拟南芥PMRP基因在叶绿体淀粉粒积累和抗寒性中的作用

doi:10.3864/j.issn.0578-1752.2016.05.003

摘要/Abstract

摘要： 【目的】分析推测的膜蛋白（putitave membrane realated protein，PMRP）在拟南芥叶绿体发育过程的作用，明确PMRP对拟南芥光合能力的影响及抗寒性分析，为改善作物光合性能及增强抗逆性提供理论依据。【方法】构建PMRP的RNAi和过表达载体，转化农杆菌EHA105，采用花絮侵染法转化野生型拟南芥（Columbia，Col-0），获得PMRP RNAi和过表达转基因拟南芥；以野生型和PMRP RNAi、过表达转基因拟南芥为材料，利用细胞生物学方法，观察PMRP表达量对拟南芥叶肉细胞叶绿体的结构和淀粉粒积累的影响；利用红外CO₂分析法测定拟南芥的光合速率，分析PMRP对拟南芥光合能力的影响。选取16 h光照、21℃条件下培养的21 d的野生型Col-0、3个过表达PMRP转基因拟南芥株系，3个PMRP-RNAi转基因拟南芥株系，用冷光源培养箱培养，先在4℃培养7 d，然后在-8℃培养1.5 h，取出放到16 h光照、21℃条件培养7 d后，分析PMRP转基因拟南芥对寒害的抗性。选取14 d的野生型Col-0、3个过表达PMRP转基因拟南芥株系和3个PMRP-RNAi转基因拟南芥株系，对其莲座叶细胞渗出液电导率进行测定。【结果】获得PMRP RNAi和过表达转基因拟南芥株系；通过测定野生型和转基因株系的莲座叶光合速率，野生型Col-0的光合速率为7.3 μmol·m^-2·s^-1，PMRP-RNAi转基因拟南芥的光合速率分别为8.8、7.8和8.5 μmol·m^-2·s^-1，PMRP表达量的降低略增强了拟南芥的光合速率。野生型Col-0的绿叶率为48%，过表达PMRP转基因拟南芥的3个株系绿叶率分别为48.6%、47.8%和49.2%，3个PMRP-RNAi转基因拟南芥的绿叶率分别为65.9%、67.4%和68.3%，表明PMRP表达量的降低增强了植物的耐寒性。在-8℃下处理30 min，野生型Col-0拟南芥莲座叶的渗出液电导率为70.67 μS·cm^-1，PMRP-RNAi拟南芥莲座叶渗出液电导率分别为48.57、45.40和52.10 μS·cm^-1。表明PMRP表达量的降低明显降低了逆境对细胞膜的损伤。通过对PMRP-RNAi转基因拟南芥和野生型拟南芥完全展开的莲座叶，进行超微结构分析，发现野生型拟南芥莲座叶细胞中，叶绿体为椭圆形，而PMRP-RNAi转基因拟南芥莲座叶细胞叶绿体变为近似圆形；在光照情况下，PMRP-RNAi转基因拟南芥叶绿体中淀粉粒的积累与野生型相似，均有明显的淀粉粒积累，但在黑暗环境中，PMRP-RNAi转基因拟南芥叶绿体中淀粉粒积累明显增多。【结论】PMRP表达量降低造成叶绿体形状由梭型变为圆球型，淀粉粒明显增多，增强了拟南芥的抗寒性。

关键词: PMRP ；抗寒性；淀粉粒；拟南芥

Abstract: 【Objective】The aim of this study was 1) to analyze the role of PMRP (Putative Membrane Related Protein) in the progress of chloroplast development in Arabidopsis, 2) to illuminate the effect of PMRP on the photosynthetic capacity and cold resistance of Arabidopsis, 3) to provide a theoretical basis for improving the photosynthetic performance and enhancement of plant cold-resistance.【Method】PMRP RNAi and over expression vector were constructed and transformed into Agrobacterium tumefaciens EHA105, and then infected the buds of wildtype Arabidopsis(Columbia, Col-0), so we obtained the PMRP RNAi and overexpressing transgenic Arabidopsis plants; with wild type, PMRP RNAi and over-expressing transgenic Arabidopsis thaliana as materials, cell biology method was used to observe the chloroplast structure and starch grain accumulation; The CO₂ infrared analytical method was used to analyze the photosynthetic rate with different PMRP expression in Arabidopsis transgenic plants; Wild-type (Col-0), overexpression PMRP transgenic Arabidopsis and PMRP-RNAi transgenic Arabidopsis were grown at 21℃ with a 16-h-light/8-h-dark cycle. After 21 days, they were transferred to 4℃cold light incubator for 7 d and then cultivated for 1.5 h at 8℃, then were grown at 21℃ with a 16-h-light/8-h-dark cycle again for 7 d, then we analyzed the chilling resistance of PMRP transgenic Arabidopsis; Wild-type (Col-0), overexpression PMRP transgenic Arabidopsis and PMRP-RNAi transgenic Arabidopsis were grown for 14 days and there rosette leaves were used to determine the conductivity of cellular exudate.【Result】PMRP RNAi and overexpressing transgenic Arabidopsis plants were obtained. The photosynthetic rate of rosette leaves in wild-type (Col-0) was 7.3 μmol·m^-2·s^-1, but the photosynthetic rate in the 3 lines of PMRP-RNAi transgenic Arabidopsis plants was 8.8, 7.8 and 8.5 μmol·m^-2·s^-1 respectively, this results showed that the photosynthetic rate was increased with the decreased PMRP expression. Under cold stress condition, the green leaves rate of wild type (Col-0) was 48%, the green leaves rate of the 3 overexpression PMRP transgenic Arabidopsis lines was 48.6%, 47.8%, 49.2% respectively, the green leaves rate of the 3 PMRP-RNAi transgenic Arabidopsis lines was 65.9%, 67.4%, 68.3% respectively. These data indicate that the decreasing expression of PMRP enhanced the tolerance of Arabidopsis to cold stress. After 30 mins treatment in -8℃ condition, the cellular exudate conductivity of the rosette leaves in wild type Arabidopsis was 70.67 μS·cm^-1, the cellular exudate conductivity of the rosette leaves in the 3 lines of PMRP RNAi transgenic Arabidopsis plants was 48.57, 45.40 and 52.10 μS·cm^-1 respectively, this indicated that the decrease of PMRP expression decreased the damage of cell membrane significantly. We analyzed the ultrastructure of the fully expanded rosette leaf of wild-type and PMRP-RNAi transgenic Arabidopsis, imaging results showed that chloroplasts are ellipse in wild type (Col-0) and was approximately circular shape in PMRP-RNAi transgenic Arabidopsis. Under light condition, the starch grains accumulation obviously in both PMRP transgenic Arabidopsis and wild type; but in the dark environment, the accumulation of starch grains in chloroplasts of PMRP-RNAi transgenic Arabidopsis was more than that of wild type Arabidopsis.【Conclusion】The decreased expression of PMRP changes the chloroplast shape from ellipse to round, increased the starch grains significantly, and enhanced the cold resistance of Arabidopsis thaliana.

Key words: PMRP, cold resistance, starch grains, Arabidopsis

高梦烛，赵亚林，闫青地，张海丽，王凤茹，董金皋. 拟南芥PMRP基因在叶绿体淀粉粒积累和抗寒性中的作用[J]. 中国农业科学, 2016, 49(5): 832-839.

GAO Meng-zhu, ZHAO Ya-lin, YAN Qing-di, ZHANG Hai-li, WANG Feng-ru, DONG Jin-gao. Analysis of the Role of the PMRP Gene in the Accumulation of Chloroplast Starch and Cold Resistance in Arabidopsis thaliana[J]. Scientia Agricultura Sinica, 2016, 49(5): 832-839.

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