【作者】 李焱；

【导师】 付凤玲； 于好强；

【作者基本信息】 四川农业大学 ， 生物化学与分子生物学， 2025， 硕士

【摘要】 目前,全球土壤盐渍化地区正在逐渐扩大,严重影响农作物生长发育和产量。因此,研究作物如何响应盐胁迫具有重要意义。胁迫相关蛋白(stress associated protein,SAP)在植物响应逆境胁迫以及生长发育等方面发挥作用。前期研究发现,玉米中存在10个ZmSAPs基因,且受到盐、干旱、ABA等多种胁迫诱导表达,其中,ZmSAP1、ZmSAP2及ZmSAP7可能调控耐盐性。因此,本研究对ZmSAP1/2/7进行生物信息学分析、组织表达模式分析与亚细胞定位鉴定。进一步,通过在拟南芥和水稻中分别进行异源过表达实验,探究ZmSAP1/2/7基因调控耐盐性的功能。主要研究结果如下:(1)ZmSAP1/2/7均属于A20/AN1型锌指蛋白。成功扩增到ZmSAP1、ZmSAP2及ZmSAP7基因,其CDS序列分别长531、486及516 bp,分别编码176、161、171个氨基酸。生物信息学分析表明,ZmSAP1和ZmSAP7的N端包含一个A20结构域,C端包含一个AN1结构域,但ZmSAP2仅具有C端的AN1结构域。(2)ZmSAP1/2/7基因在玉米各组织中均有表达,其编码蛋白均具有核定位特征。实时荧光定量PCR(q RT-PCR)结果表明,ZmSAP1/2/7基因在玉米授粉前的根、气生根、茎、花丝、苞叶、穗轴、雄穗、穗位叶等组织中均有表达,其中,ZmSAP1在穗位叶中表达量最高,在穗轴中表达量最低。ZmSAP2在根中表达量最高,在花丝中表达量最低。ZmSAP7在雄穗中表达量最高,在花丝中表达量最低。亚细胞定位实验结果证实,在烟草与玉米原生质体中,ZmSAP1/2均定位于细胞核,ZmSAP7则定位于细胞核和细胞质。(3)异源表达ZmSAP1/2/7增强转基因拟南芥的耐盐性。分别将ZmSAP1/2/7基因转化野生型拟南芥并进行表型鉴定。结果表明,正常条件下,转基因株系与野生型(wild type,WT)根长与生长状况基本一致,各生理生化指标无显著差异。盐胁迫处理下,与WT相比,ZmSAP1/2/7转基因株系的存活率、根长、生物量、相对含水量均显著增加,而相对电导率、丙二醛含量均显著降低。该结果表明,转化ZmSAP1/2/7基因可增强转基因拟南芥的耐盐性。(4)异源表达ZmSAP1/2/7增强转基因水稻的耐盐性。为进一步验证ZmSAP1/2/7基因的功能,分别将其转化水稻日本晴并进行表型鉴定。结果表明,正常生长条件下,转基因株系与WT根长无明显差异,长势一致。各项生理指标与活性氧相关指标均无显著差异。盐胁迫后,与WT相比,ZmSAP1/2/7转基因株系的存活率、根长、生物量、相对含水量、POD、SOD酶活均显著增加。但是,转基因株系的相对电导率、丙二醛含量、过氧化氢含量均显著降低。这些结果表明,转化ZmSAP1/2/7基因可通过清除活性氧进而增强转基因水稻株系的耐盐性。(5)异源表达ZmSAP1/2/7调控非生物胁迫相关基因的转录。为解析ZmSAP1/2/7基因调控耐盐性的潜在机制,对盐处理24 h的转基因水稻株系与WT进行转录组测序分析。结果显示,与WT相比,在转化ZmSAP1/2/7基因的转基因水稻中分别鉴定到4348、233及86个差异表达基因(Differentially expressed genes,DEGs),其中分别有350、16和8个DEGs被注释为与盐胁迫、渗透胁迫、氧化胁迫、脱落酸响应等非生物胁迫相关。qRT-PCR结果表明,在不同转基因株系中,正调控耐盐性相关基因OsSAPK2、OsCCA1、OsNR2、OsHSP90等上调表达,而负调控耐盐相关基因OsSDS1、OsMODD、OsbZIP5等下调表达。该部分结果表明,ZmSAP1/2/7可通过调控非生物胁迫相关基因转录,进而调控转基因株系的耐盐性。综上,本研究证实,异源表达ZmSAP1、ZmSAP2及ZmSAP7均可增强拟南芥和水稻耐盐性,为进一步研究其调控玉米耐盐性与机制奠定基础。更多还原

【Abstract】 The global soil salinization area is gradually expanding,affecting the growth and production of crops.Therefore,it is important to study how crops respond to salt stress.Stress associated proteins（SAPs）play a role in plant response to stress,plant growth and development.Previous studies showed that there were 10 ZmSAPs genes in maize,and their expression were induced by salt,drought and ABA,among which ZmSAP1,ZmSAP2 and ZmSAP7 might regulate salt tolerance.In this study,bioinformatics analysis,tissue expression pattern and subcellular localization identification of ZmSAP1/2/7 were performed.Furthermore,heterologous over-expression experiments were performed in Arabidopsis thaliana and rice to explore the function of ZmSAP1/2/7genes in regulating salt stress tolerance.The main findings are as follows:（1）ZmSAP1/2/7 belong to the A20/AN1 type zinc finger proteins.The coding sequences（CDS）of ZmSAP1,ZmSAP2 and ZmSAP7 genes were successfully amplified,and they were 531 bp,486 bp and 516 bp in length,encoding 176,161 and 171 amino acids,respectively.Bioinformatics analysis showed that ZmSAP1 and ZmSAP7contained an A20 domain in the N-terminus,and an AN1 domain in the C-terminus,but ZmSAP2 only contained an AN1 domain in the C-terminus.（2）ZmSAP1/2/7 genes were expressed in all tissues of maize,and all the encoded proteins had nuclear localization features.The results of q RT-PCR showed that ZmSAP1/2/7 genes were expressed in root,aerial root,stem,silk,husk,cob,tassel,ear leaf.The expression level of ZmSAP1 was the highest in ear leaf and the lowest in cob.The expression level of ZmSAP2 was the highest in root and the lowest in silk.The expression of ZmSAP7 was the highest in tassel and the lowest in silk.The results of subcellular localization experiments confirmed that ZmSAP1/2 were localized in the nucleus and ZmSAP7 was in the nucleus and cytoplasm.（3）Heterologous expression of ZmSAP1/2/7 enhanced the salt tolerance of transgenic Arabidopsis.The ZmSAP1/2/7 genes were transformed into wild-type Arabidopsis for phenotyping.The results showed that under normal conditions,the root length and growth status of transgenic lines and wild type（WT）lines were basically the same,and there were no significant differences in physiological and biochemical indexes.But the root length,biomass,survival rate,and relative water content in lines transformed with ZmSAP1/2/7 genes were significantly increased compared with WT under salt stress condition,while the relative conductivity and malondialdehyde content were significantly decreased compared with WT.These results showed that the transformation of ZmSAP1/2/7 genes could enhance the salt tolerance of Arabidopsis.（4）Heterologous expression of ZmSAP1/2/7 enhanced the salt tolerance of transgenic rice.To further verify the function of ZmSAP1/2/7 genes,they were transformed into rice for phenotyping.The results showed that under normal growth conditions,there was no significant difference in root length between the transgenic lines and WT,and the growth trend was the same,there was no significant difference between the physiological indexes and the reactive oxygen species-related indexes.But the survival rate,biomass,root length,relative water content,POD and SOD enzymes activities of lines transformed with ZmSAP1/2/7 genes were significantly increased compared with WT after salt stress.However,the relative conductivity,malondialdehyde content,and H₂O₂content of transgenic lines were significantly reduced.These results indicated that the transformation of ZmSAP1/2/7 genes could enhance the salt tolerance of transgenic rice lines by scavenging reactive oxygen species.（5）Heterologous expression of ZmSAP1/2/7 regulated the transcription of abiotic stress-related genes.To elucidate the mechanism of ZmSAP1/2/7 genes regulating salt tolerance,RNA-seq was performed on transgenic rice lines and WT after salt treatment with one day.The results showed that compared with WT,4348,233 and 86 DEGs were identified in the transgenic rice transformed with ZmSAP1/2/7 genes,of which 350,16and 8 DEGs were related to abiotic stresses such as salt stress,osmotic stress,oxidative stress and abscisic acid response,respectively.The results of q RT-PCR showed that the expression of salt-tolerance-related genes:OsSAPK2,OsCCA1,OsNR2,OsHSP90were up-regulated,while the expression of salt-tolerance-related genes OsSDS1,OsMODD and OsbZIP5 were significantly down-regulated.The results of this study showed that ZmSAP1/2/7 could regulate the salt tolerance of transgenic lines by regulating the transcription of abiotic stress-related genes.In conclusion,this study confirmed that ZmSAP1,ZmSAP2 and ZmSAP7 were positively regulating salt tolerance in Arabidopsis and rice,which laid a foundation for further research on their regulation to salt stress in maize.更多还原