【作者】 王楠；

【导师】 向凤宁；

【作者基本信息】 山东大学 ， 细胞生物学， 2018， 博士

【摘要】 水资源短缺及土壤盐渍化是全球性的生态问题,是限制植物生长发育以及造成农作物减产的重要原因,对农业生产产生了极大的负面影响。迄今为止,全球已有约7.6%的土壤发生了盐渍化,43%的土壤处于干旱、半干旱状态。我国的盐碱地的面积为0.27亿hm2,约占我国耕地总面积的10%,并且仍呈逐年递增的趋势,而干旱作为我国最为严重的气候灾害正严重地危害着我国的粮食和生态安全。随着人口的增加、耕地面积的日益减少,开发和利用盐渍化和干旱土地显得尤为重要。大豆(Glycine max(L.)Merill)的种子中含有丰富的蛋白、高品质的植物油、矿物质和维生素等,是一种重要的可食用经济作物。1984年,Lauchli等根据高盐条件下植物盐胁迫耐受性分级,将大豆划分为盐敏感的甜土作物,同时发现大豆的产量易受干旱等气候灾害的影响。因此,开展大豆耐盐、抗旱分子机理研究,鉴定出相关的抗逆基因具有重要的意义。转录因子(Transcription factor,TF)能直接调控植物体内抗逆基因表达,参与多种生理生化过程,从而提高植物的综合抗逆性。目前,已发现大豆基因组中约12%的基因是转录因子家族成员,其中NAC转录因子家族与MYB转录因子家族是植物中两个较大的转录因子家族。近年来,在模式植物拟南芥中研究发现,NAC与MYB转录因子家族成员在非生物胁迫应答中发挥的重要作用。大豆中有226个基因是NAC转录因子家族成员,206个基因是MYB转录因子家族成员。但由于大豆基因组庞大且复杂,有关NAC与MYB转录因子家族基因在盐及干旱胁迫应答中的研究非常有限。膜结合转录因子(membrane-bound transcription factors,MTFs)含有一段跨膜区,可直接整合在细胞内的膜结构上(如细胞质膜,内质网膜等)。当植物发育至特定的生长时期,或受到外界环境变化刺激后,膜结合转录因子便从膜上释放,转运到细胞核内行使功能。本实验室前期通过对盐及非盐条件下大豆耐盐品种圣豆9号的表达谱分析,从中分离、克隆到了一个NAC转录因子基因GmSIN1与一个MYB转录因子基因GmMYB84,对这两个基因进行了生物学特征分析并对其进行了耐逆性的初步研究。另一方面,前期通过对大豆全基因组的搜索和鉴定,分离克隆到15个GmNTLs基因并对其序列特征、表达特征进行了初步分析。本论文基于实验室前期的研究结果,以35Spro:GmSIN1,35Spro:GmSIN1 RNAi转基因大豆株系为实验材料,进一步对GmSIN1参与大豆盐胁迫应答的分子机制进行研究,确定了 GmSIN1在盐胁迫应答中直接结合的下游靶基因。同时,以35Spro:GmMYB84转基因大豆为实验材料,探究GmMYB84是如何通过调控活性氧的快速产生和清除进而参与大豆干旱胁迫应答的。另外,研究了GmNTLs家族的基因特性和功能,并对其家族基因的膜释放机制进行探究。主要研究结果包括:1)大豆GmSIN1调控耐盐性的作用机制研究:通过比较盐地、非盐地中大豆产量,发现相较于对照组,35Spro:GmSIN1转基因大豆株系获得了更高的产量(增产 15-19%)。利用 Southern blot 鉴定出35Spro:GmSIN1 T4 代纯系及35Spro:GmSIN1 RNAi阳性株系,用于进行后续表型实验。首先,比较盐、非盐条件下大豆主根相对伸长量,发现相较于野生型,35Spro:GmSIN1主根相对伸长量较长,而35Spro:GmSIN1 RNAi株系的根伸长量更短。比较野生型与35SpNro:GmSIN1转基因株系在盐及非盐下的RNA-seq表达谱,发现GmSIN1参与调控ABA及ROS生物合成途径中相关基因的表达。同时,EMSA、Chip等实验证明GmST1可以直接结合在ABA生物合成相关基因GmNCED3-1/-2、H2O2生物合成相关基因GmRBOHB-1/-2/-3启动子上游的顺式作用元件上调节其表达。同时,对RNA-seq筛选到的GmSIN1可能调节的靶基因进行启动子MEME分析,鉴定出了 GmSIN1特异性识别的新的顺式作用元件,并命名为SIN1BM。研究结果表明,GmSIN1是大豆盐胁迫响应的重要转录因子之一,以ABA依赖的方式介导盐胁迫转录组的早期响应,其通过直接调控多种下游基因进而调节ABA等的激素水平和ROS产生,提高大豆的耐盐性。2)大豆膜结合转录因子GmNTLs家族成员的基因序列特征及功能研究:通过对大豆全基因组进行搜索和鉴定,找到15个大豆中的NAC膜结合转录因子家族成员,分别命名为GmNTL1-GmNTL15。对这15个GmNTLs基因进行克隆、分别构建N-端GFP标签的瞬时表达载体,转化拟南芥原生质体,研究其亚细胞定位模式;连入pDest32载体分析其转录激活活性。同时,分析了它们的组织特异性表达及非生物胁迫下的表达模式。利用拟南芥原生质体的亚细胞定位发现,这15个GmNTLs均定位在内质网膜上。同时,利用拟南芥原生质体分析了GmNTL1、GmNTL2、GmNTL3、GmNTL4 和 GmNTL10的膜释放模式,发现GmNTL1、GmNTL2、GmNTL3、GmNTL4 受 H2O2诱导释放:GmNTL1、GmNTL3、GmNTL4、GmNTL10 受冷胁迫诱导释放。对p35S:GmNTL1、p35S:GmNTL11、p35S:GmNTL1-ΔC、p35S:GmNTL11-ΔC转基因拟南芥抗逆性分析发现,相较于野生型,p35S:GmNTL1、p35S:GmNTL11、p35S:GmNTL1-ΔC、p35S:GmNTL11-ΔC对NaCl、ABA、mannitol和H2O2的敏感性显著降低。3)大豆GmMYB84在调控耐旱性中的作用研究:干旱胁迫条件下,转基因大豆植株的存活率、根伸长、叶片含水量均优于对照组植株。非生物胁迫表达模式分析发现,GmMYB84受多种非生物胁迫诱导表达,其中受干旱诱导表达最为显著。组织特异性表达分析发现,GmMYB84主要表达部位集中在大豆根、叶中。EMSA、LUC实验结果表明:大豆GmMYB84直接结合在ROS生成基因GmRBOHB1/2启动子上游的顺式作用元件上,上调其表达,使得过表达植株相较于野生型在早期干旱胁迫下获得了更多的H2O2积累,这正是35Spro:GmMYB84植株在干旱胁迫早期表现出较强抗旱性的主要原因。随着干旱胁迫时间的增加,35Spro:GmMYB84植株快速上调过氧化物酶生成相关基因(CAT1,CAT2,CAT3,FeSOD1,FeSOD2,FeSOD3,Cu/ZnSOD1,Cu/ZnSOD2,Cu/ZnSOD3,At5g24070,At4g25980,At5g1 9890)表达,启动体内H2O2清除机制,避免过度积累的H2O2对植株产生氧化胁迫伤害。正是GmMYB84调节的这种ROS快速产生、清除的机制使得35Spro:GmMYB84转基因大豆获得了更高的干旱胁迫耐受性。研究结果,证明了 R2R3-MYB类型转录因子基因GmMYB84在干旱胁迫耐受性中的重要作用,并揭示了其抗旱的分子机理。更多还原

【Abstract】 Water scarcity and soil salinization are global ecological problems,which have a tremendous negative impact on agricultural production via limiting the growth and development of plants.So far,about 7.6%of the world’s soil has salinization and 43%of the soil in the arid or semi-arid state.The area of saline-alkali land in China is 27 million hm2,accounting for about 10%of the total cultivated land area,and is still increasing year by year.Moreover,drought,the most serious climate disaster in our country,is seriously endangering China’s food and ecological security.As the population increases,the area of arable land declines day by day and it is particularly important to develop and utilize salinized and arid lands.Application of genetic engineering to cultivate new varieties of salt-tolerant and drought-resistant crops is an important way of using salt and dry land.Soybean(Glycine max(L.)Merill)is an important edible cash crop,which is rich in protein,high-quality vegetable oils,minerals,and vitamins.In 1984,Lauchli et al.utilized the classification of plant salt stress tolerance under high salt conditions and found that soybean was a salt-sensitive sweet soil crop.At the same time,the study found that the soybean yield was easily affected by climate disasters such as drought.Therefore,molecular mechanism study of salt tolerance,drought resistance,identification of the relevant genes is of great significance.Transcription factors(TFs)can directly regulate the expression of stress-resistant genes in plants and participate in a variety of physiological and biochemical processes,thereby enhancing the plant’s comprehensive resistance.Currently,about 12%of the genes in the soybean genome have been found to be members of the transcription factor family.NAC transcription factor family and the MYB transcription factor family are two large families of transcription factors in plants.In recent years,studies in the Arabidopsis have found that NAC and MYB transcription factors play important roles in abiotic stress response.There are 226 NAC transcription factors and 206 MYB transcription factors in soybean.However,due to the large and complex genome of soybean,there are very limited researchers on the response of NAC and MYB transcription factors to salt and drought stress.Membrane-bound transcription factors(MTFs)contain a transmembrane domain that can be directly integrated into the intracellular membrane structure(eg,the plasma membrane,the endoplasmic reticulum,etc.).When plants develop to a specific growth stage or stimulated by changes in the external environment,the membrane-bound transcription factors are released from the membrane and transported to the nucleus.Up to date,no studies have been reported on the evolution and function of soybean NAC membrane-bound transcription factors(GmNTLs).In our lab,a NAC transcription factor gene GmSIN1 and an MYB transcription factor gene GmMYB84 were cloned based on expression profile analysis of soybean salt-tolerant variety Sheng Dou 9 under salt and non-salt conditions.The biological characteristics of these two genes were analyzed and the preliminary study on their resistance was carried out.On the other hand,15 GmNTLs were isolated and cloned by searching and identifying the whole genome of soybean in the early stage and their sequence characteristics and expression characteristics were analyzed preliminarily.Based on the above results,in this study,the molecular mechanism of GmSIN1 in response to soybean salt stress was further studied and how GmMYB84 participated in the response to drought stress of soybean was explored.In addition,the evolution and function of the GmNTLs family were identified and the membrane release mechanisms of their family genes were explored.The main results are as follows:1)35Spro:GmSIN1 transgenic soybeans exhibited significantly increased salt tolerance:By comparing soybean yields in saline and non-salt soybeans,35Spro:GmSIN1 transgenic soybean lines were found to achieve higher yields than the control.The important role of GmSINl in soybean salt tolerance was confirmed by overexpressing soybean field data.By comparing the relative elongation of soybean root under salt and non-salt conditions,it was found that the relative elongation of 35Spro:GmSINl was larger than that of wild-type,while the root elongation of 35Spro:GmSIN1 RNAi was smaller.By comparing the RNA-seq expression profiles of wild-type and 35Spro:GmSIN1 transgenic lines under salt and non-salinity,we found that GmSIN1 was involved in the regulation of ABA and ROS biosynthesis-related genes.Meanwhile,EMSA,ChIP and other experiments proved that GmST1 can directly bind to the cis-acting element upstream of GmNCED3-1/-2 and GmRBOHB-1/-2/-3.Promoter-MEME analysis of target genes regulated by GmST1 in RNA-seq was screened,and a new specifical GmSIN1 binding cis-acting element was identified and named as SIN IBM.The results showed that GmSIN1 was one of the most important transcription factors in response to salt stress,and mediated the early response of salt stress transcriptome in ABA-dependent manner.By directly regulating a variety of downstream genes,GmSIN1 balanced hormone levels such as ABA and ROS production and improved the salt tolerance of soybean.2)By searching and identifying the whole genome of soybean,15 NAC membrane-bound transcription factors in soybean were found.The 15 GmNTLs genes were cloned and analyzed for their tissue-specific and abiotic stress response patterns.Using subcellular localization in Arabidopsis thaliana protoplasts,all 15 GmNTLs were identified to locate on the ER.At the same time,the membrane release patterns of GmNTL1,GmNTL2,GmNTL3,GmNTL4 and GmNTL10 were analyzed using protoplasts of Arabidopsis thaliana.The Membrane release of GmNTL1,GmNTL2,GmNTL3,and GmNTL4 were triggered by H2O2.The Membrane release of GmNTL1,GmNTL3,GmNTL4,and GmNTL10 were triggered by cold.The p35S:GmNTL1,p35S:GmNTL11,p35S:GmNTL1-ΔC,p35S:GmNTL11-ΔC transgenic lines were constructed.Compared to the wild type,p35S:GmNTL1,p35S:GmNTL11,p35S:GmNTL1-ΔC,p35S:GmNTL11-ΔC exhibited significantly decreased sensitivity to NaCl,ABA,mannitol,and H2O2.3)The drought-resistant ability of 35Spro:GmMYB84 transgenic soybean lines was significantly enhanced.In soybean,the GmMYB84 transcription factor bind directly to the cis-acting element upstream of the GmRbohBl/2 up-regulates their expression,resulting in more accumulation of H2O2 in over-expressing plants compared to the wild-type under drought stress.This is the main cause for 35Spro:GmMYB84 plants showing strong drought resistance in early drought stress.With the increase of drought stress time,35Spro:GmMYB84 plants rapidly up-regulated the expression of peroxidase-related genes and activated the H2O2 scavenging mechanism in vivo to avoid oxidative stress injury caused by excessive accumulation of H2O2.Highly efficient ROS generation and scavenging were important for plant drought stress tolerance.The results demonstrated that GmMYB84,an R2R3-MYB transcription factor gene,played an important role in drought stress tolerance via ROS generation and scavenging.更多还原