| Acacia, one of the subfamily of mimosa, all were introduced from abroad, besides Taiwan Acacia, whicn was native to China. This tree species was mainly distributed in Australia and Africa, due to the reasons that most of them belonged to subtropical or tropical species. What is more, trees of this species were sensitive to low temperature, and the rate of survival after seedling hibernation was low. A implexa, somewhat more resistant to coldness, was speed-growth, quick-fell and versative tree species,. The lumberqualit of it was smooth, corrosion durable, and can be used to improve the soil and raise the land capability. With benifit from favaroble economic and ecological aspects, in recent years,it is one of the vigorously introduced and promoted tree species by Fujian province. At present, this tree species still depend mainly on seed introduction and propagation. Correspondingly, not only the tree form, amount of growth, but also the biological stress resistance, were affected by seed origin, and resulted in serious segregation of characters.In turn, the above limitation restrict the forestation speed and region expansion of Acacia implexa, and also its exploitation and application.In this study, A. implexa was taken as test materials,and the experimental program was designed according to the above problems.Through tissue culture technique and rapid propagation, the isolated culture and regeneration system were established, one new avenue which can rapidly propagate the seedling with stable and favorable characters was expected to be obtained.With coldness assessment, lines with strong coldness resistance and weak coldness resistance were expected to be screened.The corresponding change in cellular morphology after different low temperature menace could be observed by apoptosis detection technique. At last, proteomics and bioinformatics were applied to gain the differentially expressed proteins after coldness menace, and the molecular mechanism of coldness resistance was discussed with the information deriving from the protein identification.The results were as follows:1,A. implexa, with hard testa, long period of dormancy, the pre-treatment method of 15 min immersion in concentrated sulfuric acid and 15 min boiled water soak can effectively break the dormancy and raise the germination percentage of seed and germinative force improvement. The appropriate culture medium formula for adventitious bud induction from A. implexa explant was: MS+6-BA 1.0 mg·L-1+NAA 0.2 mg·L-1+ZT 1.0 mg·L-1. High concentration of hormone can result in more adventitious buds and more rapid growth, but easier to promote seedling vitrification. Through quadrature experiment, suitable proliferation subculture was obtained::MS(40 mmol·L-1, NH4+:NO3-= 1:2)+6-BA 1.0 mg-L-1+NAA 0.05 mg-L-1, and this formula not only largely decreased the vitrification rate, but raise the proliferation multiple. Then the corresponding robust stem was innoculated into the improved 1/2MS+NAA 0.1 mg-L-1 rooting culture, of which the rooting rate can reach as high as 93%, and higher concentration of NAA can significantly suppressed it. In the end, the test-tube plantlet was transplanted on to the plastic seed bed with ground substance loess:river sand= 3:2, and the survival rate was more than 70%.2,The outcome of coldness resistance assessment between three superior trees and nine seedlings(totally twelve) showed that, the physiological index,such as relative conductivity change of cytoplasma membrane, solvable sugar content, dissociate proline content, combinated with Logistic formula, can be applied to work out the half-leathl temperature of ZH-01 to be-6.11℃(most coldness resistanct) and of ZH-73 to be-0.93℃(least coldness resistance). And it also found that coldness resistanct lines can accumulated more dissociate proline and sovable sugar, and the content ascend relatively rapid, but little change in relative conductivity change of cytoplasma membrane. From above, we can deduced that when exposed to outside menace, more strong adaption ability was always associated with rapid physiological reaction.3,The microscopic observation of nucleolus,DNA electrophoresis and TUNEL detection technique were applied to discussed the cellular apoptosis after cold menace at the cellular and molecular biological level. Like rice and tobacco, there was not any positive DNA ladder existing in the cell of A. implexa;but in treatment groups, positive signal was detected in TUNEL, and the karyomorphism existed apotosis to some extent;in treatment groups, the DNA of mitochondrion and chloroplast was found to rupture prior to nucleus DNA,so the manifest sequence of the corresponding apoptosis was as follows:rupture of the DNA in mitochondrion and chloroplast→rupture of the DNA in nucleus DNA→chromatin condesion→nucleus dissortion.The research revealed that coldness menace of average intensity can induce A. implexa to apoptosis, but when the intensity exceed, it die.4,Highly efficient and stable protein isolatin method and repeatable 2D electrophoresis procedure was established in this study. The protein lysate was 7 mmol/L Urea,2 mmol/L Thiourea,4% CHAPS,40 mmol/L Trisbase,65 mmol/L DTT; the volume of spplication of sample was 1000μg/24cmIPG; the concentration of SDS-PAGE in second vertical electrophoresis was 12.5%; the protein isolation efficiency and mass spectrum identification can both be achieved, when using the Comassie Bright Blue as staining material.5. Three kinds of IPG gel, with different pH extent(pH 3-10,pH 4-7,pH 7-11) was applied in our study and the total protein of ZH-01 (resistant to coldness) and ZH-73(sensitive to coldness) were seperated. Then, the 2D electrophoresis map was analysed by ImageMaster and 38 peculiar protein spot were selected. There are 12 up-regulated,6 down-regulated,4 appended and 1 disappeared protein spot existing in ZH-01, respectively; in ZH-73, the number was 9 up-regulated,2 down-regulated,3 appended, and 1 disappeared, respectively. After, mass spectrum and database identification, the known protein can be assorted into five categories:photosynthesis related protein;lgene regulation related protein;metabolism related protein; stress resistant related protein and othat not assorted unknown protein.. As a result, the coldness resistance mechanism of A. implexa was not only due to individual reaction or the expression of little protein, but the together function of plant photosynthesis reaction system, membrane system stability, gene transcript, translation, and the energy metabolism of sugar and protein, hormone secretion, etc.From all the above, the menace below the endurance extent of plant can activate the physiological metabolism, gene regulation and active apoptosis, so raise the stress resistance and under these circumtances the injury was reversible and the body can repair itself; When the stress menace exceed the endurance threshold, the injury was irreversible and can result in damage or death.The more rapid its response to outward menace, the more adaptable the lines was. The improvement of plant stress resistance was the together function of body photosynthesis reaction system, physiological and biochemical reaction, energy metabolism, gene regulation and protein expression and so on, but not only due to one simple reason in some aspect. |
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