Study on the Drought Resistance Function of the Transcription Factor CsMYB12 Gene in Industrial Hemp

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

Abstract

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

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, 2026, 42(6): 294-303.

Figures/Tables 9

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

Fig. 1 Expressions of CsMYB12 gene under drought stressDifferent letters indicate significant differences (P0.05). The same below

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

Fig. 3 Transcriptional activating activity of CsMYB12

Fig. 4 Construction of CsMYB12 plant overexpression 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

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

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

Fig. 8 Expressions of stress-related genes in CsMYB12 transgenic tobacco* and ** refer to significant difference at P0.05 and P0.01, respectively

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