Cloning And Function Analysis Of LobHLH34 Gene From Larix Olgensis

Larix olgensis is one of the main timber species for afforestation in Northeast China.It plays an important role in the protection of ecological environment,but its growth cycle is long.Somatic embryogenesis and transgenic technology can rapidly improve the quality of forest trees,which is one of the hotspots in the field of forest breeding.bHLH(basic/helix loop helix)is one of the largest transcription factor families in plants.It can respond to the stress of low temperature,high salt,drought and various exogenous hormones,so it is widely used in the research of plant stress resistance.In this study,LobHLH34 gene was cloned from the genome of Larix olgensis.The bioinformatics method was used to analyze the physicochemical properties and function prediction of the gene,and the expression pattern of the gene under different stresses of adversity was analyzed by qRT-PCR,and then the function of the gene was verified by exogenously transforming Nicotiana tabacum and natively transforming Larix gmelinii.The main results are as follows:(1)The complete CDS sequence of LobHLH34 gene is 696 bp,encoding 231 amino acids.The theoretical isoelectric point(PI)of the protein was 7.73,the liposoluble index was 61.73,the total average hydrophilic coefficient was-0.830,and the instability coefficient was 67.17,indicating that the protein was an unstable acidic hydrophilic protein.Phylogenetic tree analysis showed that the gene was closely related to Picea asperata and Selaginella tamariscina.(2)The tissue-specific expression of LobHLH34 gene in Larix olgensis and its response to abiotic stress were analyzed by qRT-PCR.The results showed that the gene was expressed in its roots,stems and leaves,but expression of different tissue parts were different,with the highest expression in leaves and the lowest in stems.The expression of LobHLH34 was up-regulated under NaCl,PEG and ABA stress.(3)A subcellular localization expression vector was constructed and transiently transform the into xylem protoplasts of Populus trichocarpa,and the fluorescent signal was observed by laser confocal microscopy,which showed that the protein encoded by LobHLH34 gene was localized in the nucleus,consistent with the expression characteristics of transcription factors.(4)The LobHLH34 plant overexpression vector was constructed,and the gene function was verified by transformation of model plant Nicotiana tabacum through Agrobacterium-mediated method,and the stress-related physiological indexes of transgenic plants were measured under NaCl stress and PEG treatment to analyze their stress resistance.The results showed that the chlorophyll content of the transgene Nicotiana tabacum plants was higher than that of the wild type under NaCl and PEG stress,and the SOD and POD activities of the transgenic strains were higher than that of the wild type,while the MDA content was the opposite.The results showed that the transfer of the LobHLH34 gene improved the antioxidant capacity of Nicotiana tabacum and the transgenic plants also suffered less membrane damage than the wild type.(5)Eighteen resistant embryonic cell lines were obtained by transformation of Larix olgensis embryonic callus by Agrobacterium mediated method.The initial transformation rate was 83.3%,and five of the resistant cell lines with darker blue color were tested by PCR,and the results were positive.Transition culture and somatic embryo induction of transgenic resistant embryonic callus of Larix olgensis and germination of somatic embryos to obtain resistant plants.In addition,the stress-related physiological indexes of transgenic embryonic callus were analyzed under NaCl,PEG and ABA treatments,and the results showed that the salt tolerance and drought resistance of transgenic Larix embryonic callus were enhanced compared with the control.