Genome-wide Analysis Of Trihelix Family In Phyllostachys Edulis And Functional Identification Of PheGT8 And PheGT16

Moso bamboo(Phyllostachys edulis)is one of the most important forest products and non-timber forest resources in China.It is a large-scale woody bamboo with high economic,ecological and social value,and is important resource for the economic development and the protection of nature.Resistant stress,like low temperature and drought,have great influence on bamboo survival,which also be the reasons to limit the distribution of bamboo.Moso bamboo mainly distributes in the tropical and subtropical regions of south China,and few can grow in cold and arid north China.Previous studies have proved that trihelix transcription factors have great potentials in improving plant stress resistance.It is of great significance to carry out genome-wide identification and functional verification of trihelix genes of bamboo.In this study,bamboo seedlings were used as materials.Based on genomic information of moso bamboo,trihelix transcription factor family was identified and analyzed comprehensively.Furthermore,the expression of trihelix genes were analyzed under different abiotic stresses.Two trihelix genes were cloned and the transgenic function was verified under cold,drought and high salt stress.The main results were as follows:1.A total of 24 trihelix transcription factors of moso bamboo were identified.Phylogenetic analysis showed that 24 Phe GT proteins can be divided into six subfamilies: O,I(SH4),II(GT-1),III(GTγ),IV(SIP1)and V(GT-2).And each subfamily contained one,two,two,four,seven and eight members,respectively,in which,eight pairs of homologous genes were identified.The intron/exon structure and conserved motifs of Phe GTs were analyzed.The results showed that members of different subfamily had different gene structures and motifs.By analyzing cis-elements of Phe GTs’ promoters,there were amount of abiotic stress-related regulatory elements were found,such as cold,drought and hormone regulatory elements.2.Expression patterns of Phe GTs were analyzed.Most of Phe GTs showed a positive regulatory role in response to cold and drought stress,and a negative regulatory role in response to high salt stress.Tissue-specific expression profiling indicated that most Phe GTshighly expressed in stems,followed by leaves and roots.Based on transcriptome sequencing data of flower and shoot,expression profile of Phe GTs was constructed,and their expression patterns in different stages of flower development and different heights of shoot tips were analyzed.3.Two Phe GT genes,Phe GT8 and Phe GT16(gene ID: PH01000551G0750 and PH01002648G0060)were cloned.The CDS lengths of Phe GT8 and Phe GT16 were 1,317 bp and 1,200 bp,coding the proteins with molecular weights of 49.56 k D and 46.12 k D respectively.The isoelectric points of Phe GT8 and Phe GT16 were 6.18 and 6.13.Both of Phe GT8 and Phe GT16 contained a typical trihelix DNA binding domain.The expression of Phe GT8 and Phe GT16 were detected in roots,stems and leaves of moso bamboo by real-time quantitative PCR assay.Phe GT8 was strongly induced by cold and drought stress,but lightly expressed under high salt stress.Phe GT16 was strongly induced by cold stress,whose expression is quite low under drought and high salt stress.4.Phe GT8 and Phe GT16 were constructed into transient expression vectors 35S-e GFP,respectively,and transformed into tobacco leaf cells by Agrobacterium-mediated transformation.Expression positions of fusion vectors were observed by confocal microscopy.And the results showed that two Phe GTs located in the nucleus.5.Phe GT8 and Phe GT16 were constructed into expression vector p GBKT7,respectively,and transformed into yeast strain AH109.Results showed that Phe GT16 protein had transcriptional autoactivation activity,while Phe GT8 protein had no transcriptional autoactivation activity.6.The antisense chain Phe GT8 gene,antisense chain Phe GT16 gene and justice chain Phe GT16 gene were transferred into Arabidopsis thaliana,respectively.It was found that compared with wild type Arabidopsis,transgenic lines of justice chain Phe GT16 was significantly tolerant to cold and drought stress,but it could not grow normally under high salt stress;transgenic lines of antisense chain Phe GT16 was poorly tolerant to cold and high salt stress;transgenic lines of antisense chain Phe GT8 was not resistant to cold and high salt stress.In this study,trihelix transcription factor family and its members Phe GT8 and Phe GT16 genes were studied.It has important reference value for further exploration of the response regulation of trihelix gene in stress resistance.For the future,it is of practical significance to improve the resistance of bamboo plants through molecular means.