工业大麻转录因子CsMYB12抗旱功能的研究

doi:10.13560/j.cnki.biotech.bull.1985.2025-0782

摘要/Abstract

摘要：

目的 MYB转录因子家族成员在植物中数量众多，它们在调控植物的生长发育及适应外界环境胁迫中发挥重要作用。工业大麻作为经济作物，用途广泛，具有极大的开发利用潜力。揭示工业大麻转录因子基因CsMYB12的抗旱功能，为工业大麻品种的抗旱性改良及产量提升奠定基础。方法通过实时荧光定量PCR检测CsMYB12在干旱胁迫下的表达；克隆CsMYB12并对其进行生物信息学分析；通过酵母转录激活试验检测CsMYB12转录激活活性；构建CsMYB12植物表达载体转化烟草，并对转基因烟草的抗旱性进行鉴定。结果干旱胁迫可以显著诱导CsMYB12上调表达。CsMYB12开放阅读框序列长1 560 bp，编码519个氨基酸。CsMYB12蛋白分子量为5.81 kD，等电点为4.94。CsMYB12蛋白含有2个SANT结构域，是一个典型的R2R3-MYB转录因子。CsMYB12在酵母细胞中具有转录激活活性。经鉴定，共获得6株CsMYB12转基因烟草植株。干旱胁迫及复水处理后，CsMYB12转基因烟草的表现优于野生型烟草。干旱胁迫后，与野生型烟草相比，CsMYB12转基因烟草中的渗透调节物质含量、相对含水量和抗氧化酶活性增加，电解质渗透率和丙二醛含量下降。CsMYB12转基因烟草中抗逆相关基因（NtLTP、NtOsmotin、NtLEA5、NtERD10B和NtCSD）的表达量显著升高。结论 CsMYB12在烟草中的异源超表达提高了转基因烟草的抗旱性。

关键词: 工业大麻, 转录因子, MYB, 转基因烟草, 抗旱性

Abstract:

Objective There are numerous MYB transcription factor family members in plants. They play a crucial role in regulating the growth and development of plants as well as in enabling plants to adapt to external environmental stress. Industrial hemp (Cannabis sativa L.), as an economic crop, has a wide range of applications and holds great potential for development and utilization. Revealing the drought resistance function of the transcription factor gene CsMYB12 in industrial hemp may lay a foundation for improving the drought resistance and yield of industrial hemp varieties. Method The expression of CsMYB12 under drought stress was detected by real-time fluorescent quantitative PCR. CsMYB12 was cloned and subjected to bioinformatics analysis. The transcriptional activation activity of CsMYB12 was detected through yeast transcriptional activation test. The CsMYB12 plant expression vector was constructed and transformed into tobacco. The resistance of CsMYB12 transgenic tobacco to drought was identified. Result The expressions of CsMYB12 were significantly induced by drought stress. The open reading frame of CsMYB12 was 1 560 bp and encoded 519 amino acids. The molecular weight of the CsMYB12 protein was 5.81 kD and its isoelectric point was 4.94. The CsMYB12 protein contained two SANT domains and it was a typical R2R3-MYB transcription factor. CsMYB12 had transcriptional activation activity in yeast cells. Six CsMYB12 transgenic tobacco plants were identified. After drought stress and rehydration treatment, the performance of CsMYB12 transgenic tobacco was superior to that of wild-type tobacco. After drought stress, the content of osmotic regulatory substances, relative water content and antioxidant enzyme activities in CsMYB12 transgenic tobacco increased, while the electrolyte leakage rate and malondialdehyde content decreased compared with the wild-type tobacco. The expressions of stress-related genes (NtLTP, NtOsmotin, NtLEA5, NtERD10B and NtCSD) in CsMYB12 transgenic tobacco significantly increased. Conclusion The heterologous overexpression of CsMYB12 in tobacco enhances the resistance of transgenic tobacco to drought.

Key words: industrial hemp, transcription factor, MYB, transgenic tobacco, resistance to drought

翟莹, 高双, 计俊杰, 于海伟, 赵艳, 马天意, 张梅娟, 李珊珊. 工业大麻转录因子CsMYB12抗旱功能的研究[J]. 生物技术通报, doi: 10.13560/j.cnki.biotech.bull.1985.2025-0782.

ZHAI Ying, GAO Shuang, JI Jun-jie, YU Hai-wei, ZHAO Yan, MA Tian-yi, ZHANG Mei-juan, LI Shan-shan. Study on the Drought Resistance Function of the Transcription Factor CsMYB12 Gene in Industrial Hemp[J]. Biotechnology Bulletin, doi: 10.13560/j.cnki.biotech.bull.1985.2025-0782.

图/表 9

表1 PCR引物序列

Table 1 PCR primer sequences

基因 Gene	GenBank登录号 GenBank accession number	正向引物 Forward primer （5′‒3′）	反向引物 Reverse primer （5′‒3′）	用途 Use
CsMYB12	XM030631399	GAAGAAGGAAGTGGTGGTTGTGG	AGCAGTAGCAGGAACAGGAGATAC	RT-qPCR
CsEF1a	JP452083	TGTTTTGCACGGATCAGTTTG	AATGCCGACCGCTACAGTTC	RT-qPCR
CsMYB12	XM030631399	ATGGGTAGGGCTCCTTGCTG	TCAGGATAGAAGCCATGCAACC	PCR
NtActin	AB158612	TTGCTGGTCGTGATCTTACTGATTG	CAGTCTCCAACTCTTGCTCATAGTC	RT-qPCR
NtLTP	AB625593	TTAAGGGCATTGATATGGGCAACG	GTGGAGGGACTGATCTTGTAGGG	RT-qPCR
NtOsmotin	M29279	CTCCTTGCCTTGGTGACTT	ACCGCCTATGGGTGTCG	RT-qPCR
NtLEA5	AF053076	TGGCTCGCTCTTTCTCTAACTCC	CTCACTCCACCTGGCACACTAG	RT-qPCR
NtERD10B	AB049336	TCCCATTCGTCAAACCG	CCCACCAAGTATGCCAGT	RT-qPCR
NtCSD	EU123521	CCATTACCGACAAGCAGATTCCTC	CAACCCTTCCACCAGCATTTCC	RT-qPCR

图1 干旱胁迫下CsMYB12的表达不同字母表示差异显著（P<0.05）。下同

Fig. 1 Expressions of CsMYB12 gene under drought stressDifferent letters indicate significant differences (P<0.05). The same below

图2 CsMYB12核苷酸及其编码蛋白氨基酸序列蓝色代表SANT结构域；*代表终止密码子

Fig. 2 Nucleotide of CsMYB12 and amino acid sequences of its encoded proteinBlue refers to the HSANT domain; * refers to the termination codon

图3 CsMYB12转录激活活性分析

Fig. 3 Transcriptional activating activity of CsMYB12

图4 CsMYB12植物表达载体的构建及转基因烟草鉴定A：CsMYB12重组载体的构建；B：T0代转基因烟草的PCR检测；C：T0代转基因烟草的RT-qPCR检测。M：DL2000分子量标记物；1：pRI101-CsMYB12质粒双酶切；2：pRI101-CsMYB12农杆菌菌液PCR；+：pRI101-CsMYB12质粒；WT：野生型烟草；OE1-OE6：转基因烟草。下同

Fig. 4 Construction of CsMYB12 plant expression vector and identification of transgenic tobaccoA: Construction of CsMYB12 recombinant vector. B: PCR detection of T0 generation transgenic tobacco. C: RT-qPCR detection of T0 generation transgenic tobacco. M: DL2000 marker for molecular weight. 1: Double enzyme digestion of pRI101-CsMYB12 plasmid. 2: Agrobacterium tumefaciens PCR of PRI101-CsMYB12. +: PRI101-CsMYB12 plasmid. WT: Wild-type tobacco. OE1-OE6: CsMYB12 transgenic tobacco. The same below

图5 CsMYB12转基因烟草在干旱及复水处理下的表型A：停止浇水前；B：停止浇水20 d；C：停止浇水34 d；D：恢复浇水5 d

Fig. 5 Phenotype of CsMYB12 transgenic tobacco under drought and rehydration treatmentA: Before stop watering. B: Stop watering for 20 d. C: Stop watering for 34 d. D: Rehydration for 5 d

图6 CsMYB12转基因烟草干旱胁迫下的生理生化指标

图7 CsMYB12转基因烟草干旱胁迫下叶片DAB和NBT染色A：DAB染色；B：NBT染色

Fig. 7 DAB and NBT staining of CsMYB12 transgenic tobacco leaves under drought stressA: DAB staining. B: NBT staining

图8 CsMYB12转基因烟草胁迫相关基因的表达*和**分别表示在P<0.05和P<0.01水平上差异显著

Fig. 8 Expressions of stress-related genes in CsMYB12 transgenic tobacco* and ** refer to significant difference at P<0.05 and P<0.01, respectively

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