摘要
盐离子和重金属离子抑制植物正常的生长发育。过高的盐浓度会破坏植物体内的水分平衡、离子平衡和活性氧平衡；而过量的重金属离子(如Cd2+)能够通过干扰光合、呼吸、营养平衡等生理过程来抑制植物生长,甚至导致植物死亡。在我国西北干旱盐碱的荒漠地区,胡杨(Populus euphrattca)是唯一能够成林的乔木树种。胡杨叶片在长期盐处理后能够发生肉质化,起到稀释组织和细胞中盐分的作用,但盐诱导肉质化的分子机制尚不清楚。另有研究表明,胡杨对Cd2+的响应非常敏感,但其对重金属胁迫的响应和适应机理还不明确。
木葡聚糖内糖基转移酶/水解酶调控细胞壁的松弛和伸展。本实验室前期从胡杨叶片中克隆得到PeXTH全长基因,荧光定量PCR结果显示该基因在长期盐胁迫下的表达呈上调趋势,因此PeXTH很有可能参与盐诱导的胡杨叶片肉质化的发生,从而提高胡杨的抗盐性。此外,XTH基因能够响应重金属胁迫,在低浓度的Cd2+胁迫下,胡杨PeXTH基因的表达水平提高了1.2～2.1倍,因而该基因也可能参与胡杨适应Cd2+胁迫的生理过程。
本论文利用转基因技术,将PeXTH基因在模式植物烟草中过量表达,通过比较转基因烟草和野生型烟草在叶片显微结构、形态表型、生理特征等方面的差异表现,探讨了PeXTH在植物适应盐胁迫和重金属胁迫中的作用机制。主要研究结果如下：
1.PeXTH蛋白具有木葡聚糖内转糖苷酶(XET)的催化活性域DEIDFEFLG,且定位在内质网和细胞壁上。
2.PeXTH的纯化蛋白大小约为35kD,且具有体外XET活性。该蛋白发挥活性的最适温度是37℃,最适pH是6.0。
3.PeXTH通过调控组织肉质化来提高植物耐盐性。实验表明,PeXTH基因的过表达使转基因烟草的抗盐性增加,盐胁迫下转基因烟草的存活率和根长都明显高于野生型。这种变化来源于转基因烟草叶片显微结构和生理特征的改变：同野生型烟草相比,转基因烟草的肉质化程度明显提高,栅栏组织细胞排列更为紧密,叶肉细胞的胞间隙较小,从而导致转基因烟草单位叶面积水含量和鲜/干重比值这两个肉质化指标分别比野生型烟草高36%和39%；这种显微结构的变化一方面可以达到稀释盐分的效果,另一方面也有助于提高转基因烟草的叶片保水力,从而在盐胁迫下起到稀释叶组织和细胞盐浓度的作用。此外,叶肉细胞数目的增加和胞间隙的减小还有利于碳利用率和光合效率的提高。总之,PeXTH基因的过表达能够通过引发叶片肉质化来提高转基因烟草的抗盐性。
4.PeXTH还可以缓解植物的Cd2+胁迫。PeXTH基因的过表达能够使转基因烟草的抗Cd2+性提高,Cd2+胁迫下转基因烟草叶和根生长受到的抑制明显较小：Cd2+胁迫下,转基因烟草根系的Cd2+内流显著低于野生型烟草,从而使转基因烟草根尖和根成熟区中积累的Cd2+比野生型烟草少49%-58%；值得注意的是,转基因烟草根系的木葡聚糖降解活性比野生型烟草高56%～87%,导致根细胞壁中的木葡聚糖含量比野生型烟草低25%～27%。由此可见,PeXTH基因的过表达提高了转基因烟草根系的木葡聚糖降解活性,降低了根细胞壁中的木葡聚糖含量,导致Cd2+结合位点减少,从而使根系对Cd2+的吸收减弱,减少了Cd2+在根中的积累,最终缓解了Cd2+对转基因烟草的毒害。
Salt ions and heavy metals suppress plant growth and development. High salt concentrations lead to water deficit and ion toxicity, which induce oxidative damage in plants. Excess heavy metals, such as cadmium (Cd2+) interferes with a series of physiological processes, including photosynthesis, transpiration, and nutrient balance, resulting in growth retardation and eventually plant death. Being a valuable tree species that survives in saline and alkaline desert sites, Populus euphratica has great potential for genetic improvement in large-scale afforestation. During a prolonged period of salt stress, P. euphratica develops pronounced succulent leaves, which benefits the adaptation of P. euphratica to salt environments. However, the molecular mechanism of salt-induced succulence remains poorly understood. P. euphratica has been shown to be sensitive to Cd2+stress, the acclimation and adaptation processes need to be investigated.
Xyloglucan endotransglucosylase/hydrolase (XTH) is considered to be a vital factor controlling cell wall loosening and extensibility. We had cloned the full-length PeXTH gene from leaves of P. euphratica. Real-time PCR assay showed that the expression of PeXTH was up-regulated under salt conditions. Therefore, the PeXTH gene was suggested to involve in salt-induced leaf succulence and thus enhancing salt tolerance of P. euphratica. In addition, XTH is involved in the plant response to heavy metal toxicity. However, the link between XTH and Cd2+stress has not yet been established in higher plants. PeXTH expression was up-regulated by1.2-2.1fold in P. euphratica roots and leaves upon Cd2+exposure (40μM-80μM CdCL2), indicating that the PeXTH gene might play a role in Cd2+tolerance of P. euphratica.
In this study, the PeXTH gene was overexpressed into tobacco by transgenic technique. We investigated the functions of PeXTH in salinity and Cd2+tolerance by comparing the leaf anatomy, plant morphology, and physiological traits of PeXTH-transgenic and wild-type tobacco plants. The main results and conclusions are as follows:
1. The putative amino acid sequence of PeXTH protein contained the DEIDFEFLG domain, which has been proposed to be the catalytic site of xyloglucan endotrans-glucosylase (XET). In addition, PeXTH localized exclusively to the endoplasmic reticulum and cell wall.
2. Purified PeXTH protein was approximately35kD and had XET activity in vitro. The optimum temperature of the enzyme was37℃and the optimum pH was6.0.
3. Plants overexpressing PeXTH were more salt tolerant than wild-type tobacco with respect to root and leaf growth, and survival. The increased capacity for salt tolerance was due mainly to the anatomical and physiological alterations caused by PeXTH over-expression. Compared with the wild type, PeXTH-transgenic plants contained36%higher water content per unit area and39%higher ratio of fresh weight to dry weight, a hallmark of leaf succulence. However, the increased water storage in the leaves in PeXTH-transgenic plants was not accompanied by greater leaf thickness but was due to highly packed palisade parenchyma cells and fewer intercellular air spaces between mesophyll cells. In addition to the salt dilution effect in response to NaCl, these anatomical changes increased leaf water-retaining capacity, which lowered the increase of salt concentration in the succulent tissues and mesophyll cells. Moreover, the increased number of mesophyll cells reduced the intercellular air space, which improved carbon economy and resulted in a47%-78%greater net photosynthesis under control and salt treatments. Taken together, the results indicate that PeXTH overexpression enhanced salt tolerance by the development of succulent leaves in tobacco plants without swelling.
4. Tobacco plants overexpressing PeXTH were more tolerant to Cd2+stress than wild-type tobacco in terms of root and shoot growth. Transgenic lines accumulated49%-58%less Cd2+in root apical and mature regions, as compared to the wild-type. The less buildup of Cd2+in roots of transgenic lines was the result of lower influx of Cd2+under Cd2+stress. It is noting that transgenic plants displayed56%-87%higher xyloglucan degradation activity (XDA) than the wild type, leading to a25%-27%decline of xyloglucan content in the root cell walls. Therefore, overexpression of PeXTH increased the activity of XDA in transgenic plants, which enhanced the degradation of xyloglucan in the wall. The down-regulated amount of xyloglucan led to less binding sites for Cd2+and thus reduced the root Cd2+uptake and buildup in transgenic plants. Consequently, the Cd2+toxicity was eventually alleviated in transgenic tobacco.
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