Resistance Transcription Factor Transformation And Functional Verification Of Poplar

As the important renewable resources, trees are playing an important role on the maintenance and protection of the ecological environment. But with the current worsening biotic and abiotic stress conditions, their yield, quality and productivity of forest trees have been severely affected. Consequently, it has become the hot topic to cultivate new resistant variety of forest tree with resistant, improve arid, semi-arid and saline wasteland, promote the development and utilization of land resources and improve environmental resources. Genetic engineering techniques can improve the trait of trees from the genetic level to overcome the shortcomings of traditional tree breeding with cycle long, tedious process and others, and open a new avenue for the genetic improvement of forest tree breeding. In this study, Populus × euramericana cl ?Lingfeng 2‘ was used as the experimental, three resistance transcription factors gene zinc finger protein（ZxZF）, ABA response element binding protein（ckAREB） and jasmonic acid ethylene-responsive element（JERF36） were transferred to Populus × euramericana cl ?Lingfeng 2‘ in multiple combinations using Agrobacterium tumefaciens-mediated method for first time. We verified the resilience of transgenic poplars by measuring physiological indicators; Na⁺, K⁺, H⁺ ion flow and the expression of downstream genes. We hope to obtain transgenic popular with multi-resistance. It has theoretical guidance significance for cultivation of new varieties of genetically engineered trees efficiently by apply transcription factor gene in tree breeding, the strategies of combination genetic engineering with traditional breeding and the study of mechanisms of transcription factors in transgenic poplar with multiple combinations. The main results are described as follows:1. Successfully constructed transgenic plant expression vector with double transcription factors and three transcription factors, respectively. ZxZF from Zygophyllum xanthoxylum, ckAREB from Caragana Korshinski and JERF36 from Lycopersicon esculentum, i were transferred to Populus × euramericana cl ?Lingfeng 2‘ in multiple combinations. The vector with double gene is pBIJEAR and the vector with three genes is pMDCZXARJE. Provide genetic material for future study.2. Establishment of new varieties of poplar efficient regeneration system and genetic transformation system, overcome the technical problems of low regeneration rate in Aigeiros tree. Populus × euramericana cl ?Lingfeng 2‘ was used as the experimental, which was cultivated by Chinese academy forestry. The optimum medium for differentiation of leaves and petiole was: MS ⁺ 6-BA 0.5 mg ? L^{-1 +} NAA 0.05 mg ? L^-1. The optimum mediumof rooting was:1/2 MS ⁺ IBA 0.01 mg ? L^{-1 +} NAA 0.01 mg ? L^-1. The selection pressure of Kanamycin was 30 mg ? L^-1, but Hygromycin was 5 mg ? L^-1. When using this system for the regeneration of tissue culture,the differentiation rate of leaves, petioles highest and the rooting rate were up to 100.00%, the average per regeneration bud up to 20 of leaves, and 11 of petioles. Transformation for Populus × euramericana cl ?Lingfeng 2‘ using this system, 1581 resistant plants were obtained. Through PCR, qRT-PCR molecular detection, 53 lines transgenic plant with successful integration of exogenous gene and stable expression were obtained, including 41 transgenic plant with single exogenous gene, which contained 7 transgenic plants expressing ZxZF, 6 transgenic plants expressing ckAREB, 28 transgenic plants with JERF36, and 12 transgenic plants expressing double genes（JERF-ckAREB）.3. Transformations of transcription factor genes can improve the resistance of transgenic poplars, and more than one regulatory pathway transcription factors were better than one regulatory pathway transcription factors. To validate the transgenic poplars resilience, the physiological detection to simulated drought（20% PEG） and salt（150 mM NaCl） stress to transgenic plants was conducted. The results showed that LJ6 performed better under salt stress, the content of proline, activity of SOD and POD were higher than non-transgenic lines 5.39%、150% and 24.07%, respectively. And the membrane permeability were lower 26.55%. The transgenic plants were showed a lower resistance in a simulated drought stress conditions; transfer lines GA51 showed good perform under simulated drought stress, the Fv/Fm, RWC, content of proline and activity of SOD and POD were higher than non-transgenic lines, proline was significantly higher than non-transgenic lines of 187.56%, while membrane permeability and MDA levels were lower than non-transgenic lines, and the lines showed some resistance under salt stress conditions; the transgenic line expressing double genes（JERF-ckAREB） has a excellent performance, the content of chlorophyll and proline were higher 13.8% and 17.05% than transgenic lines expressing JERF, under simulated drought and salt stress respectively. The content of MDA were lower 54.27% and 42.84% than expressing JERF, under simulated drought and salt stress respectively. The content of proline, activity of SOD and POD were higher 32.62%, 12.21%, 84.76% than transgenic lines expressing ckAREB. We determination of ion flow of Na⁺, K⁺ and H⁺ of transgenic poplar roots by non-invasive micro-measurement techniques(scanning ion-selective microelectrode technique. Compared with non-transgenic plants, Na⁺, H⁺ of transgenic lines（LAJ12, LAJ29） expressing double genes efflux and K⁺ influx significantly, Na⁺, K⁺ of transgenic lines expressing ckAREB efflux, and H⁺ influx significanrly under simulated drought stress; the rate of Na⁺ efflux of transgenic lines GAJ39（expressing double genes） and LJ42（expressing JERF） raised, and K⁺ influx weakened with the tend of efflux, the tend of H⁺ efflux is significant under salt stress. This is indicates that, the proton pump activity of transgenic plants was stronger than non-transgenic lines under stress, and transgenic lines expressing double genes were stronger than transgenic lines expressing single genes. The drought and salt tolerance of transgenic poplars have been improved, resistance of transgenic lines expressing double genes were higher than which expressing single gene.4. The exogenous transcription factors can regulate the expression of stress resistance related genes in transgenic plants effective, and the integration of multiple transcription factors transformation regulate gene expression importing more stable. Verification transcription factor regulating function of transgenic lines obtained above by qRT-PCR stress related genes. Abiotic stress-related gene expression of transgenic plants showed that: the import of the transcription factor genes can inordinately promote up-regulated expression of adversity stress gene（Enolase, P5 CS, SPDS and WCOR413-like protein）. In transgenic lines GAJ39 with two exogenous gene, abiotic stress-related gene‘s expression amounts are higher than non-transgenic plants and single transgenic lines in normal water, drought stress simulated by PEG and salt stress conditions. The SPDS gene of transgenic lines GA51 indicated high expression levels under stress simulated by PEG and salt stress, while its WCOR413-like protein gene was only under drought stress. Expression amounts of P5 CS gene of transgenetic lines LJ42 was higher under drought stress conditions. WCOR413-like protein of each transgenic lines were down-regulated expression in normal water conditions, whereas expression was different（up-regulated or down-regulated） under drought simulated by PEG and salt stress conditions. Generally speaking, Abiotic stress-related gene expression amount of transgenic plants were higher than non-transgenic plants and transgenic lines with exogenous gene. Consequently, we can conclude that the function of the two transcription factors may have cross action, and they can coordinately regulate abiotic stress-related gene expression, and then enhance ability of drought and salt tolerance of transgenic plant.