Analysis of germination characteristics and differential gene expression in neo-tetraploid rice under NaCl stress
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摘要:目的
评价新型四倍体水稻在不同浓度NaCl处理下的种子萌发特性、幼苗建成期形态指标和根部细胞结构差异，评估盐胁迫相关基因的表达差异。
方法在不同浓度NaCl (0、50、100、150、200和250 mmol/L)处理下，以‘华多1号’为研究材料，统计其种子在萌发时间、发芽势、发芽率和萌发指数指标的差异。在幼苗形成期，统计和比较‘华多1号’在不同浓度NaCl处理下的总根数、幼苗苗长、根长、幼苗鲜质量、含水率和根冠比参数的差异。利用WE-CLSM和塑料半薄切片技术观察根尖组织细胞的形态。利用基因组重测序、生物信息学分析和qRT-PCR技术对‘华多1号’与双亲相关的盐胁迫基因进行分析和定量验证。
结果当NaCl浓度升高时，‘华多1号’种子发芽率和萌发指数呈递减趋势，但相对盐害率和平均发芽时间呈递增趋势。当NaCl浓度高达250 mmol/L时，‘华多1号’种子相对盐害率为35%，表明其具有较强的耐盐性。在NaCl处理8~20 d后，‘华多1号’幼苗随着NaCl浓度升高，总根数呈减少趋势，同时幼苗苗长、根长、幼苗鲜质量和含水率呈下降趋势，但根冠比呈上升趋势。NaCl胁迫会抑制‘华多1号’幼苗生长，导致其根尖细胞体积变小，细胞皱缩和细胞排列松散；盐胁迫条件下，根尖伸长区的表皮和外皮层的细胞会增厚，中间皮层的细胞层数增加，中柱直径缩小，木质部导管分化数目增加。在‘华多1号’中发现有85个盐胁迫基因在CDS区中较双亲存在差异，包括53个盐敏感相关基因、32个盐胁迫抗性相关基因。qRT-PCR结果表明：与对照相比，‘华多1号’的4个盐敏感基因(LOC_Os03g16900、LOC_Os06g48510、LOC_Os02g34810和LOC_Os04g32920)和2个耐盐性基因(LOC_Os03g20090和LOC_Os11g26790)表达量显著上升。
结论该研究可为后续系统评价四倍体水稻种质资源的耐盐性、揭示四倍体水稻的耐盐性调控机理，以及后续筛选耐盐多倍体水稻品种提供理论依据。
Abstract:ObjectiveThe purpose of this research was to evaluate differences in seed germination characteristics, morphological indexes, and root cell structure in ‘Huaduo1’ under different concentrations of NaCl and to evaluate the gene expression differences of salt stress related genes in ‘Huaduo1’.
MethodUsing ‘Huaduo1’ as the research material, the differences in seed germination time, germination potential, germination rate, and germination index under various concentrations of NaCl (0, 50, 100, 150, 200, and 250 mmol/L) were analyzed. The differences in total root number, seedling length, root length, seedling fresh weight, water content, and root-to-head ratio of ‘Huaduo1’ at seedling forming stage under various concentrations of NaCl were summarized and compared. Root tip cell morphology of ‘Huaduo1’ was observed by the WE-CLSM and semi-thin section analysis. Genomic resequencing, bioinformatics analysis, and qRT-PCR analysis were conducted to analyze and quantify the expression of the salt stress genes in ‘Huaduo1’ related to its parents.
ResultThe seed germination rate and germination index of ‘Huaduo1’ decreased with increased NaCl concentration, while the relative salt damage rate and average germination time increased. ‘Huaduo1’ showed a strong salt tolerance with the relative salt damage rate reached 35% under 250 mmol/L NaCl treatment. After 8 to 20 days of NaCl treatment, the total root number of ‘Huaduo1’ seedlings decreased with increased NaCl concentration. Meanwhile, the seedling length, root length, fresh weight and water content of seedlings under NaCl stress showed a decreasing trend, and the root-to-head ratio showed an increasing trend. NaCl stress inhibited the growth of the ‘Huaduo1’ seedlings, resulting in smaller cell volume, cell shrinkage, and loose cell arrangement in the root tip elongation zone. Moreover, the cells of the epidermis and outer cortex layer in the root tip elongation zone were thickened, the number of cell layers in the mesocortex increased, the diameter of the middle column decreased, and the number of xylem ductal differentiation increased under salt stress. Totally 85 salt stress-related genes of ‘Huaduo1’ showed variance in the CDS region compared with its two parents, including 53 salt sensitivity related genes and 32 salt stress tolerance related genes. The qRT-PCR results showed that the expressions of four salt sensitivity genes (LOC_Os03g16900, LOC_Os06g48510, LOC_Os02g34810, and LOC_Os04g32920) and two salt tolerance genes (LOC_Os03g20090 and LOC_Os11g26790) were significantly increased compared with the control.
ConclusionThe study can be used to evaluate the salt tolerance of tetraploid rice germplasm, reveal the regulation mechanism of salt tolerance in tetraploid rice, and provide a theoretical basis of the salt-tolerant germplasm selection in polyploidy rice.
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Keywords:
- Neo-tetraploid rice /
- NaCl stress /
- Seed germination /
- Gene expression
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图 1 ‘华多1号’种子在不同浓度NaCl处理至7 d后的萌发情况比较
Figure 1. Comparison of seed germination of ‘Huaduo1’ after seven days under different NaCl concentrations
A~F：0、50、100、150、200、250 mmol/L NaCl
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图 2 ‘华多1号’种子在不同浓度NaCl处理下的萌发特性
各小图中，柱子上方的不同小写字母表示差异显著(P<0.05，LSD法)
Figure 2. Germination characteristics of ‘Huaduo1’ under different NaCl concentrations
In each figure, different lowercase letters on bars indicate significant differences (P<0.05, LSD method)
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图 3 ‘华多1号’幼苗在不同浓度NaCl处理下的根部生长情况
各小图中，柱子上方的不同小写字母表示差异显著(P<0.05，LSD法)
Figure 3. Root growth statistics of ‘Huaduo1’ seedlings under different NaCl concentrations
In each figure, different lowercase letters on bars indicate significant differences (P<0.05, LSD method)
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图 4 ‘华多1号’在不同浓度NaCl处理下的幼苗长度及总鲜质量的变化情况
各小图中，柱子上方的不同小写字母表示差异显著(P<0.05，LSD法)
Figure 4. Changes of ‘Huaduo1’ seedling length and total fresh weight under different NaCl concentrations
In each figure, different lowercase letters on bars indicate significant differences (P<0.05, LSD method)
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图 5 ‘华多1号’幼苗在不同浓度NaCl处理下的含水率及根冠比变化情况
各小图中，柱子上方的不同小写字母表示差异显著(P<0.05，LSD法)
Figure 5. Changes of water content and root/shoot ratio in ‘Huaduo1’ seedlings under different NaCl concentrations
In each figure, different lowercase letters on bars indicate significant differences (P<0.05, LSD method)
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图 6 不同浓度NaCl处理下根尖细胞的形态差异
A~D分别为0、50、100和150 mmol/L的NaCl溶液处理后的根尖细胞，标尺= 20 µm；E~H分别为A~D对应的分生区细胞，标尺= 40 µm；I~L分别为A~D对应的伸长区细胞，标尺= 40 µm；M~P分别为A~D对应的伸长区细胞，标尺= 100 µm
Figure 6. Morphological differences of root tip cells under different NaCl concentrations
A−D: Root tip cells under NaCl concentration of 0, 50, 100, and 150 mmol/L, respectively, bar = 20 μm; E−H: Root tip meristematic zone cells corresponding to A−D respectively, bar = 40 μm; I−L: Root tip elongation zone cells corresponding to A−D respectively, bar =40 μm; M−P: Root tip elongation zone cells corresponding to A−D respectively, bar =100 μm
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图 7 不同浓度NaCl处理下根尖伸长区横切面差异
A、B分别表示0 mmol/L NaCl溶液中处理8和20 d；C、D分别表示在50 mmol/L NaCl溶液中处理8和20 d；E、F分别表示100 mmol/L NaCl溶液中处理8和20 d；G、H分别表示150 mmol/L NaCl溶液中处理8和20 d；A1~H1：分别对应图A~H的根尖伸长区中柱结构细节；绿色和红色箭头分别表示盐胁迫处理下8和20 d后的根尖伸长区木质部；A~H：标尺=100 μm，A1-H1：标尺=20 μm
Figure 7. Transection of root tip elongation zone under different NaCl concentrations
A, B: Transection of root tip elongation zone under the 0 mmol/L NaCl treatment after the 8th day and 20th day; C, D: Transection of root tip elongation zone under the 50 mmol/L NaCl treatment afterthe 8th day and 20th day; E, F Transection of root tip elongation zone under the 100 mmol/L NaCl treatment after the 8th day and 20th day; G, H: Transection of root tip elongation zone under the 150 mmol/L NaCl treatment after the 8th day and 20th day; A1−H1: Detailed diagram of the column structure in the root tip elongation zone corresponding to figure A−H; The green arrow indicates the xylem of the root tip elongation zone under salt stress treatment after the 8th day, and the red arrow indicates the xylem of the root tip elongation zone under salt stress treatment after the 20th day; Bar = 100 μm in figure A−H, bar = 20 μm in figure A1−H1
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图 8 ‘华多1号’及双亲在各染色体的基因组变异情况
图C中，‘华多1号’分别与T44-4x和T45-4x的SNPs(粉红色)和InDels(浅绿色)的差值显示在纵轴上，正数表示H1和T44-4x之间的变化，负数表示H1和T45-4x之间的差异
Figure 8. Genomic variation of ‘Huaduo1’ and its parents on each chromosome
In figure C, the difference of SNPs (pink) and InDels (light green) in ‘Huaduo1’ is listed on the vertical axis, compared with T44-4x and T45-4x, respectively, the positive numbers represent the change between ‘Huaduo1’ and T44-4x, and the negative numbers represent the differences between ‘Huaduo1’ and T45-4x
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图 9 ‘华多1号’中基因组变异基因的GO富集分析
Figure 9. GO enrichment analysis of genomic variant genes in ‘Huaduo1’
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图 10 ‘华多1号’中盐胁迫相关基因的差异比较
“*”和“**”分别表示处理间存在0.05和0.01水平的显著差异(t检验)
Figure 10. Expression comparison of salt stress related genes in ‘Huaduo1’
“*” and “**” indicate significant differences between treatments at the levels of 0.05 and 0.01, respectively(t test)
表 1 盐胁迫相关基因的qRT-PCR引物序列
Table 1 Primer sequence for qRT-PCR of salt stress related genes
基因名称
Gene name引物序列 (5'→3')
Primer sequenceLOC_Os03g16900 F: ATAATGAAGCATCCGATT R: ATATGAGCAACAGAACAC LOC_Os03g20090 F: ACAACGAGATCAAGAACTACTG R: TTGAACTGCTGGCTGTTG LOC_Os11g26790 F: TCCAGCTCAAGCTCGTCT R: AGCTTCTCCTTGATCTTCTCCTT LOC_Os04g32920 F: TCCTCTCCCTCATCCTCT R: CATTGTCGTTGGCGTAGA LOC_Os02g34810 F: GCTTATCCAACCAATCAA R: ACATCAACTCGTCCATAT LOC_Os06g48510 F: ACACCTCGTTGCCTCATA R: ATAGCCACCATCCAGATAGTT Ubiquition F: ACCACTTCGACCGCCACTACT R: ACGCCTAAGCCTGCTGGTT 
下载: 导出CSV
表 2 不同浓度NaCl胁迫对‘华多1号’种子发芽时间的影响1)
Table 2 Effects of salt stress with different NaCl concentration on seed germination time of ‘Huaduo1’
c(NaCl)/(mmol·L−1) 发芽起始时间/d
Germination initiation time发芽终止时间/d
Germination termination time平均发芽时间/d
Average germination time0 4 5 4.25±0.030a 50 4 5 4.36±0.009a 100 4 6 4.77±0.113b 150 4 7 4.97±0.154b 200 4 7 5.19±0.182c 250 5 8 6.81±0.409d 1)同列数据后不同小写字母表示差异显著(P<0.05，LSD法)
1)Different lowercase letters in the same column indicate significant differences (P<0.05, LSD method )
下载: 导出CSV
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