草业学报 ›› 2022, Vol. 31 ›› Issue (3): 71-84.DOI: 10.11686/cyxb2020557
收稿日期:
2020-12-09
修回日期:
2021-01-06
出版日期:
2022-03-20
发布日期:
2022-01-15
通讯作者:
魏玉清
作者简介:
Corresponding author. E-mail: weiyuqing@126.com基金资助:
Zhi-heng WANG(), Yu-qing WEI(), Yan-rong ZHAO, Yue-juan WANG
Received:
2020-12-09
Revised:
2021-01-06
Online:
2022-03-20
Published:
2022-01-15
Contact:
Yu-qing WEI
摘要:
探究甜高粱响应干旱与盐胁迫的生理学差异及其分子机制,分析甜高粱在干旱和盐胁迫下的不同基因调控机制和代谢通路,可为饲草作物甜高粱抗逆栽培和育种提供依据。以辽甜1号甜高粱幼苗为材料,使用10%的PEG-6000和0.9%的NaCl模拟中度干旱和盐胁迫,胁迫2和7 d时进行光合气体交换参数、内源激素含量、有机渗透调节物质含量和抗氧化物酶活性测定,同时对幼苗叶片进行转录组测序及生物信息学分析,采用 qRT-PCR 方法对测序结果进行验证。结果表明:与对照相比,干旱和盐胁迫均显著影响甜高粱幼苗叶片生理指标的变化,但盐胁迫对甜高粱幼苗叶片光合参数和内源激素生长素、细胞分裂素含量抑制程度均高于干旱胁迫,可溶性糖含量在干旱胁迫下显著高于盐胁迫,抗氧化物酶活性和脱落酸含量低于盐胁迫,说明甜高粱对干旱和盐胁迫响应的生理机制不同;利用转录组测序技术鉴定出甜高粱叶片在处理2 d时,干旱和盐胁迫下分别有922和2047个上调基因以及975和1714个下调基因,处理7 d时分别鉴定到157和795个上调基因以及54和722个下调基因;同时鉴定出40个干旱胁迫响应基因和493个盐胁迫响应基因,基于GO富集分析发现甜高粱幼苗在干旱和盐胁迫下响应基因均显著富集于植物逆境响应相关通路,KEGG富集分析发现,干旱胁迫响应基因显著富集于内质网加工和剪切体代谢通路,盐胁迫响应基因显著富集在植物激素信号转导代谢通路;对光合作用、植物激素信号转导、抗氧化物酶和淀粉与蔗糖代谢通路相关差异基因进行分析发现,这些差异基因的表达模式与生理指标变化趋势吻合。因此,甜高粱在转录水平上对盐胁迫的适应稳态落后于干旱胁迫,中度胁迫下甜高粱幼苗对盐胁迫的耐受性要低于干旱胁迫,可溶性糖在甜高粱幼苗抵御干旱胁迫中发挥重要作用,植物激素信号转导和抗氧化物酶活性变化的共同调控是甜高粱幼苗对抗盐胁迫的关键。
王志恒, 魏玉清, 赵延蓉, 王悦娟. 基于转录组学比较研究甜高粱幼苗响应干旱和盐胁迫的生理特征[J]. 草业学报, 2022, 31(3): 71-84.
Zhi-heng WANG, Yu-qing WEI, Yan-rong ZHAO, Yue-juan WANG. A transcriptomic study of physiological responses to drought and salt stress in sweet sorghum seedlings[J]. Acta Prataculturae Sinica, 2022, 31(3): 71-84.
基因ID Gene ID | 正向引物 Forward primer (5′-3′) | 反向引物 Reverse primer (5′-3′) |
---|---|---|
gene11095 | CAAGGAGATCCTCAAGATCG | AACTTGAGGGGCTTCACCAC |
gene13164 | ACAAGCTGACTGGCGTCACT | GAGGAACTCGTGTGTCCCAG |
gene13763 | GACATCACGGGCCTCTACAT | ATGACGTACTTGGCCTCGAT |
gene17162 | ATGTGGCTCATCGACGAACT | TACGACAGGCCTTCCTGCT |
gene17374 | AATAGACCCAACTCCTGAAC | CTTCATCAAGGCTTCCACTG |
gene25496 | TCGTCATCGAGGCCTACAAG | AGGAGGGAGTTGGTGGACTT |
gene25654 | ACTCTACGAGAAGCACGAGG | TTCTTCTTCTCGTGGTGCTC |
gene31707 | GCTCATCTGCCAGGAGTACG | GTTGAGCGACTTGTAGACGG |
gene4250 | ACGTCGAGGACAATGAGACC | AGCTCGAGTACATGCAGTAG |
gene8166 | GTGGGTTATTCTTGGACACT | CTCCTATGCAGGCAATAAGC |
表1 差异基因引物序列
Table 1 Differential gene primer sequences
基因ID Gene ID | 正向引物 Forward primer (5′-3′) | 反向引物 Reverse primer (5′-3′) |
---|---|---|
gene11095 | CAAGGAGATCCTCAAGATCG | AACTTGAGGGGCTTCACCAC |
gene13164 | ACAAGCTGACTGGCGTCACT | GAGGAACTCGTGTGTCCCAG |
gene13763 | GACATCACGGGCCTCTACAT | ATGACGTACTTGGCCTCGAT |
gene17162 | ATGTGGCTCATCGACGAACT | TACGACAGGCCTTCCTGCT |
gene17374 | AATAGACCCAACTCCTGAAC | CTTCATCAAGGCTTCCACTG |
gene25496 | TCGTCATCGAGGCCTACAAG | AGGAGGGAGTTGGTGGACTT |
gene25654 | ACTCTACGAGAAGCACGAGG | TTCTTCTTCTCGTGGTGCTC |
gene31707 | GCTCATCTGCCAGGAGTACG | GTTGAGCGACTTGTAGACGG |
gene4250 | ACGTCGAGGACAATGAGACC | AGCTCGAGTACATGCAGTAG |
gene8166 | GTGGGTTATTCTTGGACACT | CTCCTATGCAGGCAATAAGC |
图1 干旱和盐胁迫下甜高粱幼苗叶片光合气体交换参数的变化同一时间的不同字母表示差异显著(P<0.05),下同。Different letters at the same time indicate significant differences (P<0.05),the same below.
Fig.1 Changes in leaf photosynthetic parameters of sweet sorghum seedlings under drought and salt stress
样品名称 Sample ID | 过滤后数据 Clean reads | 错误率 Error rate (%) | Q20值 Q20 (%) | Q30值 Q30 (%) | GC含量 GC content (%) | 定位到参考基因组上的clean reads数 The number of clean reads mapped to the reference genome |
---|---|---|---|---|---|---|
CK2_1 | 57425630 | 0.0228 | 98.89 | 96.45 | 56.48 | 55361409(96.41%) |
CK2_2 | 60983370 | 0.0226 | 98.97 | 96.66 | 56.99 | 58918196(96.61%) |
CK2_3 | 51111204 | 0.0226 | 98.98 | 96.66 | 56.47 | 49100413(96.07%) |
CK7_1 | 56728540 | 0.0241 | 98.35 | 95.14 | 57.72 | 54902246(96.78%) |
CK7_2 | 53934386 | 0.0242 | 98.31 | 95.06 | 57.05 | 52136670(96.67%) |
CK7_3 | 51439608 | 0.0239 | 98.45 | 95.34 | 55.14 | 49452077(96.14%) |
D2_1 | 52216072 | 0.0226 | 98.97 | 96.65 | 54.97 | 50289972(96.31%) |
D2_2 | 57679448 | 0.0227 | 98.95 | 96.55 | 55.20 | 55522573(96.26%) |
D2_3 | 57022218 | 0.0227 | 98.95 | 96.59 | 55.94 | 54992698(96.44%) |
D7_1 | 57220412 | 0.0240 | 98.38 | 95.20 | 56.24 | 55260378(96.57%) |
D7_2 | 52471316 | 0.0242 | 98.29 | 94.98 | 56.94 | 50624415(96.48%) |
D7_3 | 48330116 | 0.0239 | 98.42 | 95.28 | 56.13 | 46730432(96.69%) |
S2_1 | 55413052 | 0.0226 | 98.97 | 96.70 | 56.01 | 53508304(96.56%) |
S2_2 | 53001756 | 0.0227 | 98.96 | 96.61 | 55.75 | 51205821(96.61%) |
S2_3 | 50589156 | 0.0226 | 98.98 | 96.67 | 55.49 | 48505209(95.88%) |
S7_1 | 51841776 | 0.0241 | 98.33 | 95.05 | 56.38 | 50074578(96.59%) |
S7_2 | 52991606 | 0.0238 | 98.47 | 95.44 | 56.75 | 51135623(96.50%) |
S7_3 | 55635952 | 0.0239 | 98.41 | 95.27 | 55.05 | 53377926(95.94%) |
表2 样品测序和数据比对统计
Table 2 Sample sequencing and data comparison statistics
样品名称 Sample ID | 过滤后数据 Clean reads | 错误率 Error rate (%) | Q20值 Q20 (%) | Q30值 Q30 (%) | GC含量 GC content (%) | 定位到参考基因组上的clean reads数 The number of clean reads mapped to the reference genome |
---|---|---|---|---|---|---|
CK2_1 | 57425630 | 0.0228 | 98.89 | 96.45 | 56.48 | 55361409(96.41%) |
CK2_2 | 60983370 | 0.0226 | 98.97 | 96.66 | 56.99 | 58918196(96.61%) |
CK2_3 | 51111204 | 0.0226 | 98.98 | 96.66 | 56.47 | 49100413(96.07%) |
CK7_1 | 56728540 | 0.0241 | 98.35 | 95.14 | 57.72 | 54902246(96.78%) |
CK7_2 | 53934386 | 0.0242 | 98.31 | 95.06 | 57.05 | 52136670(96.67%) |
CK7_3 | 51439608 | 0.0239 | 98.45 | 95.34 | 55.14 | 49452077(96.14%) |
D2_1 | 52216072 | 0.0226 | 98.97 | 96.65 | 54.97 | 50289972(96.31%) |
D2_2 | 57679448 | 0.0227 | 98.95 | 96.55 | 55.20 | 55522573(96.26%) |
D2_3 | 57022218 | 0.0227 | 98.95 | 96.59 | 55.94 | 54992698(96.44%) |
D7_1 | 57220412 | 0.0240 | 98.38 | 95.20 | 56.24 | 55260378(96.57%) |
D7_2 | 52471316 | 0.0242 | 98.29 | 94.98 | 56.94 | 50624415(96.48%) |
D7_3 | 48330116 | 0.0239 | 98.42 | 95.28 | 56.13 | 46730432(96.69%) |
S2_1 | 55413052 | 0.0226 | 98.97 | 96.70 | 56.01 | 53508304(96.56%) |
S2_2 | 53001756 | 0.0227 | 98.96 | 96.61 | 55.75 | 51205821(96.61%) |
S2_3 | 50589156 | 0.0226 | 98.98 | 96.67 | 55.49 | 48505209(95.88%) |
S7_1 | 51841776 | 0.0241 | 98.33 | 95.05 | 56.38 | 50074578(96.59%) |
S7_2 | 52991606 | 0.0238 | 98.47 | 95.44 | 56.75 | 51135623(96.50%) |
S7_3 | 55635952 | 0.0239 | 98.41 | 95.27 | 55.05 | 53377926(95.94%) |
图5 差异表达基因统计和韦恩图CK、干旱胁迫、盐胁迫在2和7 d时的样本分别命名CK2、CK7、D2、D7、S2、S7。The samples of CK, drought stress, and salt stress at 2 and 7 days were named CK2, CK7, D2, D7, S2, S7, respectively. DEG:差异表达基因Differential expression gene.
Fig.5 DEG statistics graph and Venn diagram
图7 干旱和盐胁迫响应差异基因KEGG富集分析纵轴表示通路名称,横轴表示富集因子,点的大小表示此通路中基因个数,而点的颜色对应于不同的P-adjust范围。The vertical axis indicates the pathway name, the horizontal axis indicates the rich factor, the size of the dots indicates the number of genes in the pathway, and the color of the dots corresponds to the different P-adjust ranges.
Fig.7 KEGG enrichment analysis of DEG in response to drought and salt stress
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