【作者】 杨勇；

【导师】 蒋德安；

【作者基本信息】 浙江大学 ， 植物学， 2007， 博士

【摘要】 盐胁迫是严重影响植物生长,导致农作物减产的主要非生物因子之一。为了研究植物的耐盐机制及功能,本文通过不对称体细胞杂交技术,将野生大豆(Glycine cyrioloba ACC547)的耐盐特性引入到了栽培大豆(G.max Melrose)中,综合分析比较了亲本及其体细胞杂交后代的耐盐特性,利用这些材料筛选到了野生大豆特异的ndhH基因并研究了该基因及其生理功能在大豆盐胁迫耐性中的可能作用。主要结果如下:Glycine cyrtoloba ACC547是生长在澳大利亚海滩上的一种野生大豆,具有较强的耐盐性能;G.max Melrose是一个高产栽培大豆品种,但对盐胁迫较为敏感。利用不对称体细胞杂交技术,获得了一批ACC547和Melrose的原生质体融合再生株系。随机扩增多态性DNA(randomamplified polymorphic DNA,RAPD)和扩增片段长度多态性(amplified fragment lengthpolymorphism,AFLP)分析结果都表明这些再生株系为野生大豆和栽培大豆的体细胞杂交后代。这些杂交后代在表型上获得了两个亲本的一些特征,耐盐性实验表明有些杂交后代也从野生大豆获得了较高的耐盐特性。对两个稳定遗传的体细胞杂交后代S111-9和S113-6及其亲本的耐盐性鉴定表明,与栽培大豆相比,杂交后代及其野生大豆亲本具有较强的耐盐性能。盐胁迫严重地影响了栽培大豆的生长,表现为叶片出现失绿坏死,生物量和光合速率严重下降;而盐胁迫下两个杂交后代的叶片失绿坏死程度和生物量的降低都比其栽培种亲本轻,但要重于其野生亲本,然而其光合速率的下降程度与野生大豆无显著的差别,这表明体细胞杂交后代部分地获得了其野生大豆亲本的耐盐性能。在盐处理后期,野生大豆亲本根茎中的Na~+和CI含量与栽培大豆差异不大,但叶中这两种离子的含量明显少于栽培大豆,而K~+含量和K~+/Na~+比值要远大于栽培大豆。这表明降低植株体内尤其是叶片中的盐离子含量,增强叶片对K~+的选择性吸收,减轻盐分对叶片的伤害,维持较高的光合性能,是野生大豆具有较高耐盐特性的部分原因。尽管体细胞杂交后代的叶片K~+含量和K~+/Na~+明显高于栽培大豆,但其叶中和整个植株体内的Na~+和Cl~-含量都要远大于其野生大豆亲本,而与其栽培亲本无显著差别。因此,同野生大豆相比,杂交后代较高的盐胁迫适应能力可能主要不在于其减少体内盐离子的能力,而在于其对盐胁迫的耐受性。在体细胞杂交后代从野生大豆亲本获得的AFLP条带中,发现有一个AFLP条带(ASH1)与叶绿体NAD(P)H脱氢酶(NAD(P)H dehydrogenase,NDH)H亚基的基因ndhH具有很大的同源性。通过设计ASH1特异引物进行PCR鉴定,证明了ASH1代表了野生大豆亲本特异的ndhH基因并通过PCR扩增和DNA测序获得了其全长序列。野生大豆特异的ndhH与栽培大豆的ndhH的比对结果表明,二者在其所推测蛋白的氨基酸序列上只有五个氨基酸残基的差别,野生大豆特异的NDH-H蛋白在三级构型上于相应的位置缺少了一个β转角。与栽培大豆相比,盐胁迫诱导了野生大豆亲本类囊体膜结合的约45kD蛋白的合成,其在分子量上与野生大豆亲本特异的NDH-H类似。盐胁迫下野生大豆亲本的‘作用光关闭后叶绿素荧光瞬时上升’的程度和P700的重还原速率以及利用不同电子受体和供体所测定的PSI活性,都要远高于栽培大豆亲本,这表明盐胁迫下野生大豆的NDH介导的围绕PSI的环式电子传递(cyclic electron flow around PSI,CEF1)速率要高于栽培大豆。基于以上这些结果,我们推测野生大豆亲本特异的ndhH能够受盐胁迫的特异诱导而高丰度表达,其所编码的产物参与组成了一个特异的NDH,介导了盐胁迫下高效的光合环式电子传递。另外我们还发现,由于获得了野生大豆亲本特异的ndhH基因,体细胞杂交后代的CEF1活性也受盐胁迫诱导而显著提高,这可能是杂交后代具有较高耐盐特性的一个重要原因。盐胁迫下,体细胞杂交后代及其野生大豆亲本的叶片毫秒延迟荧光(ms-DLE)强度明显升高,叶绿素荧光的非光化学淬灭(NPQ)尤其是高能态非光化学荧光淬灭(qE)也维持在较高的水平;叶片内活性氧的积累明显少于栽培大豆;叶绿体结构受损较轻,PSⅡ的最大光化学效率(Fv/Fm)下降也较少。而栽培大豆叶片的ms-DLE强度、NPQ和qE在盐处理下显著下降;活性氧的生成明显增强,叶绿体受损较为严重,Fv/Fm明显降低。这些结果表明杂交后代及其野生大豆亲本的CEF1活性有利于其叶片跨类囊体膜的pH梯度(△pH)的生成和维持,从而激发了其叶片内过剩光能的耗散机制来保护光合机构免受活性氧的损害;而由于不具备高效的CEF1活性,栽培大豆的这种保护机制较弱。此外,杂交后代及其野生大豆亲本盐胁迫下叶片中光诱导的ATP合成明显高于栽培大豆,表明野生大豆及其杂交后代较高的CEF1活性推动了ATP的积累。我们还发现野生大豆的Na~+大多定位于液泡,这是否预示着这种ATP的积累参与了Na~+的液泡区域化,尚待进一步研究。更多还原

【Abstract】 Salt stress is one of the major abiotic stresses that depress plants growth and drastically limit crop production.To study the mechanisms for plant tolerance to salt stress,salinity-resistant traits of a wild soybean species Glycine cyrtoloba ACC547 were introduced to a cultivated soybean G.max Melrose using asymmetric somatic hybridization.The capacity for salt tolerance was compared in the parents and their hybrid descendants,and a wild-soybean-special-ndhH gene was discovered with these materials and the possible function of this gene in the tolerance of soybean to salt stress was studied.The main results obtained were shown as follow:Glycine cyrtoloba ACC547,with high tolerance to salinity,is a wild soybean species native to saline soils in Australian beach.G.max Melrose is a high-yielding soybean cultivar but susceptible to salinity.A series of regenerated lines were obtained from the fused protoplast using asymmetric somatic hybridization.RAPD(random amplified polymorphic DNA)and AFLP(amplified fragment length polymorphism)analysis revealed that these regenerated plants were hybrids between ACC547 and Melrose.The hybrid descendants possessed several morphological features of their parents and some of them also possessed higher salinity-tolerant traits of the wild soybean detecting with salinity-tolerance experiments.It was shown that two of the stable hybrid descendants(S111-9 and S113-6)and their wild parent had higher salinity-tolerance in comparison with the cultivated soybean.Salt stress dramatically depressed the growth of cultivated soybean whose leaves lost green with necrotic lesions,biomass and photosynthetic rate decreased markedly.Both necrotic lesions in leaves and decline in biomass are much lighter in hybrid descendants than in cultivated soybean,but more serious than in wild soybean.However,the decline in photosynthetic rate was not significantly different between hybrid descendants and their wild parent.These results suggested that the descendants inherited partially tolerance to salt stress from the wild species.In the late time of saline treatment,it had no significant difference in root and stem Na~+ and Cl~-concentrations between the wild and cultivated soybean,but the latter owned much higher concentrations of the two ions and much lower K~+ content and K~+/Na~+ ratio in leaves.These results indicated that the partial reason for salt tolerance in wild species was the exclusion of Na~+ and Cl~-from the whole plant and especially from the leaves,the enhancement in K~+ selectivity of leaves,the alleviation in damage of salt to leaves,and the coupled maintenance in photosynthesis.Although possessing higher leaf K~+ content and K~+/Na~+ ratio than their cultivated parent,the descendances showed higher Na~+ and Cl~- contents in both leaves and whole plant than their wild parent with no significant difference from the cultivated soybean.It was suggested that the higher adaptation of the descendances to salt stress might not be due to the capacity for excluding toxic ions from their bodies, but to their tolerance to salinity.One of the AFLP bands which the descendants obtained from their wild soybean parent--ASH1 was found to have highly similar sequence to ndhH,a gene for the H-subunit of chloroplast NAD(P)H dehydrogenase(NDH).PCR analysis with special primers for ASH1 demonstrated that ASH1 represented an ACC547-special ndhH gene,and the whole sequence of this gene was obtained through PCR amplification and DNA sequencing.Multi-alignment of the deduced protein sequences indicated only 5 amino acids in ACC547-special NDH-H different from soybean NDH-H,and the ACC547-special NDH-H lacked oneβcorner in related site in three-dimensional model.In wild soybean,salt stress induced the synthesis of an approximately 45kD thylakoid-membrane-bound protein which was similar to ACC547-special NDH-H in molecular weight.Under salt-stress conditions,the post-illumination transient increase in chlorophyll fluorescence,the rate of p700~+ rereduction,and PSI activity detected with different electron donors and accepters,were all much higher in wild than in cultivated soybean,indicating that NDH-mediated cyclic electron flow around PS I(CEF1)was accelerated by salt stress more greatly in wild than in cultivated soybean.Based on these results,we suspected ACC547-special ndhH could be induced by salinity to express with high level,and its product might be a part of a special NDH complex which mediated efficient CEF1 under salt stress.Moreover,salt stress also prompted CEF1 activity in the descendants due to its derivation of ndhH gene from their wild parent,which might make great contributions to the high salinity-tolerance of the descendants.Under salt tress,Ms-delayed light emission(ms-DLE)was markedly strengthened, non-photochemical quenching of chlorophyll fluorescence(NPQ),particularly high-energy state non-photochemical quenching(qE)were still maintained at a high level,less reactive oxygen species (ROS)was generated in the leaves,chloroplast remained the integrated ultrastructure,and there was higher PSⅡmaximal photochemical efficiency(Fv/Fm)in the descendants and their wild parent. Comparatively,salt treatment significantly decreased ms-DLE,NPQ,qE and Fv/Fm,enhanced the accumulation of ROS,and disrupted the chloroplast structure in Melrose.These results suggested that the higher CEF1 activity via ACC547-special NDH in the wild and hybrid soybeans contributed to the generation and maintenance of proton gradient across thylakoid membranes(△pH)and hence to the efficient thermal dissipation of excess light energy,which protected photosynthetic apparatus from ROS damage.However,this protective mechanism functioned less due to the absence of the efficient CEF1 in cultivated soybean.In addition,light-induced ATP synthesis in leaves was higher in the descendants and their wild parent than in their cultivated parent under salt stress,which indicated that the higher CEF1 activity accelerated the generation of extra ATP in the wild and hybrid soybeans.We also discovered that most of Na~+ were localized in vacuoles in wild soybean,and further study was required to elucidate whether the extra ATP participated the compartmentation of Na~+ into vacuoles.更多还原