Burkholderia sp. GD17对水稻幼苗镉耐受的调节

doi:10.13560/j.cnki.biotech.bull.1985.2021-0915

摘要/Abstract

摘要：

探究根内生菌Burkholderia sp. GD17在水稻对镉（cadmium,Cd）胁迫应答中的调节作用,为减少Cd的积累、提高植株的Cd耐受性奠定基础。以接种GD17和未接菌的幼苗为材料,在添加Cd条件下,分析其生理生化代谢和相关基因表达。接菌5 d后,根中GD17菌的数量达到3.6×10⁶ CFU/g根鲜重,且在植株生长过程中内生菌数量维持在这一级别。Cd暴露（20和40 mg/kg土壤）20 d后,接种GD17植株的干（鲜）重显著高于未接菌植株。接种GD17和未接菌植株根中的Cd含量无显著差异,但前者地上部的Cd含量是后者的43%。Cd胁迫下,接种GD17根和叶片的丙二醛含量分别是未接菌植株的78%和64%。与未接菌植株相比,接种GD17叶片的超氧化物歧化酶活性降低,但过氧化物酶和过氧化氢酶活性均升高。接种GD17有效阻止了光合作用的损伤,如减缓Cd引发的总叶绿素含量、净光合速率和气孔导度的下降,以及胞间CO₂浓度的升高。叶绿素荧光成像进一步反映了GD17对光合作用的保护性影响。与未接菌植株相比,接种GD17植株根中所有分析的与Cd耐受相关基因的表达水平均显著升高。根部接种GD17可系统性地减轻Cd对水稻幼苗的伤害,其可能的机理包括：降低Cd的根-茎运输、减轻Cd引发的氧化伤害和对光合作用的损伤;上调Cd耐受相关基因在根中的表达。基于此,GD17菌株在低Cd水稻生产中具有潜在的应用价值。

关键词: 镉胁迫, 伯克霍尔德菌, 水稻, 氧化胁迫, 光合作用, 叶绿素荧光

Abstract:

This work is to explore the regulatory role of Burkholderia sp. GD17 in the response of rice to cadmium（Cd）stress,thus laying a foundation for reducing Cd accumulation and increasing the Cd-tolerance of rice plant. GD17-inoculated（+GD17）and non-inoculated plants with Cd addition were used as study material,and their physiological and biochemical parameters,and gene expression were analyzed. The number of GD17 reached 3.6×10⁶ CFU/g of fresh root at 5 d after inoculation,and maintained a certain size of the endophytic population during plant growth. Following exposure to Cd（20 and 40 mg/kg soil）for 20 d,the dry and fresh weights of the shoots and roots of GD17-innoculated（+GD17）plants significantly were higher than that of non-inoculated seedlings. There was no significant difference in Cd contents of the roots between +GD17 and non-inoculated rice plants,while Cd content in the +GD17 shoots was only 43% of that in the non-inoculated ones. The contents of malondialdehyde in the roots and leaves of shoots of +GD17 rice plants were the 78% and 64% of those in non-inoculated plants under Cd stress. The activity of superoxide dismutase markedly decreased in the +GD17 shoots than in the non-inoculated ones,but the activities of peroxidase and catalase increased. GD17 addition efficiently inhibited the damages from photosynthesis,such as reduced the decrease of total chlorophyll content,net photosynthetic rate and stomatal conductance,and the increase of the intercellular CO₂ concentration from Cd stress. The Cd-induced photosynthetic impairment and GD17-conferred restorative effect were also reflected on the changes of revealed by chlorophyll fluorescence parameters. The expressions of the genes related to Cd absorption,chelation,transport,and vacuolar sequestration presented a higher level in the roots of +GD17 plants than in non-inoculated ones. These data suggest that root inoculated with GD17 may systematically alleviate Cd damage to rice seedlings,and the involved mechanisms might be associated with reducing root-shoot transport of Cd,regulating antioxidant defense from Cd,preventing Cd-induced photosynthetic impairment,and increasing the expressions of Cd tolerance-related genes. This work provided a scientific basis for the potential application of GD17 strain in low-Cd rice production.

Key words: cadmium stress, Burkholderia, rice, oxidative stress, photosynthesis, chlorophyll fluorescence

祖国蔷, 胡哲, 王琪, 李光哲, 郝林. Burkholderia sp. GD17对水稻幼苗镉耐受的调节[J]. 生物技术通报, 2022, 38(4): 153-162.

ZU Guo-qiang, HU Zhe, WANG Qi, LI Guang-zhe, HAO Lin. Regulatory Role of Burkholderia sp. GD17 in Rice Seedling’s Responses to Cadmium Stress[J]. Biotechnology Bulletin, 2022, 38(4): 153-162.

图/表 7

表1 基因表达分析特异性引物

Table 1 Primer sequences used for the analysis of gene expression

基因 Gene	基因ID Gene ID	功能 Function	引物序列 Primer sequence（5'-3'）	产物长度 Length/bp
ABCG5	Os03g0281900	介导Cd进入液泡 Transporter pumping Cd into vacuole	F：AGGAGGACCTTCTGTACCCG R：CAGCATGATCGGGTTGTGGA	245
HMA1	Os06g0690700	介导Cd进入液泡 Transporter pumping Cd into vacuole	F：CCGTAGCCTTCCCACTTGTC R：ACCGCCCTCCAATGAGTTTC	140
HMA3	Os07g0232900	介导Cd进入液泡 Transporter pumping Cd into vacuole	F：CTGGCTCTGGTGATGCTTGT R：CCCAACCAGATGGAACGAGT	219
MT1a	Os11g0704500	Cd络合和活性氧清除 Cd chelation and ROS scavenging	F：TGCGGAAAGAAGTACCCTGAC R：CCTCAAACTGCTGCGCCTTC	100
MT1c	Os12g0571100	Cd络合和活性氧清除 Cd chelation and ROS scavenging	F：ATGTCGTGCGGTGGAAGTT R：TTGCAGTAGTGGTGGTGGTG	109
NRAMP5	Os07g0257200	调节Cd的根-茎转运 Regulating Cd root-shoot transport	F：GGCCTCCAAAAATACGGGGT R：GCAAGAACAGATTGTGGGGC	217
PCS1	Os05g0415200	介导络合素-Cd复合物的形成 Mediating the formation of phytochelatin -Cd complex	F：TGGATGTCGCTCGCTTCAAA R：CATGAACCCCCTGAGAAGCC	107
PCS2	Os06g0102300	介导络合素-Cd复合物的形成 Mediating the formation of phytochelatin -Cd complex	F：GCAACTGGTCTACTCAGGGG R：GCTTTCATCTCTGCAACTCTGC	101

图1 GD17菌在根中的定殖效率（以菌落形成单位表示）在种子催芽时加入GD17菌悬液,对照以蒸馏水代替。图中数据以3次独立生物学试验的平均值±标准差表示（n=3）;每次试验取材于5株植株的根。图中不同的小写字母表示在P<0.05具有显著性差异。下同

Fig. 1 Colonization efficiency of GD17 inside roots as indicated by colony-forming units（CFU） GD17 inoculation was performed during seed germination,CK was replaced with distilled water. The data were collected from three replicated experiments（n = 3）with 5 plants used in each batch of experiment,and represented as means ± SD. Bars with different lower-case letters indicate significant differences at P<0.05. The same below

图2 GD17和（或）Cd胁迫对植株生长和Cd含量的影响图中数据以3次独立生物学试验的平均值±标准差表示（n=3）;每次试验取材于20株20 d龄的植株。A和B：茎叶鲜重和干重;C和D：根鲜重和干重;E：地上部形态的代表性图片;F和G：根和地上部Cd含量

Fig. 2 Effects of GD17 and（or）Cd stress on plant grow-th and Cd contents Twenty-day-old plants were evaluated. The data were collected from three replicated experiments（n=3）with 20 plants used in each batch of experiment,and represented as means ± SD. A and B：Fresh and dry weight of shoots. C and D：Fresh and dry weight of roots. E：Representative pictures showing the morphology of aboveground part. F and G：Cd contents in roots and shoots

图3 GD17和（或）Cd胁迫对植株丙二醛含量和抗氧化酶活性的影响 A和B：根和地上部的丙二醛（MDA）含量;C和D：根和地上部超氧化物歧化酶（SOD）活性;E和F：根和地上部过氧化物酶（PRX）活性;G和H：根和地上部过氧化氢酶（CAT）活性

Fig. 3 Effects of GD17 and（or）Cd stress on malondialde-hyde（MDA）content of plants,and activities of antioxidation enzymes A and B：Malondialdehyde contents in roots and shoots. C and D：Superoxide dismutase activity in roots and shoots. E and F：Peroxidase activity in roots and shoots. G and H：Catalase activity in roots and shoots

图4 GD17和（或）Cd胁迫对光合作用相关参数的影响 A：总叶绿素含量;B：净光合速率;C：气孔导度;D：胞间CO2浓度

Fig. 4 Effects of GD17 and（or）Cd stress on photosynth-esis-related parameters A：Total chlorophyll content. B：Photosynthetic rate（Pn）. C：Stomatal conductance（Gs）. D：Intercellular CO2 concentration（Ci）

图5 GD17和（或）Cd胁迫对叶绿素荧光参数的影响 Fv/Fm：PSII最大光化学效率;ФPSII：PSII实际光化学效率;ETR：电子传递效率;NPQ：非光化学荧光猝灭系数

Fig. 5 Effects of GD17 and（or）Cd stress on chlorophyll fluorescence parameters Fv/Fm：Maximal photochemical efficiency of PSII. ФPSII：Actual photochemical efficiency of PSII. ETR：Electron transport rate. NPQ：Non-photochemical quenching of fluorescence

图6 GD17和（或）Cd胁迫对Cd耐受相关基因根中表达的影响

Fig. 6 Effects of GD17 and（or）Cd stress on the expres-sion of Cd tolerance-related genes in roots

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