【作者】 李澄；

【导师】 金竹萍；

【作者基本信息】 山西大学 ， 植物学， 2020， 硕士

【摘要】 气孔是植物表皮的特有结构,其孔径大小和密度分布直接决定植物叶片光合作用的效率和作物产量,是农业生产中很重要的形态学指标。气孔的发育过程受到严格的“单细胞间隔原则”调控,其中表皮模式因子（Epidermal pattern factors,EPFs）是关键环节,EPF1和EPF2为负调控子,EPFL9为正调控子。气体信号分子H₂S（hydrogen sulfide,H₂S）参与植物生长发育和响应逆境胁迫的诸多过程,其中H₂S调节气孔运动的作用机制备受关注。相对而言,H₂S信号对气孔发育的影响却鲜有报道。因此,开展H₂S对叶片气孔发育调节机制的研究,为深入了解H₂S气体信号在植物响应逆境胁迫过程中的作用机制提供更多思路。本研究以模式植物拟南芥为材料,探究气体信号H₂S参与拟南芥EPFs多肽调节气孔发育的作用机制,主要结果如下:1.H₂S主要生成酶编码基因缺失的突变体lcd、des1和lcd/des1与野生型WT相比,叶片气孔密度显著降低;WT+HA与野生型WT相比,新生叶气孔密度也显著降低;WT+NaHS和WT+HT处理组的新生叶气孔密度与野生型WT相比无明显变化;lcd/des1+NaHS与lcd/des1相比,新生叶气孔密度得到恢复。lcd、lcd/des1和WT+HA与WT相比,新生叶气孔指数有降低的趋势;des1、WT+NaHS、lcd/des1+NaHS和WT+HT与WT相比,新生叶气孔指数有升高的趋势;但所有处理组的气孔指数无显著性差异。与WT相比,所有处理组的新生叶保卫细胞的大小无显著变化。以上结果说明,H₂S信号参与调控植物叶片的气孔发育过程。2.与WT相比,突变体lcd、des1、lcd/des1、WT+HA和WT+HT的新生叶的H₂S含量和产率显著降低。与WT相比,WT+NaHS、lcd/des1+NaHS的新生叶的H₂S含量显著升高。与WT相比,lcd/des1+NaHS的新生叶的H₂S产率显著降低,WT+NaHS的新生叶的H₂S产率有升高的趋势,但无显著性差异。以上结果说明,新生叶的H₂S含量和产率变化可能是导致新生叶气孔发育出现差异的原因。3.生理浓度NaHS处理WT 0.5 h时,EPFs编码的基因变化趋势比较明显,EPF1和EPF2基因呈显著下降趋势,而EPFL9呈显著升高趋势。突变体lcd、des1和lcd/des1与野生型WT相比,新生叶片EPF2基因表达呈显著升高趋势;lcd/des1+NaHS与野生型WT相比,新生叶片EPFL9基因表达呈显著升高趋势。以上结果说明,EPFs的编码基因会在短时间内响应H₂S信号,内源H₂S与外源H₂S的参与对拟南芥EPFs的基因响应不同。4.拟南芥表皮模式因子EPFs的原核表达载体的构建、EPFs多肽的诱导表达和纯化。以拟南芥cDNA为模板,克隆EPF1、EPF2和EPFL9基因,得到重组质粒pET28a-EPF1、pET28a-EPF2和pET28a-EPFL9;将重组质粒分别转入大肠杆菌BL21（DE3）进行异丙基β-D-硫代半乳糖苷（IPTG）诱导表达。优化后的表达条件:IPTG诱导浓度分别为0.5、0.3和0.05 mM;最适诱导温度分别为28℃、28℃和16℃;最适诱导时间分别为16 h、16 h和20 h;经Ni琼脂糖凝胶柱纯化获得融合蛋白,大小分别为18 kDa、19 kDa和14.5 kDa左右。5.H₂S处理体外表达纯化的EPFs融合蛋白,经生物素开关法进行巯基化检测。结果表明EPF2和EPFL9蛋白的巯基化信号于对照比显著升高,EPF1蛋白未见显著变化。说明H₂S在调节拟南芥气孔发育中,可能主要是通过巯基化修饰EPF2和EPFL9蛋白发挥作用的。综上所述,H₂S信号通过EPFs多肽参与到拟南芥气孔发育的调节作用中,H₂S调节EPFs的转录表达,巯基化修饰EPF2和EPFL9蛋白,进而影响拟南芥气孔的发育,提高拟南芥叶片下表皮的气孔密度。为后续深入研究气体信号H₂S对植物气孔发育的作用机制提供基础数据,对增加作物产量、增强植物抗逆性有重要意义。更多还原

【Abstract】 Stomata,a unique structure of plant epidermis,whose pore size and density distribution directly determine the photosynthetic efficiency of plant leaves and crop yield,so it is an important morphological indicators in agricultural production.Stomatal development is regulated by the strict "single-cell spacing principle".Epidermal pattern factors（EPFs）are the key links,EPF1 and EPF2 are negative regulators,and EPFL9 is a positive regulator.Hydrogen sulfide（H₂S）signal is involved in growth and development,and stress resistance in plant.The role of H₂S in regulating stomatal movement has attracted more and more attention.In contrast,the effect of H₂S signal on stomatal development has been rarely reported.Therefore,research on the regulation mechanism of leaf stomatal development by H₂S will provide more ideas for understanding the mechanism of H₂S gas signal in the plant response to stress.In this study,the model plant Arabidopsis was used as the material to explore the mechanism of H₂S signal involved in the regulation of stomatal development via EPFs peptides.The main results are as follows:1.H₂S mutants lcd,des1,and lcd/des1,with a major loss of enzyme-encoding genes,whose stomatal density of leaves were significantly reduced compared with wild-type WT;compared with wild-type WT,WT+HA significantly reduced new leaves stomatal density;The stomatal density of new leaves in the WT+Na HS and WT+HT treatment groups did not change significantly compared with wild-type WT;compared with lcd/des1,the stomatal density of new leaves in the lcd/des1+Na HS recovered.Compared with WT,the stomatal index of new leaves in the lcd,lcd/des1 and WT+HA had a tendency to reduce,the stomatal index of new leaves in the des1,WT+Na HS,lcd/des1+Na HS and WT+HT had a tendency to increase,but there was no obvious difference of the stomatal index in the all treatment groups.Compared with WT,there was no significant difference in the size of new leaves guard cells in all treatment groups.The above results indicate thatH₂S signal regulates stomatal development in Arabidopsis.2.Compared with WT,the H₂S content and H₂S production rate of the new leaves in mutants lcd,des1,lcd/des1,WT+HA and WT+HT were significantly reduced.While,the H₂S content of new leaves of WT+Na HS and lcd/des1+Na HS was significantly increased.Compared with WT,the H₂S production rate of the new leaves of lcd/des1+Na HS was significantly reduced,and the H₂S production rate of the new leaves of WT+Na HS had a tendency to increase,but there was no obvious difference.The above results indicate that the changes in H₂S content and H₂S production rate of the new leaves may be the cause of the differences in the development of the stomata of the new leaves.3.When physiologically concentrated Na HS treated WT for 0.5 h,the changes of EPFs-encoded genes were more obvious,EPF1 and EPF2 genes showed a significant downward trend,and EPFL9 showed a significant upward trend.Compared with wild-type WT,the mutant lcd,des1,and lcd/des1 showed a significant increase in the expression of EPF2 in the new leaves;compared with wild-type WT,lcd/des1+Na HS showed a significant increase in the expression of EPFL9 in the new leaves.The above results indicate that the genes encoding EPFs will respond to H₂S signals in a short time,and the participation of endogenous H₂S and exogenous H₂S will have different genetic responses to Arabidopsis EPFs.4.Construction of EPFs prokaryotic expression vector,induction expression and purification of EPFs polypeptides.Using Arabidopsis c DNA as a template,EPF1,EPF2 and EPFL9 genes were cloned to obtain recombinant plasmids p ET28a-EPF1,p ET28a-EPF2 and p ET28a-EPFL9;recombinant plasmids were transferred into E.coli BL21（DE3）for isopropylβ-D-thiogalactoside（IPTG）induction and expression.Optimized expression conditions: IPTG-induced concentrations were 0.5,0.3,and 0.05 m M respectively;optimal induction temperatures were 28 ℃,28 ℃,and 16 ℃;optimal induction times were 16 h,16 h,and 20 h,respectively;The fusion protein was purified by Ni agarose gel column and the size was about 18 k Da,19 k Da and 14.5 k Da,respectively.5.EPFs fusion protein expressed and purified in vitro by H₂S treatment was tested for S-sulfhydration by biotin switch method.The results showed that the S-sulfhydration signals of EPF2 and EPFL9 proteins were significantly higher than those of the control,and there was no significant change in EPF1 protein.It is suggested that H₂S may play an important role in the regulation of stomatal development in Arabidopsis thaliana through the modification of EPF2 and EPFL9 proteins by S-sulfhydration.In summary,H₂S signals are involved in the regulation of stomatal development in Arabidopsis thaliana through EPFs polypeptides,H₂S regulates the transcriptional expression of EPFs,and S-sulfhydration modifies EPF2 and EPFL9 proteins,which in turn affects the development of Arabidopsis stomata and improves Arabidopsis thaliana Stomatal density of leaf epidermis.It provides basic data for further research on the mechanism of the gas signal H₂S on the development of plant stomata,which is of great significance to increase crop yield and enhance plant stress resistance.更多还原