【作者】 何文锦；

【导师】 林思祖； 陈由强；

【作者基本信息】 福建农林大学 ， 森林培育， 2007， 博士

【摘要】 相思类树种(Acacia)是含羞草亚科,其中除台湾相思一种原产于我国外,其余皆从国外引进。该树种主要分布于澳大利亚及非洲等地,大多为热带与亚热带树种,对低温敏感,幼苗过冬成活率低。灰木相思(Acacia implexa Benth.)是较抗寒的品种之一,属速生短轮伐期多用途树种,其材质光滑、平整,耐腐蚀,可改良土壤提高地力,具良好的经济、生态效益,是近年福建省大力引进推广的树种之一。目前,该类树种主要靠引进种子来繁殖,无论是树形、生长量还是抗逆性等都受种源影响,出现严重的性状分离现象,上述局限性限制了灰木相思造林推广速度及引种区域的扩大,影响了这一优良树种的开发利用。本课题选用灰木相思作为研究材料,针对以上问题设计实验方案。通过组织培养技术建立灰木相思优树离体培养再生体系,期望能为快速繁育出具稳定优良性状的种苗提供一条新途径；并运用抗寒性评估技术筛选出抗寒性强和抗寒性弱的品系；通过细胞凋亡检测技术观察抗寒品系在不同低温胁迫后细胞形态上的相应变化；应用蛋白质组学和生物信息学技术,研究经寒胁迫诱导出的表达差异的蛋白,对与寒胁迫相关的特异蛋白进行鉴定分析,探讨其抗寒的分子机制。研究结果如下：1、灰木相思种子种皮坚硬,休眠期长,采用浓硫酸浸泡15 min+沸水浸泡15 min的预处理方式能有效打破休眠,提高种子的发芽率和发芽势。适宜灰木相思优树外植体不定芽诱导的培养基配方为：MS+6-BA 1.0 mg·L-1+NAA 0.2 mg·L-1+ZT 1.0 mg·L-1。高浓度的激素可产生较多不定芽且生长快,但更易促进组培苗玻璃化。通过正交实验筛选出适合灰木相思增殖继代的培养基：MS(总氮含量40 mmol·L-1,NH4+:NO3-=1:2)+6-BA 1.0 mg·L-1+NAA 0.05 mg·L-1,该配方不但大幅度减少了玻璃化苗的发生,而且提高了增殖倍数。将继代增殖的健壮茎段接入改良1/2MS+NAA 0.1 mg·L-1生根培养基中,生根率可达93％,而较高浓度的NAA则有明显的抑制作用。将生根试管苗移栽于基质为干净的黄心土：河沙=3：2的塑料大棚苗床中,成活率可达70%以上。2、对3个优树和9个种子苗共12无性系进行抗寒性评估,通过不同低温胁迫过程中不同无性系的细胞质膜相对电导率、可溶性糖含量及游离脯氨酸含量的变化等生理指标,并结合Logistic方程计算出12个无性系的半致死温度,筛选出其中ZH-01最抗寒半致死温度为-6.11℃,而ZH-73最不抗寒,半致死温度为-0.93℃。发现抗寒性强的品系比寒敏感品系能积累更多的游离脯氨酸和可溶性糖,含量上升幅度也更大,而细胞质膜的相对电导率则变化相对更小。说明在外界胁迫时植物体的生理反应越快,其适应能力就越强。3、运用细胞核显微观察、DNA电泳、TUNEL检测等技术,从细胞分子生物学水平探讨寒胁迫诱导灰木相思的细胞凋亡情况。灰木相思细胞如水稻、烟草等细胞一样都未出现明显的DNA ladder;但处理组在TUNEL检测中呈阳性信号,且细胞核形态也出现了凋亡特征,都说明有细胞凋亡发生,在处理组中还发现线粒体和叶绿体的DNA比核DNA先发生断裂,所以推断灰木相思细胞凋亡的表现顺序可能是：叶绿体和线粒体中的DNA断裂→细胞核中的DNA断裂→染色质浓缩—→核变形。研究结果说明中等强度的寒胁迫能诱导灰木相思的细胞凋亡,当胁迫强度超过植物体忍耐限度时,将导致植物体死亡。4、建立适合于灰木相思总蛋白质分离的稳定高效、重复性好的双向电泳体系：蛋白裂解液为7 mmol/L Urea,2 mmol/L Thiourea,4%CHAPS,40 mmol/L Trisbase,65 mmol/L DTT;蛋白上样量为1000μg/24cmIPG胶条；第二向垂直电泳SDS-PAGE浓度为12.5%；选用考马斯亮兰染色,可以兼顾蛋白质分离和质谱分析鉴定的效果。5、分别选用pH 3-10、pH 4-7、pH 7-11的三种不同pH范围的IPG胶条,对经寒胁迫诱导的抗寒品系ZH-01和寒敏感品系ZH-73的总蛋白进行分离。经ImageMaster软件对两组双向电泳图谱进行分析,共选取38个特异蛋白。抗寒品系ZH-01蛋白图谱中出现的差异蛋白点,表达上调12个,下调6个；胁迫后新增4个,消失1个；寒敏感品系ZH-73经寒胁迫诱导出现的差异蛋白点,表达上调9个,表达下调2个,胁迫后新增3个,胁迫后消失1个。这些特异蛋白进行质谱分析及数据库鉴定后,其中已知蛋白大致可以分为5类：光合作用相关蛋白、基因调控相关蛋白、代谢相关蛋白、耐逆相关蛋白以及其它未分类的蛋白。这说明灰木相思的抗寒机制不是关于个别反应或少数蛋白表达的结果,而是关于植物光合反应系统、膜系统稳定性、基因的转录、翻译过程、糖、蛋白等能量代谢、激素分泌等多方面共同作用的结果。研究结果初步说明：在植物体可忍耐范围内的中低强度的胁迫,能激活植物体的生理代谢、基因调控及主动发生细胞凋亡等一系列反应来提高机体的抗逆性,这时造成的损伤是可逆的,机体可自行修复；如果逆境胁迫超过植物体可忍耐的某一阈值,造成的损伤将是不可逆的,最终导致植物体受害严重甚至死亡。对外界的胁迫抗逆性越强的品系做出的一系列抗逆反应越快,其适应能力也越强。植物体的抗逆性的提高不是某个方面的原因,而是机体内光合反应系统、生理生化反应、能量代谢、基因调控,蛋白表达等多方面共同作用的结果。更多还原

【Abstract】 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.更多还原