Identification And Expression Profiling Of Lhc Genes In Ramie

Ramie is an important fiber crop in the world,and it is also an important raw material crop in Chinese import and export trade.With changes in the land environment of large-scale cultivation,the influence of abiotic stress on ramie growth becomes more significant.Using molecular breeding techniques to improve ramie stress resistance and improve fiber quality has always been an important direction of ramie genetic breeding.However,the research on gene functions such as plant morphogenesis and photosynthesis is still in the exploration stage.Photosynthesis is an important life process for organisms to obtain energy,and capturing and transmitting light energy is the beginning of plant energy utilization.In higher plants,Lhc protein is an antenna protein for light energy absorption and transmission,and its coding gene Lhc family genes play an indispensable role in the establishment of light morphology and the maintenance of plant life in response to stress.Ramie has the characteristics of rapid growth,can be harvested for three times a year,and has strong environmental adaptability,which is closely related to its high assimilation efficiency.In this study,the ramie cultivar "Huazhu 5" was used as the material.Based on the 3 transcriptome databases established in the previous study,the Bn Lhc genes were identified.The expression pattern of Bn Lhc genes was explored,which lay the foundation to identify and analyze of ramie light-harvesting protein and the green tissue specific genes.The main results are as follows:(1)From the three transcriptome databases,14 ramie Lhc gene CDSs were screened and identified,and the two gene families Lhca and Lhcb contained 6 and 8 each.The full length of the gene coding region(from 5'ATG start to stop codon)was cloned.Cluster analysis of the two family members and the corresponding amino acid sequences in Arabidopsis,poplar and mulberry,respectively,found that the ramie Lhca and Lhcb genes include 5 and 7 subfamilies,respectively,which are named according to the classification(BnLhca1,BnLhca2.1,BnLhca2.2,BnLhca3,BnLhca4 and BnLhca5;BnLhcb1.1,BnLhcb1.2,BnLhcb2,BnLhcb3,BnLhcb4,BnLhcb5,BnLhcb6 and BnLhcb7).Using the full-length gene sequence as a template,the structure of the genes was analyzed and cloned,which was used to carry out follow-up analysis.(2)Analyze the biological information amino acid sequences of BnLhca and BnLhcb protein,such as transit peptides,transmembrane domains and conserved domains to obtain 14 Motif elements,of which,Motif1,Motif5,Motif7 and Motif8 are conserved domains of BnLhcb protein sequences.There are 11 genes with obvious chloroplast transit peptides at the N-terminus;the remaining Motif elements distributed in different subfamilies play their respective functions,and also predict protein physicochemical properties,subcellular localization and tertiary structure.All Lhc genes were compared and confirmed in the genomic sketch database,and the area of the 5'non-coding region(about 2000 bp)upstream of the 14genes ATG was analyzed and predicted,and the promoter position range was determined to obtain the distribution of cis-acting elements such as light-responsive elements.(3)The expression pattern of ramie Lhc gene in different parts of ramie and light-dark cycle was explored by qRT-PCR.Firstly,the expression of the two gene families Lhca and Lhcb in leaves,stems(tender shoots and mature stems)and roots was studied,and all genes were found to have the highest expression in green tissues(leaf and stem).The expression levels in roots and mature stems are weak.Then,in the experiment simulating the diurnal light-dark cycle(16 h light / 8 h darkness),it was found that the expression level during the day was significantly higher than that at night,and a clear bell-shaped curve distribution with time over 24 hours was presented.(4)According to the spatiotemporal expression pattern,the expression of Lhc gene in ramie leaves during the day under different abiotic stresses was explored.It was found that the expression of BnLhca and BnLhcb gene families were significantly affected by high temperature,low temperature,drought and high salt stress.The expression levels of BnLhca were down-regulated under four stress conditions;the expression levels of BnLhcb1.2,BnLhcb2 and BnLhcb3 were up-regulated under low-temperature stress,but under other stresses,their expression levels were down-regulated.(5)Fourteen ramie Lhc gene promoter region cis-acting elements were analyzed.The results showed that conserved elements AAGAA-motif,TATA-box,CAAT-box and GATAbox were distributed on them,as well as homeopathic elements related to response of light,drought and low temperature.The ramie BnLhcb1.1 gene ATG upstream 1634 bp DNA sequence(BnLhcb1.1pro-1634)and three truncated fragments of different lengths(BnLhcb1.1pro-1159,BnLhcb1.1pro-855 and BnLhcb1.1pro-344)were cloned and transferred to Arabidopsis thaliana to verify the results of GUS histochemical staining of different strains at seedling stage.It was found that cotyledon-specific expression characteristics began to appear on the 4th day after the seedlings of each strain germinated.Truncated to 344 bp can still drive the GUS reporter gene and can be stably expressed in cotyledons and true leaves during the growth period of 4 d-10 d.