Functional Analysis Of Flowering Regulation Of Glycine Max GmHLH63 In Transgenic Soybean

Soybean has been an important food and cash crop in China because of its rich oil content and plant protein that is beneficial to human absorption.At the same time,soybean is a very common short-day crop,which has a relatively sensitive response to changes in the light time,which results in the limitation of the range of suitable varieties of soybean varieties and greatly limits the possibility of introduction in cross-region.Therefore,the study on the functions of soybean flowering regulatory genes and the molecular mechanisms involved in them is of great significance and value for accelerating soybean variety improvement and increasing soybean yield and quality.The results of bioinformatics analysis showed that the GmHLH63 gene was located on the chromosome 6 of soybean and it was 550 bp in length and encoded 110 amino acids.The promoter cis-acting elements found in the upstream gene regulatory region contained AE-box,AT1,GAG,and GT1.There are numerous promoter elements that may respond to the photoperiod.The GmHLH6 protein contains a conserved DNA domain consisting of 34 amino acids and belongs to the HLH family.It shares high homology with the bHLH transcription factor in many dicotyledonous plants.Protein structure predictions indicate that GmHLH63 is a hydrophobic protein with no transmembrane domain and no signal peptide sequence.The predicted location is on the nucleus.The expression pattern of the gene was analyzed by qRT-PCR and it was found that the gene was expressed in various organs of soybean,and the expression level was relatively high in the root and single leaf and three compound leaves.The expression rhythm during the growth period indicated that the results showed that the expression level of the gene increased during the later period of vegetative growth,which may be related to the transition from vegetative growth to reproductive growth.Under short-day conditions,the expression of this gene was higher than that of long-day sunlight,and there was no significant change in the expression level,and no significant peak was observed.Comparing the expression pattern of the gene under long-day and short-day conditions,the time when the expression peaked changed,which proved that the gene responded to the photoperiod and the expression pattern changed.Construction of Fusion Vector p1302-eGFP-GmHLH63 by DNA Recombination Technology to inserted the sequence of GmHLH63 into the p1302 vector upstream of GFP to construct a fusion expression vector.The subcellular localization of tobacco epidermal cells revealed that the GmHLH63 protein was localized in the nucleus.The sequence of GmHLH63 was inserted into pGBKT7 for yeast transformation and also showed transcriptional self-activation activity.This is consistent with the characteristics of transcription factors.In summary,the gene is a bHLH family transcription factor.Inserted theequence of GmHLH63 into the pTF101 overexpression vector and the recombinant overexpression vector was introduced into the recipient Huachun 6 and overexpressed in soybean to obtain 4 transgenic events.The phenotypic analysis of the transgenic plants showed that under the long-day conditions,compared with Huachun 6 as control,the transgenic lines showed more obvious phenomenon of early flowering.In this study,through the overexpression of the target gene,the function of GmHLH63 gene was preliminarily identified,which may be involved in the photoperiodic flowering regulation of soybean.This provides a theoretical basis for further understanding of the mechanism of soybean flowering regulation.