转录组测序分析青稞对啶磺草胺的耐药性分析

蔡青; 翁华

转录组测序分析青稞对啶磺草胺的耐药性分析

蔡青^1,,
翁华^2, ,

1.
青海大学，青海　西宁　810016
2.
青海大学农林科学院，青海　西宁　810016

基金项目: 青海省科技厅基础研究项目（2023-ZJ-930M）

详细信息

作者简介:
蔡青（1999 —），女，硕士研究生，研究方向：杂草治理与利用（E-mail：2663146708@qq.com）

通讯作者:
翁华（1979 —），女，硕士，副研究员，研究方向：杂草治理与利用（E-mail：wenghua_0872@163.com）

中图分类号: S512
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出版历程
- 收稿日期: 2023-08-22
- 修回日期: 2024-02-09
- 网络出版日期: 2024-05-08

Analysis of resistance of hulless barley to pyroxsulam by transcription

CAI Qing^1
,,
WENG Hua^{2
, ,}

1.
Qinghai University, Xining, Qinghai　810016, Chian
2.
Academy of Agricultural and Forestry Sciences, Qinghai University, Xining, Qinghai　810016, Chian

摘要

摘要: 目的挖掘青稞代谢啶磺草胺可能涉及的重要基因，明确青稞对啶磺草胺的代谢解毒机制，为啶磺草胺的科学使用和耐受除草剂的青稞品种选育提供理论基础。方法以啶磺草胺处理0 d、1 d 和6 d的敏感青稞品种青0160以及耐药青稞品种青0306的叶片为材料，借助高通量测序技术进行转录组测序结果分析。结果 GO富集分析结果表明，啶磺草胺处理前后差异表达基因均显著富集在光合作用上。KEGG富集分析表明，啶磺草胺处理1 d后，谷胱甘肽代谢途径中抗氧化剂相关基因以及苯丙素和吡啶生物碱生物合成途径中与应激相关基因显著富集；啶磺草胺处理6 d后，与维持细胞功能的一系列生物过程及氨基酸的生物合成和代谢相关的基因显著富集。进一步分析表明，两个品种中的SOD21972、POD55052、CAT15170、DHAR59510、APX42784、GR34873、GSTs849、GSTs36979、GSTs31507、GSTs15106、GSTs20762表达上调，PSⅡ55705、Cc31194、Cc17547、Cc17551、CYP12424受到抑制；其中POD55052在敏感青稞中表达量高于耐药青稞，SOD21972、CAT15170、DHAR59510、APX42784、GR34873、GSTs849、GSTs36979、GSTs31507、GSTs15106、GSTs20762、PSⅡ55705、Cc31194、Cc17547、Cc17551、CYP12424在耐药青稞中表达量高于敏感青稞，这些基因可能参与了青稞耐啶磺草胺机制。qRT-PCR相对表达量和转录组测序结果变化趋势基本一致，证明转录组测序结果具有可靠性。结论本研究结果明确了青稞对啶磺草胺的代谢解毒机制，为青稞耐受啶磺草胺提供分子基础，对啶磺草胺安全用药及选育青稞耐除草剂品种有重要意义。
- 青稞 /
- 啶磺草胺 /
- 转录组 /
- 耐药基因
Abstract: : Objective To explore the important genes involved in the metabolism of pyroxsulam in hulless barley, and to clarify the metabolic detoxification mechanism of hulless barley to pyroxsulam so as to provide a theoretical basis for the scientific use of pyroxsulam and the breeding of hulless barley varieties tolerant to herbicides. Method The leaves of sensitive hulless barley variety 'Qing0160' and drug resistant hulless barley variety 'Qing0306' treated with pyroxsulam for 0 d, 1 d and 6 d were used as materials, and the transcriptome sequencing results were analyzed by means of high throughput sequencing technique. Result The results of GO enrichment analysis showed that the differentially expressed genes were significantly enriched in photosynthesis before and after pyroxsulam treatment. KEGG enrichment analysis showed that genes related to antioxidants and stress related genes in glutathione metabolic pathway and phenylpropanol and pyridine alkaloid biosynthesis pathway were significantly enriched after 1 day treatment, and genes related to a series of biological processes maintaining cell function and amino acid biosynthesis and metabolism were significantly enriched after 6 days treatment. Further analysis shows that the expressions of SOD21972, POD55052, CAT15170, DHAR59510, APX42784, GR34873, GSTs849, GSTs36979, GSTs31507, GSTs15106 and GSTs20762 were up-regulated, while PSⅡ55705, Cc31194, Cc17547, Cc17551 and CYP12424 were inhibited. The expression of POD55052 in sensitive hulless barley was higher than that in drug resistant hulless barley SOD21972, CAT15170, DHAR59510, APX42784, GR34873, GSTs849, GSTs36979, GSTs31507, GSTs15106, GSTs20762, PSⅡ55705, Cc31194, Cc17547, Cc17551, CYP12424 in drug resistant hulless barley were higher than sensitive hulless barley. These genes may be involved in the mechanism of resistance to pyroxsulam in hulless barley. The change trend of qRT-PCR relative expression was basically the same as that of transcriptome sequencing, which proved that the transcriptome sequencing result was reliable. Conclusion The results of this study made clear the metabolic detoxification mechanism of hulless barley to pyroxsulam which provides a molecular basis for the tolerance of hulless barley to pyroxsulam, and is of great significance for the safe use of pyroxsulam and the breeding of herbicide tolerant varieties of hulless barley.
- hulless barley /
- pyroxsulam /
- transcriptome /
- drug resistance gene

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图 1 敏感、耐药青稞对啶磺草胺的响应

A：处理前；B：处理1 d后；C：处理6 d后。

Figure 1. response of sensitive and drug-resistant hulless barley to pyroxsulam

A: before treatment; B: after 1 day treatment; C: after 6 days treatment.

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图 2 基因表达间的相关性矩形分析

Figure 2. Rectangular analysis of the correlation between gene expression

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图 3 RNA-seq数据概述A: R0 vs S0 volcano; B：R1 vsS1 volcano；C：R6 vsS6 volcano；D：The number of DEGs in each comparison combination

A：R0 vs S0 火山图；B：R1 vsS1 火山图；C：R6 vsS6 火山图；D：每个比较组合的差异表达基因数

Figure 3. Overview of RNA-seq data

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图 4 差异表达基因的GO富集分析

A：R0vsS0的GO富集分析；B：R1vsS1的GO富集分析；C：R6vsS6的GO富集分析

Figure 4. GO enrichment analysis of the DGEs

A: Analysis of R0vsS0 GO enrichment; B: Analysis of R1vsS1 GO enrichment; C: Analysis of R6vsS6 GO enrichment

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图 5 抗氧化系统相关基因表达量分析

Figure 5. Analysis of gene expression related to antioxidant system

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图 6 代谢酶GSTs相关基因表达量分析

Figure 6. Analysis of the expression of GSTs-related genes of metabolic enzymes

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图 7 光合作用相关基因表达量分析

Figure 7. Analysis of expression of photosynthesis-related genes

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图 8 qRT-PCR验证基因表达

Figure 8. qRT-PCR verification of gene expression

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表 1 Real-time qRT-PCR分析中所用引物序列信息

Table 1. Primer sequence information used in Real-time qRT-PCR analysis

基因名称 gene name	正向引物（5–3′） Sense primer（5–3′）	反向引物（5–3′） Anti-sense primer（5–3′）
gene_07062	CCAGTCAAGCATCAAAAGCA	GCAGAAAGGCCACTGTTTTC
gene_12424	CAAGGAGAGCGGTGCTAAAC	AGCTCCTCTTGCAGCTTCTG
gene_26867	GACGGAGATGTGCAGAGTGA	GTGCTGGTTTCACCACCTTT
gene_59076	ACCGCAGCATAGCAGAAGAT	AAGGGAGAAGCACCAGTTCA
ACTIN (hulless barley)	CTATTCAGGCCGTGCTTTCC	CCAGCGAGATCCAAACGAAG

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表 2 差异表达基因显著富集KEGG途径

Table 2. KEGG-pathway Significantly enriched of the DGEs

途径注释 description of pathway	差异表达基因 DEGs	途径注释 description of pathway	差异表达基因 DEGs
R0vsS0		R6vsS6
碳代谢 Carbon metabolism	139	核糖体 Ribosome	109
光合生物中的碳固定 Carbon fixation in photosynthetic organisms	41	氨基酸生物合成 Biosynthesis of amino acids	90
丙酮酸代谢 Pyruvate metabolism	43	碳代谢 Carbon metabolism	101
氨基酸生物合成 Biosynthesis of amino acids	113	乙醛酸和二羧酸代谢 Glyoxylate and dicarboxylate metabolism	36
乙醛酸和二羧酸代谢 Glyoxylate and dicarboxylate metabolism	42	光合作用-天线蛋白 Photosynthesis - antenna proteins	16
甘氨酸、丝氨酸和苏氨酸代谢 Glycine, serine and threonine metabolism	39	光合生物的碳代谢 Carbon fixation in photosynthetic organisms	33
氧化磷酸化 Oxidative phosphorylation	63	光合作用 Photosynthesis	32
柠檬酸循环（TCA循环） Citrate cycle (TCA cycle)	33	甘氨酸、丝氨酸和苏氨酸代谢 Glycine, serine and threonine metabolism	32
光合作用 Photosynthesis	40	丙氨酸、天冬氨酸和谷氨酸代谢 Alanine, aspartate and glutamate metabolism	23
光合作用-天线蛋白 Photosynthesis - antenna proteins	16	精氨酸生物合成 Arginine biosynthesis	17
丁酸代谢 Butanoate metabolism	14	莨菪烷、哌啶和吡啶生物碱生物合成 Tropane, piperidine and pyridine alkaloid biosynthesis	9
磷酸戊糖途径 Pentose phosphate pathway	31	2-氧代羧酸代谢 2-Oxocarboxylic acid metabolism	22
R1vsS1		丙酮酸代谢 Pyruvate metabolism	28
核糖体 Ribosome	50	烟酸和烟酰胺代谢 Nicotinate and nicotinamide metabolism	11
光合作用-天线蛋白 Photosynthesis -antenna proteins	9

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