【作者】 李静；

【导师】 陈火英；

【作者基本信息】 上海交通大学 ， 园艺学， 2018， 博士

【摘要】 土壤盐渍化是严重危害植物生长的非生物胁迫之一,我国耕地总面积中有近三分之一的盐渍化土地,还有约1亿公顷的滩涂、盐碱荒地。因此,研究植物的耐盐分子调控机制对盐渍化地区农作物的可持续生产具有重要意义。茄子（Solanum melongena L.）是我国主要的茄果类蔬菜作物之一,在露地及设施中均广泛种植。截至目前,关于盐胁迫对茄子影响的研究主要集中在生长形态以及生理生化指标方面,而茄子的耐盐分子机理尚不清楚。因此为推动茄子在盐渍化地区可持续生产,本论文对耐盐型茄子品种进行了筛选并对其耐盐分子机理进行了探究。主要结论如下:1)为避免基因型间种子自身发芽缺陷的影响,选取发芽势一致的茄子种子进行盐胁迫处理。结果发现,随着NaCl处理浓度的增加,茄子生长受抑制程度明显增加,但不同基因型之间存在较大的差异。利用隶属函数分析法综合多个生长指标对19份茄子材料发芽期的耐盐性进行了排序。基于发芽期的实验结果,挑选发芽期耐盐性表现不同的8份茄子材料进行苗期的耐盐性鉴定,发现茄子的耐盐性在发芽期和苗期之间不存在显著相关性。值得注意的是,无论是发芽期还是苗期,茄子材料竹丝茄（编号118）均表现出很高的耐盐性,而茄子材料贵州红茄（编号30）则均表现出较强的盐敏感性。2)对耐盐型茄子No.118和盐敏感型茄子No.30进行盐胁迫处理发现,与No.30相比,No.118的生长受抑制程度较小且其叶片中具有较高的K⁺/Na⁺比。综上,我们将这两个茄子材料重命名为ST118和SS30。为探究茄子的耐盐分子机制,利用比较转录组学的方法对盐胁迫处理下两个茄子品种的根系和叶片进行分析。结果发现茄子盐胁迫响应机制存在基因型特异性和组织特异性的特点。在ST118和SS30中均被鉴定为差异表达的基因具有几乎相同的表达模式,因此推测在ST118中特有的差异表达基因可能与其高耐盐性表型密切相关。进一步分析发现,一些参与盐胁迫响应的转录因子在ST118和SS30中存在不同的表达模式,包括两个NACs、一个WRKY、一个MYB和一个COL基因。另外值得注意的是,在这些差异表达基因中,共发现7个编码K⁺转运蛋白或通道蛋白的基因,而编码Na⁺转运蛋白的基因仅有SOS1。而且AKT1、KAT1和SOS1仅在No.118的叶片中被显著上调表达。因此,盐胁迫条件下,K⁺转运蛋白可能在维持耐盐茄子品种细胞中K⁺和Na⁺平衡与分布方面发挥重要作用。3)对茄子耐盐功能候选基因SmAKT1进行克隆,并进行氨基酸序列比对和进化树分析,发现SmAKT1蛋白与其它物种中的AKT1具有较高的序列相似性。另外,亚细胞定位分析发现SmAKT1仅在细胞膜上表达;组织特异性分析发现SmAKT1在茄子根系中表达量最高,这些结果预示着SmAKT1可能介导根系细胞对K⁺的吸收。SmAKT1可以被低钾和盐胁迫处理诱导表达,且其在两种胁迫下具有相似的表达变化模式。将SmAKT1转入酵母钾吸收缺陷型菌株和拟南芥akt1突变体后,不仅能够恢复受体在低钾环境下的生长能力,而且还能够增强受体的抗盐性,说明SmAKT1不仅具有K⁺转运活性还在提高植物耐盐性方面发挥重要作用。4)CBL作为第二信使Ca²⁺的响应元件,可以通过与CIPK互作调控多种离子转运蛋白的活性。前人研究发现CBLs和CIPKs家族基因是AKT1的调控因子。本论文基于已公布的茄子基因组,成功克隆了5个SmCBLs和15个SmCIPKs基因,并通过保守性与进化树分析进行身份确认。组织表达特异性分析发现5个SmCBLs和12个SmCIPKs主要在茄子根系中表达,且它们的表达受到不同离子胁迫的诱导或抑制。另外,通过酵母双杂交技术和双分子荧光互补实验探究SmCBLs和SmCIPKs之间的互作模式,结果发现多种新的CBL-CIPK互作组合。本论文为研究CBL-CIPK复合体在茄子耐盐性方面的作用机制奠定了基础。更多还原

【Abstract】 Soil salinization is one of the most crucial abiotic stresses that limit plants growth.There are approximately one-third of the total irrigated agricultural land is salinized and about 100 million ha land is tidal flats and saline-alkali wasteland in China.Studying the salt-tolerant molecular mechanisms in plants is very beneficial for the sustainable production of crops in salinized areas.Eggplant(Solanum melongena L.)is one of the main solanaceous vegetable cultivated and consumed worldwide.The existing researches in eggplant were focused on salt-induced morphological,biochemical and physiological changes,while salt-tolerant molecular mechanisms in eggplant unclearly.Therefore,in order to promote the sustainable production of eggplant in salinized areas,the salt-tolerant eggplant materials were screened out and the salt-tolerant molecular mechanisms were explored in this paper.The main results are as follows:1)In order to avoid seed itself defects,uniformly germinated eggplant seeds were selected and treated with salt stress.With the increase of NaCl concentration,the growth of eggplants was inhibited more seriously.But significant differences were found among different eggplant materials.The subordinate function method was used to comprehensive four indicators to sort the salt tolerance of the 19 eggplant materials.Based on the results of early embryo growth stage,the salt tolerance on the seedling stage of part eggplant varieties were studied.The results suggested that the relationship of salt tolerance between the two growth stages was not close.Strikingly,on both the two growth stages,the eggplant material Zhusiqie(No.118)showed highest salt tolerance,while Hongqie(No.30)showed salt sesentivity.2)Salt-tolerance analysis between the salt-tolerant eggplant No.118 and salt-sensitive eggplant No.30 showed that less damaged on growth and higher K~+/Na~+ratios in No.118 leaves than those of No.30.Consequently,they were re-named the two eggplant materials as ST118 and SS30,respectively.Then,comparative-transcriptome analysis was used as a powerful approach to understand the salt-response mechanisms in the leaves and roots of SS30 and ST118.And it revealed that genotype-specific and organ-specific manners exist in eggplant in response to salt stress.Strikingly,the genotype-specific differentially expressed genes(DEGs)in ST118 were considered crucial to its higher salt-tolerance,because the expression patterns of common DEGs in the leaves/roots of the two eggplant genotypes were almost the same.Further analysis found that five transcription factors,which have been reported to be in response to elevated external salinity,showed different expression pattern between ST118 and SS30,including two NACs,one WRKY,one MYB and C2C2-CO-like.In addition,there were seven K~+transporters while only one Na~+transporter were significantly regulated by salt stress,and the AKT1,KAT1 and SOS1 were only up-regulated in the leaves of ST118.These results implied that the K~+transporters were very important for maintaining K~+and Na~+homeostasis and distribution in plants under salt conditions.3)The salt tolerant candicate gene SmAKT1 was cloned from eggplant.The amino acid sequence alignment and phylogenetic tree analysis showed that SmAKT1and AKT1s from other plant species were highly conserved.In addition,the subcellular localization assay in tobacco leaves showed that SmAKT1 was only expressed at the plasma membrane,and the tissue-specific analysis found that it was mainly expressed in eggplant roots.These results indicated SmAKT1 could regulate K~+absorption in cells.Both the low-K~+and salt stress could induce SmAKT1expression,and the expression patterns under the two stresses were similar.SmAKT1could not only complement the growth of yeast K~+defect mutant strain and rescue the low-K~+-sensitive phenotype of Arabidopsis akt1 mutant under low-K~+stress,but also enhance the growth of them under salt stress.These results demonstrated that SmAKT1 not only has K~+uptake activity but also play an important role on improving salt-tolerance.4)Calcineurin B-like proteins(CBLs)are calcium sensors and control the affinities and activities of numerous ion transporters with CBL-interacting protein kinases(CIPKs).And it has been reported that CBLs and CIPKs gene families were the regulators of AKT1.Here,a total of 5 SmCBL and 15 SmCIPK genes were firstly identified from eggplant genome database.And the amino acid sequence alignment and phylogenetic tree analysis were used for further confirmation.The tissue-specific analysis found that 5 SmCBLs and 12 SmCIPKs were mainly expressed in eggplant roots,and they were induced or inhibited by different ion stresses.The yeast two-hybrid(Y2H)assay and bimolecular fluorescence complementation(BiFC)assay were used to explore the interaction network between SmCBLs and SmCIPKs,and some new CBL-CIPK complexes were found which have never been discovered in any other plant species.In summary,this study provided the theoretical foundation for studying the mechanisms of SmCBL-SmCIPK complexes in improving the salt tolerance of eggplants.更多还原