samples with highconcentration and good purity were prepared by acetone precipitation for three times andcentrifugation for 1h at a speed of 15000r/min under 10—15℃. IEF was conducted at avoltage raised step by step. Hydration time was 15h, eliminating salt lasted 3h and voltagerising 6.5h, the final voltage reached 8000V. The first step of equilibration was 20min, and thesecond was 15min, thus, vertical streaks of SDS-PAGE was less obvious. SDS-PAGE wascarried out by get (T=12%) under a steady electric current of 15mA for 45min. The currentwas raised to 35mA when sample run out of the gel and indicator condensed to a line.7. After analyzing with 2-DE software, approximately 600 proteins per 2-DE gel wereisolated with molecular weights ranging from 10kD to100 kD and PI ranging from 3 to 10.About 53 spots were expressed differently, with 31 at a higher level and 22 at a lower level. 7stronger expressed proteins of high levels observed in a secondary PEAT gel were excised andidentified using peptide mass fin-reprinting and matrix-assisted laser desorption ionizationtime-Of-flight mass spectrometry. 4 identified proteins and their putative functions werepresented. Three of them, spot no. 1,3,6 were identified as ribulose-1, 5-bisphosphatecarboxylase,and spot no. 7 was identified as S-adenosyl-L-methioine sythetase. 8. Resistance to cotton verticillium wilt and enzyme activity were determined whencotton seedling in greenhouse was treated with PEAT. Disease incidence rate and diseaseindex dropped to some extent when seedling was treated with PEAT at 4 differentconcentrations. 3ug/ml was the best concentration with a control efficiency of 66.7%. Theactivities of phenylalanine amonnialyase(PAL) , peroxidase (POD) , SOD, PPO, chitineseof cotton treated with PEAT and inoculated with Verticilliumdahiliae increased significantly,while the content of MDH decreased. These changes coincide with the expression of APE-induced resistance exactly. Therefore, POD, PAL, SOD, PPO and chitinese were considered tobe involved in the resistance to Verticillium dahliae when cotton seedling.was induced byPEAT.9. Two recombinant expression vectors, pBI121-pem^g1 and PCAMBIA-pem^g1 weresuccessfully constructed by utilizing protein elicitor gene pem^g1.Through pollen tube pathwaymethod, the variety of Z-1, Z-2, Z-3 were transformed, and transgenic plants were gained.Among them, 48 containing pBI121-pem^g1 which were resistant to Kan and 10 containingPCAMBIA-pem^g1 which were resistant to bar. The breeding material was enriched.. The genepem^g1 constructed in the expressive vector PBI121 and PCABIM3301 was PCRed and theresult sshowed that the target gene was transformed into the cotton cultivars.Diseaseresistance identification will be carried out further.