Study On The Regulation Function Of Photoperiod Response Gene StH2A On Growth

Different plants respond differently to photoperiod,and little is known about how plants respond to photoperiod at the molecular level.In this paper,the expression profiles of different photoperiod transcripts were obtained by sequencing the potato leaves with different photoperiod treatments.The expression profiles were analyzed,and the photoperiod response genes were identified and screened.The photoperiod response gene StH2A full-length cDNA was cloned.The expression of antisense StH2A cDNA in the Nicotiana benthamiana was studied by the genetic engineering pathway.The results showed that H2A was involved in the top-level maintenance and regulation of flowering.The results were as follows:1.The differential gene was identified as "nucleic acid binding protein" by transcriptome sequencing,GO function annotation and differential expression gene analysis.Compared with the expression of RPKM in these photoperiod genes,20 genes with significant expression of photoperiod were screened,and qRT-PCR confirmed its expression with the photoperiod.The genes with H3.2,H2A and TP were further studied.2.Cloning the full-length cDNA of H3.2,H2A and TP genes.In order to achieve the excess or inhibitory expression of the genes in vivo,through sub-cloning method,the recombinant strain DH5?-pC-35S-H3.2+/-,DH5?-pC-35S-H2A+/-,DH5?-pC-35S-TP+/-and GV3101-pC-35S-H3.2+/-,GV3101-pC-35S-H2A+/-,GV3101-pC-35S-TP+/-were obtained.3.Using the Agrobacterium tumefaciens-mediated pathway,the antisense StH2A was transformed and screened to obtain the h2a-2 mutant lines.And h2a-2 showed the phenotype of terminal deficiency and flowering delay.The results of H2A protein ELISA showed that the content of H2A protein in leaves of h2a-2 strain decreased by 23.7%compared with WT,indicating that the expression of NbH2A was inhibited by the expression of antisense StH2A.4.The genes of CONSTANS(CO),FLOWING LOCUS T(FT)and EARLY FLOWERING 4(ELF 4)were identified by searching the genome database.Design their specific primers based on their nucleic acid sequence,leaf mRNA as the template.The results of RT-PCR showed that the expression of three flowering genes in h2a-2 strain showed different degree of down-regulation,indicating that NbH2A was involved in the transcription of three flowering genes,and CO,FT and ELF4 are linked to each other through H2A.5.Twenty-two genes of NbH2A were identified,and the sequence analysis and phylogenetic tree analysis showed that four of the NbH2A were highly similar to the predicted amino acid sequence of StH2A in this study and had N-terminal poly-alanine motif,antisense StH2A at least inhibited the expression of N-terminal poly-alanine NbH2A.6.The results of RT-PCR showed that the accumulation of NbH2A mRNA in the leaves of h2a-2 strain was significantly less than that of the control.The accumulation of other non-polyalanine NbH2A mRNA was more than that of the control.Indicating that the expression of other NbH2A genes at the transcriptional level compensate for the down-regulated poly alanine NbH2A.It is concluded that the expression of antisense StH2A in vivo the tobacco inhibits the accumulation of poly alanine NbH2A mRNA.Although the accumulation of other non-polyalanine NbH2A mRNA increased,did not compensate for the decrease in the accumulation of H2A protein.H2A protein accumulation decreased,leading to the lack of top dominant and flowering delayed phenotype,indicating that the poly-alanine-encoding NbH2A gene positive regulation of flowering and growth of the Nicotiana benthamiana.