【作者】 何漪；

【导师】 蒋德安；

【作者基本信息】 浙江大学 ， 植物学， 2012， 博士

【摘要】 大豆作为重要的经济作物,是人类植物蛋白和植物油的重要来源。同时,全世界约20%的可耕地和接近一半的灌溉地都受到不同程度的盐碱威胁。因此,揭示大豆在盐胁迫条件下的光合生理机制,提高大豆的抗逆性及其光合能力自然成了这一研究领域的重要问题。叶绿体NAD(P)H脱氢酶复合体(NAD(P)H dehydrogenase (NDH) complex)是起源于藻类,并最早在烟草中被发现的一个蛋白复合体。在蓝藻和高等植物中,NAD(P)H脱氢酶复合体在光下还原PQ并参与围绕PSI的环式电子传递,在逆境条件下为光合作用提供额外的ATP,进而增强植物对逆境环境的适应能力。因此,为了明确NDH复合体在提高大豆盐耐受性中的作用,我们用栽培大豆Melrose及其体细胞杂交后代S111-9作为研究材料,并通过运用两种酵母双杂交技术,研究了NDH复合体各亚基间的互作关系,以及NDH复合体与PSI复合体之间的互作关系。同时,我们还研究了盐胁迫对两种大豆的光合生理特性、NDH复合体介导的CEF1活性、NdhB和NdhH以及Rubisco的含量和亚细胞定位的影响。结果摘要如下：通过运用酵母双杂交技术,我们首次发现了组成NDH复合体的NdhB与4个亲水亚基NdhH、NdhI、NdhJ和NdhK之间,NdhH与NdhI和NdhK之间,NdhD与NdhK之间的互作关系。从而明确了NdhB亚基在NDH复合体结构组成中的核心地位,以及NdhH亚基可能是NDH复合体亲水部分的核心亚基的结论。此外,我们还发现了S111-9中ndhB含有一个由36个氨基酸组成的类囊体转运肽。同时,我们的研究结果还首次表明,PSI复合体的两个核心亚基PsaA(?)PsaB,分别与NdhH、NdhI和NdhJ以及NdhH和NdhI存在互作关系。通过比较Melrose和S111-9在盐胁迫条件下的表型和光合生理特性,我们发现S111-9具有比Melrose更强的盐耐受性。同时,PSI和PSII光化学活性、作用光关闭后叶绿素荧光的瞬时上升和P700＋再还原速率等参数都表明,高的NDH复合体介导的CEF1活性是造成其具有更强的盐耐受性的主要原因。而GFP融合蛋白共表达、NdhB和NdhH的含量和亚细胞定位的结果表明,S111-9中ndhB所含有的类囊体转运肽,可能在其NdhB亚基迁移定位的过程中起到重要的作用,从而造成了S111-9中NDH复合体的的积累,进而使其在盐胁迫条件下具有高的NDH复合体介导的CEF1活性。此外,Rubisco的含量和亚细胞定位的结果也表明,S1l1-9的强盐耐受性可能也与Rubisco的含量和活性存在一定的联系。与此同时,我们还意外地发现盐胁迫会影响大豆叶绿体中淀粉粒的降解和运输。而盐胁迫条件下,Melrose和S111-9的叶片中有效磷含量的变化表明,有效磷含量的变化与叶绿体中淀粉粒的降解和运输存在密切的联系。综上所述,我们的研究结果首次阐明了NDH复合体各亚基间及其与PSI复合体之间的互作关系。同时,进一步明确了NDH复合体介导的CEF1在提高大豆抗耐盐能力中的作用。而S111-9中ndhB所含有的类囊体转运肽的作用,也将为我们更加深入地研究NDH复合体的生理功能提供新的视角。更多还原

【Abstract】 Soybean is an important economic crop, and the main source of plant protein and oil for human being. Meanwhile, about 20% available cultivated land and 1/2 irrigation field have been affected by saline at the different levels in the worldwide. Therefore, studying the photosynthetic physiological mechanisms of soybean under salt stress, and improving the ability of stress tolerance and photosynthesis of soybean will be important topic in this research field.Chloroplast NAD(P)H dehydrogenase (NDH) complex is originated from cyanobacteria, and was first found in tobacco. In cyanobacteria and higher plants, NDH complex reduces PQ in the light and is involved in the cyclic electron flow around PSI (CEF1), supplying extra ATP for photosynthesis and enhancing the fitness to environment, particularly under environmental stress conditions. So, in order to define the roles of NDH complex in improving the stress tolerant of soybean, G. max Melrose (cv.Melrose) and somatic hybrid descendants S111-9 were used in our research. According to the analysis by two type of yeast two hybrid, we study the relationships between all subunits that consist of NDH complex, and the relationships between NDH complex and PSI complex. At the same time, we also study the photosynthetic physiological characteristics, activities of CEF1 induced by NDH, the amounts and subcellular localization of NdhB, NdhH and Rubisco in Melrose and S111-9 under salt stress. And results are as follows:According to yeast two hybrid, we first find that NdhB subunit interacts with four hydrophilic subunits NdhH, NdhI, NdhJ and NdhK respectively, NdhH subunit interacts with NdhI and NdhJ, and NdhD subunit interacts with NdhK. Based on these results, we suggest that NdhB subunit is a core subunit in NDH complex and NdhH subunit may be a core subunit in the hydrophilic part of NDH complex. In addition, we also find that a thylakoid transit peptide of ndhB with 36 amino acids in S111-9. And our results first show that PsaA and PsaB, two core subunits in PSI complex, interact with NdhH, NdhI and NdhJ, and NdhH, NdhI respectively.Compared with the phenotype and photosynthetic physiological characteristics in Melrose and S111-9 under salt stress, we find that S111-9 have a higher salt tolerance than Melrose. Meanwhile, the results of photochemical efficiency of PSI and PSII, postillumination transient increase and re-reduction of P700+, which all show that higher activity of CEF1 induced by NDH is the main reason for having high salt tolerance in S111-9. And the results of GFP fusion protein coexpression, the amounts and subcellular localization of NdhB and NdhH, which also infer that the thylakoid transit peptide of ndhB in S111-9 may play an important role in the translocation and localization of NdhB subunit, and resulting in the accumulation of NDH complex, further improving the activity of CEF1 under salt stress. Besides, the results of the amounts and subcellular localization of Rubisco always suggest that the high salt tolerance of S111-9 may be related to the amounts and activity of Rubisco.Meanwhile, we still accidentally find that salt stress will affect the degradation and transport of starch in soybean’s chloroplast. Under salt stress conditions, the results of the contents of available phosphorous also suggest that there is a close relationship between the Pi concentrations and the degradation and transport of starch in chloroplast.In conclusion, our results first clarify the interaction relationship between the subunits of NDH complex, NDH complex and PSI complex. Moreover, we also confirm that the CEF1 induced by NDH functions in improving the salt tolerance of soybean. And the function of thylakoid transit peptide of ndhB in S111-9 also will give us a new vision for deeply studying the physiological function of NDH complex.更多还原