干旱胁迫对转PtPIP2;8基因84K杨苗木光合、生长和根系结构的影响

doi:10.17521/cjpe.2020.0058

摘要/Abstract

摘要：

为研究水通道蛋白PtPIP2;8基因功能, 了解其不同表达水平的转基因84K杨(Populus alba × P. glandulossa)应对干旱胁迫的响应, 该文以转PtPIP2;8 84K杨抑制表达株系(抑制表达)、野生型(WT)和转PtPIP2;8 84K杨超表达株系(超表达)为试验材料, 测定PtPIP2;8表达水平、根系导度、光响应曲线、气体交换参数、生长及根系形态指标。结果显示: (1) WT植株PtPIP2;8仅在根系表达; 超表达植株PtPIP2;8除在根部显著表达外, 在茎和叶片中也显著表达; 抑制表达植株PtPIP2;8仅在根部有微量表达, 表达量分别是WT和超表达植株的1/20和1/80。(2)根系结构分析发现, 超表达植株总根长、总根表面积、总根体积、总根尖数显著低于WT和抑制表达植株, 根系导水率显著高于WT和抑制表达植株, 表明PtPIP2;8参与了植物根系水分运输, 提高了水分运输效率。(3)正常水分条件下, 抑制表达植株苗高、叶面积显著低于WT和超表达植株, 根冠比显著高于WT和超表达植株。干旱胁迫后, 抑制表达植株净光合速率(P_n)、气孔导度(G_s)下降幅度小, 仍能维持较高的P_n。气体交换参数显示抑制表达植株P_n、G_s日变化为“单峰”型, 属气孔因素引起的净光合速率下降;WT和超表达植株P_n、G_s日变化为“双峰”型, 干旱胁迫后, 抑制表达植株P_n略微下降, WT和超表达植株P_n均下降, 尤其是13:00、15:00下降显著, 表明WT和超表达植株对干旱胁迫更加敏感, 干旱对其影响更大。(4)干旱胁迫后, 抑制表达植株相对生长速率、总生物量降低的最少, 根冠比最高; 总根表面积、总根体积、总根尖数显著高于WT植株。表明PtPIP2;8直接参与水分运输并提高水分运输效率, 其转化影响了植株根系发育和生长。超表达植株根系发育的下降和叶面积的增大减弱了它的抗旱性, 而抑制表达植株矮小, 降低的叶面积, 增加的根系生长和根冠比提高了它的抗旱能力。从研究结果来看, 水通道蛋白提高了水分跨膜运输效率, 而非水通道蛋白导水机制对干旱有较强的耐受性。

关键词: 干旱胁迫, 转基因杨树, 光合特性, 根系结构

Abstract:

Aims In this study, changes in growth, photosynthesis and root structure in response to drought were tested in transgenic poplar 84K (Populus alba × P. glandulosa) seedlings with different expression levels of aquaporin gene (PtPIP2;8). The function of aquaporin gene PtPIP2;8 and its response to drought stress were analyzed.
Methods We selected PtPIP2;8 silencing line of poplar 84K, PtPIP2;8 overexpressing line of poplar 84K and wildtype (WT) as the experimental materials. The Real-time fluorescence quantitative PCR technique was used to detect the PtPIP2;8 expression in roots, stems and leaves. Root hydraulic conductance was measured by high pressure liquid flow meter. The photosynthetic light-response curve, and gas exchange parameters were measured by a LI-6400 photosynthetic system. Growth indexes were determined, and the root length, root surface area, root volume and total root tips were scanned and then analyzed with the root analysis software.
Important findings The results showed that: (1) The gene PtPIP2;8 was mainly expressed in the root system in WT, while its significant expression occurs not only in roots, but also in stems and leaves in PtPIP2;8 overexpressing poplar lines. The PtPIP2;8 RNAi-silence poplar lines only showed weak expression of PtPIP2;8 in the root, and the expression level were 1/20 and 1/80 of WT and overexpression line, respectively. (2) The root structure analysis showed that overexpression lines had significantly lower total root length, total root surface area, total root volume and total root tips than RNAi-silence line and WT, but higher root hydraulic conductance compared with RNAi-silence line and WT. These results showed that the aquaporin gene PtPIP2;8 participated in plant water transport and improved water transport efficiency. (3) Under normal water conditions, RNAi-silence lines showed lower plant height and leaf area but higher root-shoot ratio compared with overexpression line and WT. After drought stress, RNAi-silence lines only slightly decreased the net photosynthetic rate (P_n) and stomatal conductance (G_s), and maintained a relatively high P_n. Diurnal changes of P_n and G_s in RNAi-silence lines showed a single-peak pattern, in which the decrease of photosynthesis was caused by stomatal limitation. Diurnal changes of P_n in both overexpression lines and WT had a two-peak pattern, indicating the non-stomatal limitation of photosynthesis. Drought stress slightly decreased P_n of RNAi-silence lines, while largely decreased P_n of overexpression line and WT decreased, especially at 13:00 and 15:00, indicating that overexpression line and WT were more sensitive to drought stress compared with the RNAi-silence lines. (4) Under drought stress, RNAi-silence line showed the least decline in relative growth rate and total biomass, and the highest root-shoot ratio among the three poplar lines. The total root surface area, total root volume and total root tips of RNAi-silence line were significantly higher than those of WT. The results suggest that aquaporin gene PtPIP2;8 directly participates in the water transport and helps to improve the water transport efficiency, thus the transformation of aquaporin PtPIP2;8 gene may affect root development and growth of plants. Overexpression lines weaken their drought resistance with decreased root development and increased leaf area, while RNAi-silence line increases its drought resistance with reduced leaf area, increased root growth and root-shoot ratio. The results of this study indicate that aquaporin improves the efficiency of water transport across membranes, while the non-aquaporin water-conducting mechanism has greater tolerance to drought.

Key words: drought stress, transgenic poplar, photosynthetic characteristics, root structure

刘丽燕, 冯锦霞, 刘文鑫, 万贤崇. 干旱胁迫对转PtPIP2;8基因84K杨苗木光合、生长和根系结构的影响. 植物生态学报, 2020, 44(6): 677-686. DOI: 10.17521/cjpe.2020.0058

LIU Li-Yan, FENG Jin-Xia, LIU Wen-Xin, WAN Xian-Chong. Effects of drought stress on photosynthesis, growth and root structure of transgenic PtPIP2;8 poplar 84K (Populus alba × P. glandulosa). Chinese Journal of Plant Ecology, 2020, 44(6): 677-686. DOI: 10.17521/cjpe.2020.0058

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链接本文: https://www.plant-ecology.com/CN/10.17521/cjpe.2020.0058

https://www.plant-ecology.com/CN/Y2020/V44/I6/677

图/表 9

表1 实时荧光定量PCR引物序列

Table 1 Primer sequences for real-time PCR

基因名称 Gene name	上游引物 Forward primer	下游引物 Reverse primer
PtACTINrt	5′-AAACTGTAATGGTCCTCCCTCCG-3′	5′-GCATCATCACAATCACTCTCCGA-3′
PtPIP2;8	5′-GAGACTGTGAGGGACTACCAGGA-3′	5′-AATACCAAGAATGCCAACACCAC-3′

图1 84K杨转基因和野生型植株PtPIP2;8基因的相对表达量(平均值+标准偏差)。不同小写字母表示转基因和野生型植株不同部位之间相对表达量差异显著(p < 0.05)。

Fig. 1 Relative quantity of PtPIP2;8 gene expression in transgenic and wildtype poplar 84K (mean + SD). Different lowercase letters indicate significant differences in relative expression levels between different parts of transgenic and wildtype plants (p < 0.05).

图2 正常水分条件下84K杨转基因和野生型植株根系导水率变化(平均值+标准偏差)。不同小写字母表示转基因和野生型植株之间根系导水率存在显著差异(p < 0.05)。

Fig. 2 Changes of root hydraulic conductivity of transgenic and wildtype poplar 84K under normal watering condition (mean + SD). Different lowercase letters indicate significant differences in transgenic and wildtype plants (p < 0.05).

表2 正常水分条件下84K杨转基因和野生型植株生物量变化(平均值±标准偏差)

Table 2 The change of biomass of transgenic and wildtype poplar 84K under normal water condition (mean ± SD)

株系 Line	基径 Basal diameter (mm)	苗高 Height (cm)	根冠比 Root-shoot ratio	叶面积 Leaf area (cm²)	比叶面积 Specific leaf area (cm²·g^-1)
抑制表达 RNAi-silence	4.93 ± 0.26^c	71.60 ± 4.57^b	0.54 ± 0.05^a	534.35 ± 32.47^b	254.33 ± 14.32^a
野生型 Wildtype	5.62 ± 0.43^a	82.55 ± 4.70^a	0.50 ± 0.03^b	565.23 ± 25.05^a	262.57 ± 20.49^a
超表达 Overexpression	5.02 ± 0.46^bc	83.85 ± 4.99^a	0.32 ± 0.02c	576.75 ± 13.31^a	240.61 ± 11.19^a

图3 正常水分条件下84K杨转基因和野生型植株根系形态变化(平均值+标准偏差)。写字母表示转基因和野生型植株之间存在显著差异(p < 0.05)。

Fig. 3 Changes in root morphological traits of transgenic and wildtype poplar 84K under normal water condition (mean + SD). ferent lowercase letters indicate significant differences between transgenic and wildtype plants (p < 0.05).

图4 84K杨转基因和野生型植株净光合速率的光响应曲线(平均值±标准偏差)。正常水分条件下净光合速率的光响应曲线。B, 干旱胁迫后净光合速率的光响应曲线。

Fig. 4 Light response curve of net photosynthetic rate in transgenic and wildtype poplar 84K (mean ± SD). Light response of net photosynthetic rate under normal watering conditions. B, Light response of net photosynthetic rate after drought stress.

图5 84K杨转基因和野生型植株净光合速率日变化(平均值±标准偏差)。 A, 正常水分条件下净光合速率日变化。B, 干旱胁迫后净光合速率日变化。

Fig. 5 Diurnal variations of net photosynthetic rate in transgenic and wildtype poplar 84K (mean ± SD). A, Diurnal variations of net photosynthetic rate under normal watering conditions. B, Diurnal variations of net photosynthetic rate after drought stress.

表3 干旱胁迫后84K杨转基因和野生型植株相对生长速率及生物量变化(平均值±标准偏差)

Table 3 Changes of relative growth rate and biomass of transgenic and wildtype poplar 84K after drought stress (mean ± SD)

株系 Line	基径 Basal diameter (%)	苗高 Height (%)	地上生物量 Aboveground biomass (%)	地下生物量 Belowground biomass (%)	根冠比 Root shoot ratio	叶面积 Leaf area (%)
抑制表达 RNAi-silence	97 ± 3^a	94 ± 3^a	93 ± 2^a	84 ± 5^a	0.5 ± 0.02^a	68 ± 4^a
野生型 Wildtype	92 ± 3^b	88 ± 4^b	86 ± 7^b	77 ± 4^b	0.4 ± 0.03^b	44 ± 2^b
超表达 Overexpression	92 ± 2^b	87 ± 4^b	77 ± 3^c	76 ± 5^b	0.3 ± 0.02^c	43 ± 2^b

图6 干旱胁迫后转基因和野生型84K杨根系形态变化(平均值+标准偏差)。不同小写字母表示转基因和野生型植株之间存在显著差异(p < 0.05)。

Fig. 6 Changes of root morphological traits of transgenic and wildtype poplar 84K after drought dress (mean + SD). Different lowercase letters indicate significant differences between transgenic and wildtype plants (p < 0.05).

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