Functional Study Of CiMYB32 In Response To Drought Stress Of Chrysanthemum Indicum Var. Aromaticum

Drought stress seriously affects the morphological structure,photosynthesis,physiological and biochemical processes of plants,which is an important factor restricting the application of garden plants.With the rapid development of molecular biology technology,using genetic engineering technology to cultivate drought-resistant garden plants is an important means to improve the application range of garden plants and the quality of garden landscape.Therefore,it is of great theoretical and practical value for cultivating drought-resistant garden plants to explore the key genes and study their drought-resistant mechanism and regulation mechanism.Lignin can not only reduce water loss in plants,but also promote water transport in xylem and resist drought stress.A large number of MYB transcription factors not only play an important role in responding to drought stress in plants,but also have been proved to affect plant tolerance to drought stress by regulating lignin synthesis.Chrysanthemum indicum var.aromaticum is an excellent landscaping plant with advantages of rapid growth and strong stress resistance.It is an excellent material for exploring drought resistance genes and studying drought resistance mechanism in plants.In this study,CiMYB32 gene of C.indicum var.aromaticum was screened and cloned,bioinformatics analysis was conducted on it,CiMYB32 gene plant expression vector was constructed and transformed into tobacco,drought resistance and lignin content of transgenic tobacco were studied,and the interaction protein of CiMYB32 was explored by yeast two-hybrid technology.Specific research results are as follows:1.Cis-acting element analysis was conducted on the promoter sequence of CiMYB32 gene with a length of 2000 bp,and it was found that the promoter contained stress response elements and MYB binding site elements during drought induction,etc.It was speculated that the gene might respond to drought stress and interact with other MYB transcription factors during drought stress.CiMYB32 gene was cloned and its ORF reading frame sequence length was774 bp,encoding 257 amino acids.The gene was logged on NCBI(OQ032509)and bioinformatics analysis was performed.Fluorescence quantitative PCR results showed that the expression level of this gene was the lowest in the leaves and the highest in the roots of C.indicum var.aromaticum.2.The plant expression vector pBI121-CiMYB32-GFP was constructed,and the plant expression vector was transferred into agrobacterium susceptible state,and the CiMYB32 gene was transferred into tobacco leaves by agrobacterium infection method.After resistance screening and fluorescence quantitative detection,it was proved that CiMYB32 gene had been successfully transferred into tobacco and expressed in vivo.Subcellular localization results indicated that CiMYB32 was localized in the nucleus.3.The results of 14 d natural drought stress test showed that the growth of wild-type tobacco at 6 weeks of age was better than that of transgenic tobacco.The physiological indexes showed that the proline content,SOD,POD and CAT activities of transgenic tobacco were significantly lower than those of wild-type tobacco,and the relative electrical conductivity and malondialdehyde content were significantly higher than those of wild-type tobacco.At 21 days of natural drought stress,almost all transgenic tobacco leaves were wilted and yellow,while wild-type tobacco had 2-3 wilted green leaves at the top.The recovery of wild-type tobacco was better than that of transgenic tobacco after 7 days of rehydration,and the physiological indexes were basically consistent with those obtained after 14 days of natural drought stress.Further observation found that the stems of the transgenic tobacco tilted and softened,the stem compressive strength and puncture strength of transgenic tobacco were lower than those of wild-type tobacco,and the lignin content of transgenic tobacco was significantly reduced.These results indicated that exogenous gene CiMYB32 negatively regulated drought resistance and lignin synthesis in tobacco.4.Transcriptional activity analysis showed that CiMYB32 had no transcriptional activation activity.The results of yeast two-hybrid showed that CiMYB32 protein interacts with CiMYB4 protein as well as with itself.