【作者】 孙红梅；

【导师】 焦锋；

【作者基本信息】 西北农林科技大学 ， 特种经济动物饲养， 2020， 博士

【摘要】 桑树(Morus alba)根系发达,枝繁叶茂,生命力旺盛,具有较强的环境适应性,因此在世界范围内分布广泛。在我国,桑树作为生态防护林选用树种,可起到防沙固土,减少地表径流,保持原生态土壤结构的作用。另一方面,因桑树生长迅速,地上部分生物量大,被应用到自然生态修复系统中,例如修复被重金属污染的土壤、多石的贫瘠荒漠以及戈壁沙漠化区域等。桑树具有顽强的生存能力,源于其所拥有的独特生理特性,近年来在植物遗传学应用领域备受关注,涉及桑树抗逆基因资源挖掘及开发利用等研究。本研究从桑树中克隆得到3个干旱差异表达基因:叶绿体干旱胁迫诱导蛋白编码基因(chloroplast drought-induced stress protein of 32 k Da,Ma CDSP32),IAA-氨基酸水解酶编码基因(ILR1-5(IAA-amino acid hydrolase ILR1-like 5,Ma ILR1)和抗坏血酸过氧化物酶(ascorbate peroxidase,Ma APX2))进行基因克隆、表达分析及基因功能分析,通过对目标序列的生物信息学分析、表达特性分析、转基因植株的抗逆性分析以及在干旱胁迫下的转录组数据分析,实现对目标基因在植物响应环境胁迫条件下分子功能的探索,为在未来以生物技术工程为手段改善植物的环境适应性和在实际生产中发挥桑树品种质资源优势等工作铺垫基石。本研究取得的主要结果如下:1.三个基因的克隆及亚细胞定位桑树中Ma CDSP32、Ma ILR1和Ma APX2基因的编码区长度分别为909 bp、1302 bp和750bp,分别编码303、434和250个氨基酸。预测结果显示,三个编码蛋白的亲水性和稳定性良好。Ma CDSP32编码的蛋白质定位在叶绿体基质中;Ma APX2编码的蛋白酶定位在细胞质内,主要富集在细胞膜和细胞核附近;Ma ILR1编码一个膜蛋白,为分泌型蛋白,定位在细胞膜上。2.三个基因在不同生长条件下的表达模式Ma CDSP32基因主要在桑树成熟叶片中表达,在桑树不同种间具有表达偏好性,但与基因组倍性无关;一天内,Ma CDSP32在15:00时达到表达高峰,呈生物钟节律表达规律;非生物胁迫和外源激素可诱导Ma CDSP32表达水平发生变化,但在叶片和根中表达模式不同。Ma ILR1基因主要在幼叶中表达,在桑树不同种间具有表达偏好性,但与基因组倍性无关;Ma ILR1无明显生物钟表达规律;非生物胁迫和外源激素可诱导Ma ILR1表达水平发生变化,但在叶片和根中表达模式不同。Ma APX2基因在地上部分表达量一致,在桑树不同种间具有表达偏好性,但与基因组倍性无关;一天内,Ma APX2在12:00时达到表达高峰,呈生物钟节律表达规律;非生物胁迫和外源激素可诱导Ma APX2表达水平发生变化,但在叶片和根中表达模式不同。3.Ma CDSP32瞬时表达桑叶的基因功能研究瞬时过表达Ma CDSP32的离体桑叶相比于未转化桑叶,在自然晾干过程中失水量增加。PEG模拟的水分胁迫下,桑叶中Ma MRSB、Ma P5CS、Ma PRX、Makds A、Ma DREB和Ma MAPK基因的表达水平以及ROS的积累量受Ma CDSP32过表达影响发生改变;盐胁迫下,桑叶中Ma WRKY、Makds A、Ma DREB和Ma MAPK基因的表达水平也受到Ma CDSP32过表达影响发生改变。这些结果表明Ma CDSP32会影响胁迫相关基因的表达水平,并参与植物非生物胁迫应答过程。4.Ma CDSP32在转基因烟草中的基因功能研究Ma CDSP32稳定转化烟草获得的转基因株系(OE-2和OE-7)与野生型烟草相比,在自然晾干过程其离体叶片失水量增加。转基因烟草对干旱敏感性增加,叶片中ROS积累和MDA含量增加,多种抗氧化酶活性降低,脯氨酸和可溶性糖积累减少;但复水后转基因植株的生长恢复能力增强,ROS积累减少,MDA含量减少,多种抗氧化酶活性增强,脯氨酸和可溶性糖积累增加。胁迫期间,转基因植株中Nt CAT、Nt ADC2、Nt LEA5和Nt MSRB基因的表达水平相对野生型发生改变。此外,Ma CDSP32通过促进转基因烟草种子和幼苗中Nt MSRB表达上调,减少ROS积累,显著增加了种子在渗透胁迫下的萌发率和幼苗生长。在盐胁迫下,叶绿素含量在Ma CDSP32转基因植株中增加,而在野生型植株中减少。这些结果揭示Ma CDSP32主要是通过参与氧化还原途径调节植物的抗逆性。5.Ma CDSP32在转基因拟南芥中的基因功能研究Ma CDSP32转基因拟南芥株系(OE-3和OE-9)与野生型拟南芥和At CDSP32突变拟南芥株系(mt-1和mt-2)相比,在干旱胁迫期间OE株系萎蔫较严重,ROS积累和MDA含量较多,SOD、POD和APX抗氧化酶活性较低。但OE株系相比野生型和突变体株系,在复水后成活率较高。在干旱和盐胁迫下,植株中不同Ma CDSP32或At CDSP32表达水平会影响At SOD、At POD、At CAT、At PRX、At APX2和At GR基因的表达水平。盐胁迫处理后,突变体株系的叶绿素含量相比野生型和OE株系降低。在渗透胁迫下OE拟南芥种子的萌发率较野生型和突变体种子显著增加。此外,OE拟南芥株系出现早花现象,其中At SOC1基因表达较野生型和突变体显著上调。这些结果表明Ma CDSP32主要是通过参与逆境应答的氧化还原途径影响植物的逆境生理和种子萌发过程,且Ma CDSP32通过影响At SOC1基因表达参与植物开花时间调节。6.Ma CDSP32过表达烟草干旱胁迫转录组学分析转基因烟草的转录组数据分析显示,在干旱处理期间,OE与WT株系差异表达基因主要集中在催化活性、核酸结合转录、转录因子活性、光合作用相关膜组件、糖类代谢进程和肽链内切酶调节因子酶活性等生理过程方面;复水后,OE与WT株系差异表达基因主要集中在高分子生物合成、氮化合物生物合成、催化活性和水解酶活性等生理过程方面。进一步分析发现,这些差异表达基因在苯丙素的生物合成、光合作用-触角蛋白、角质素,软木脂和蜡质的生物合成、核糖体和光合系统的固碳作用等通路中上调基因最多;这些差异表达基因在内质网蛋白加工、半乳糖代谢、倍半萜和三萜生物合成和脂肪酸延伸等通路中下调基因最多。此外,与WT株系相比,OE株系中在半胱氨酸和甲硫氨酸代谢途径、淀粉和糖类代谢途径、油菜素类固醇代谢途径、植物激素信号传导代谢途径和光合元件固碳作用代谢途径,以及抗氧化酶活性、脯氨酸合成代谢和糖合成代谢等方面所涉及的差异表达基因均出现干旱期间下调而复水后上调的变化趋势。推测这些差异表达基因是Ma CDSP32基因在干旱期间和复水后参与调节植物抗旱性相互作用的关键位点。综上,本研究得到的主要结果表明,桑树中的干旱持续表达基因Ma CDSP32通过参与植物抗逆过程中的ROS代谢调节过程,增强了植株的抗旱性,揭示了Ma CDSP32参与提高植物旱后恢复能力的分子机制。这项研究为桑树品种质资源优势的开发利用筛选了基因资源,为以分子生物技术手段改善植物的环境适应性和生存能力提供了新思路。更多还原

【Abstract】 Mulberry(Morus alba)is highly adaptable to environment and widely distributed all over the world,since they have developed roots,luxuriant foliage and vigorous vitality.In China,mulberry is selected as an ecological protection forest tree species to effectively prevent sand and soil,reduce surface runoff and maintain the original ecological soil structure.On the other hand,mulberry,due to its rapid growth and large abovementioned biomass,is used in a variety of natural ecological restoration systems,such as the remediation of soils contaminated by heavy metals,stony barren deserts and desertification areas.The indomitable survival ability of mulberry is due to its unique physiological characteristics,which has attracted much attention in the field of plant genetics in recent years,involving in the research on the exploration,development and utilization of the resistance gene resources in mulberry.In this study,three drought differentially expressed genes were cloned from mulberry leaves:(chloroplast drought-induced stress protein of 32 k Da,Ma CDSP32,IAA-amino acid hydrolase ILR1-like 5,Ma ILR1 and ascorbate peroxidase,Ma APX2)in mulberry.We have used bioinformatics analysis,expression characteristics analysis,transgenic plants stress tolerance analysis and transcriptome data analysis,mainly to explore the molecular functions of Ma CDSP32 in response to drought stress,and to pave the way for improving the environmental adaptability of plants by biotechnology engineering in the future and taking advantage of mulberry germplasm resources in actual production cornerstone.The main results of this study are as follows: 1.Gene cloning and subcellular localization.The encoding sequences length of Ma CDSP32,Ma ILR1 and Ma APX2 genes from mulberry were 909,1302 and 750 bp,encoding 303,434 and 250 amino acids,respectively.All the three proteins are predicted hydrophilic and stable.The protein encoded by Ma CDSP32 was located in the chloroplast stroma.Ma APX2 encodes a protease that is located in the cytoplasm,and mainly enriched near the membrane and nucleus;Ma ILR1 encodes a membrane protein,it’s a secreted protein and is located on the cell membrane.2.The genes expression pattern under different conditions in mulberry.The Ma CDSP32 was mainly expressed in mature leaves of mulberry,and showed expression preference among different mulberry cultivars while has nothing to do with the plant ploidy.Ma CDSP32 reached its peak expression at 15:00 within one day,presenting a circadian rhythm expression pattern.The Ma CDSP32 expression level changed with abiotic stress and exogenous hormones treatment,but they were different in leaves and roots.The Ma ILR1 was mainly expressed in young leaves;it showed expression preference among different mulberry cultivars while has nothing to do with the plant ploidy.Ma ILR1 expression had no obvious circadian rhythm.The Ma ILR1 expression level changed with abiotic stress and exogenous hormones treatment,but they were different in leaves and roots.The Ma APX2 expression level was consistent in the aboveground tissues;it showed expression preference among different mulberry cultivars while has nothing to do with the plant ploidy.Ma APX2 reached its peak expression at 12:00 within one day,presenting a circadian rhythm expression pattern.The Ma ILR1 expression level changed with abiotic stress and exogenous hormones treatment,but they were different in leaves and roots.3.Study on gene function of Ma CDSP32 transiently transforming in mulberry.The Ma CDSP32 transiently overexpressed mulberry leaves showed increased water loss compared with the wild-type mulberry leaves during the natural desiccation.Besides,the gene expression of Ma MRSB,Ma P5 CS,Ma PRX,Makds A,Ma DREB and Ma MAPK,and also the accumulation of ROS were affected by overexpressed Ma CDSP32 under PEG treatment.What’s more,the gene expression of Ma WRKY,Makds A,Ma DREB and Ma MAPK were affected by overexpressed Ma CDSP32 under Na Cl treatment.These results suggested that Ma CDSP32 could affect the expression level of stress-related genes and participate in plant abiotic stress response.4.Study on Ma CDSP32 gene function in transgenic tobacco.Compared with the wild-type tobacco,Ma CDSP32 overexpressed lines(OE-2 and OE-7)showed increased water loss of detached leaves during the natural desiccation process.Transgenic tobacco showed increased sensitivity to drought,the ROS accumulation and MDA content in leaves increased,the activity of various antioxidant enzymes and the contents of proline and soluble sugar decreased compared with the wild-type.However,the recovery ability of transgenic tobacco enhanced after post-drought rewater,the ROS accumulation and MDA content in leaves decreased,the activity of various antioxidant enzymes and the contents of proline and soluble sugar increased compared the wild-type.Moreover,expression levels of Nt CAT,Nt ADC2,Nt LEA5 and Nt MSRB in transgenic lines were also affected by Ma CDSP32 overexpressing under drought stress.Chlorophyll content increased in transgenic tobacco while decreased in wild-type under salt stress.Additional,Ma CDSP32 significantly increased the seed germination rate and seedling growth of transgenic tobacco under osmotic stress by increasing Nt MSRB expression and reducing ROS accumulation.These results suggested that Ma CDSP32 regulates plant stress tolerance mainly by participating in the redox pathway.5.Study on Ma CDSP32 gene function in transgenic Arabidopsis.Compared with the wild-type Arabidopsis plants and the At CDSP32 loss function mutant lines(mt-1 and mt-2),Ma CDSP32-overexpressing Arabidopsis lines(OE-3 and OE-9)showed increased severe wilting phenotype,increased ROS accumulation and MDA content,lower activities of SOD,POD and APX enzymes during drought process.However,the survival rate of OE Arabidopsis plants was the highest compared to that of wild-type and mutant lines after rewatering.The gene expressions of At SOD,At POD,At CAT,At PRX,At APX2,and At GR were affected by overexpressing of Ma CDSP32 or loss function of At CDSP32 under drought and salt treatments.The chlorophyll content of At CDSP32 mutant lines was reduced compared to the wild-type and OE lines under salt stress.The germination rate of OE Arabidopsis seeds significantly increased than the other two genotypes under osmotic stress.In addition,OE Arabidopsis lines showed early flowering and the gene expression of At SOC1 was significantly higher than the other two genotypes.These results indicated that Ma CDSP32 affects the plant’s stress physiology and seed germination rate mainly through the redox pathway involved in the stress response,and Ma CDSP32 participated in plant flowering time regulation by affecting At SOC1 expression.6.Transcriptome analysis in transgenic tobacco of Ma CDSP32 under drought stress.The transcriptome data analysis of transgenic tobacco showed that the drought-differentially expressed genes of OE and wild-type lines were mainly in the physiological processes such as catalytic activity,nucleic acid binding transcription,transcription factor activity,photosynthetic membrane module,carbohydrate metabolism process and peptide chain enzyme regulatory factor,et al.;after rewatering,the differentially expressed genes of OE and wild-type lines were mainly in the physiological processes such as polymer biosynthesis,nitrogen biosynthesis,catalytic activity and hydrolase activityand,et al..Further analysis revealed that these differentially expressed genes were most up-regulated in the pathways of phenylpropanoid biosynthesis,photosynthesis-tentin,keratin,biosynthesis of cork resin and wax,carbon fixation of ribosomes and photosynthetic systems,while were most down-regulated in endoplasmic reticulum protein processing,galactose metabolism,sesquiterpene and triterpene biosynthesis,and fatty acid extension.In addition,the differentially expressed genes of OE lines involving in cysteine and methionine metabolism pathways,starch and sugar metabolism pathways,brassinosteroid metabolism pathways,plant hormone signaling metabolism pathways,and photosynthetic element carbon fixation,as well as antioxidant enzyme activity,proline anabolism,and sugar anabolism were down-regulated during drought stress and up-regulated after rewatering.These results suggested that these differentially expressed genes are likely the key sites for the interaction with Ma CDSP32 in modulating drought tolerance under drought stage and after rewatering.Taken together,the evidences show that the drought sustained expression gene Ma CDSP32 in mulberry enhances plant drought tolerance by participating in the ROS metabolic regulation proces,revealing the molecular mechanism that Ma CDSP32 involved in to improve plant post-drought recovery ability.This research screened genetic resources for the development and utilization of mulberry germplasm resources,and provided new ideas for improving plant environmental adaptability and viability with molecular biotechnology.更多还原