Study On The Growth Regulation Function Of LcHDZ1 Gene On Lotus Corniculatus L. Under Drought Stress

Drought seriously affects the yield and quality of herbage,which is one of the important limiting factors for the development of grass industry.Plant Homeodomain-leucine Zipper（HD-ZIP）transcription factors are a class of specific proteins composed of the homologous domain（HD）and leucine zipper（LZ）domains.HD domain specifically binds to DNA,and LZ domain is the motifs of dimerization.They work together to regulate downstream target genes to participate in plant growth and development process and abiotic stress response.In this paper,the LcHDZ1 gene of HD-ZIP transcription factor was cloned from Lotus corniculatus L.,and was studied of its function in plant growth under drought stress including heterologous and overexpressed expression in model plants Arabidopsis thaliana and Lotus corniculatus L.respectively.The main research results are as follows:The 801 bp LcHDZ1 gene was cloned,encoding 261 amino acids,containing typical HD and LZ domains,belonging to the HD-ZIPI subfamily.Cis-acting elements include a core promoter involved in transcription initiation（TATA-box）,development of associated photo responsive elements（Box-4,G-box,GT1-motif）,a drought-inducing element in response to stress（MBS）,a response to ABA and Me JA element（ABRE）and（TGACG-motif,CGTCA-motif）respectively.LcHDZ1 was mainly expressed in the roots and leaves tissues in Lotus corniculatus L..It was induced by exogenous ABA and PEG6000 drought stress,and localized in the nucleus.The results showed that LcHDZ1 gene is related to plant growth regulation under stress.Six transgenic plants（TP）were obtained from Arabidopsis thaliana by Agrobacterium mediated flower dipping method.It was indicated that TP11,TP9 and TP4 plants with high,medium and low LcHDZ1 gene expression showing stronger drought stress symptoms than their wild-type parents（WT）under natural drought stress in the growth and development analysis of T₃ generation TP Arabidopsis thaliana.The germination rate and root length of TP11,TP9 and TP4 were lower than those of WT（P<0.05）,and the contents of MDA and Pro were higher than those of WT（P<0.01 or P<0.05）.Meanwhile,water loss rate and relative electrical conductivity of leaves were also increased.Further studies showed that the transcription of NCED3,a key gene for ABA synthesis in TP11,was inhibited（P<0.01）.The expression of ABI1,ABI2 and ABI5 genes encoding ABA negative regulator PP2Cs and stomatal regulator MYB60was also induced（P<0.01）.The transfer of LcHDZ1 gene increases drought sensitivity in Arabidopsis thaliana,which is related to ABA and stomatal regulation.Four TP plants were obtained through Agrobacterium-mediated genetic transformation of LcHDZ1 gene in the target plant of Lotus corniculatus L..The TP7,TP9 and TP5 plants with high,medium and low LcHDZ1 gene expression were treated with drought stress.It was found that under natural drought stress,TP root showed more severe drought stress symptoms,but the underground root system（root number and root length）had no significant effect on drought stress growth.After 9 days of simulated drought treatment with 30%PEG6000,the relative chlorophyll content was not significantly different from that of WT.The plant height of TP7 plants with high expression of LcHDZ1 gene was significantly lower than that of WT,and the contents of malondialdehyde（P<0.01）and proline in root and leaf tissues of three TP lines were higher than those of WT.The relative electrical conductivity（P<0.01）and water loss rate of leaves were higher than those of WT.The relative electrical conductivity of TP7root tissue with the highest LcHDZ1 expression was significantly higher than that of WT（P<0.05）.Gene differential expression analysis of PEG6000 simulated drought treatment for 24 h showed that the transcription of PP2C in TP was induced by drought（P<0.01）,while ABA receptor signaling gene PYL,ABA activated protein kinase and its targeted protein genes SnRK2.1,SnRK2.2,ABF transcription were inhibited（P<0.01or P<0.05）.LcHDZ1 gene may be involved in drought stress regulation in aboveground and underground parts of rhizome by inhibiting ABA signaling.By studying the heterologous and excessive expression of LcHDZ1 in Arabidopsis thaliana and Lotus corniculatus L.,It was found that LcHDZ1 gene is involved in the morphological and physiological regulation under drought stress through LcHDZ1-mediated ABA synthesis and signal transduction pathway.It is speculated that LcHDZ1gene negatively regulates drought stress response of Lotus corniculatus L.by mediating ABA pathway.