Cloning And Functional Analysis Of Key Transcription Factor ClPHR1 Responding To Low Phosphorus Stress In Cunninghamia Lanceolata

Cunninghamia lanceolata is an important fast-growing tree species in China.The lack of available phosphorus in soil caused by multipe generations of continuous planting is the important factor limiting the yield of Chinese fir plantation.Therefore,screening the genotype of Chinese fir is the important direction to solve the problem of soil available phosphorus deficiency.Studies on Arabidopsis and rice have shown that the PHR1 transcription factor is centrally located in the phosphorous starvation signaling network of plants and regulates the functional expression of a series of low phosphorus stress related genes such as phosphorus transporters,microRNA and transcription factors.The study of PHR1 transcription factor in will helps to understand the sequence characteristics and functional characteristics of PHR1 transcription factor,understands the adaptation and regulation mechanism of Chinese fir to low phosphorus stress,and lays a foundation for the efficient utilization of Chinese fir germplasm resources.However,there has been no research report on the key transcription factor PHR1 in response to low phosphorus stress in Chinese fir,which is the key to reveal the regulation mechanism of Cunninghamia lanceolata to low phosphorus stress.Therefore,in this study,the clones M32 of Chinese fir,which is screened out by previous research of tutor group,is used as the test material.The full length cDNA sequence of PHR1 transcription factor was obtained by using RACE full-length cloning technology,and the protein sequence and structure characteristics of the PHR1 were analyzed,and the temporal and spatial expressing characteristics of the PHR1 gene under low phosphorus stress were analyzed.The subcellular localization of CIPHR1 gene and the genetic transformation in Arabidopsis thaliana were studied by constructing an exogenous expression vector,and analyzed the effect of CIPHR1 gene overexpression on Arabidopsis phosphorus transporter genes expression.The results will help to understand the sequence and functional characteristics of CIPHR1 transcription factor,and lay a foundation for the efficient utilization of Chinese fir germplasm resources.The main conclusions are as follows:1.Based on known PHR1 conserved sequences and sequence similarity principle,the full-length CIPHR1 sequence of Chinese fir was obtained by RACE full-length cloning.The full-length gene is 1954 bp,which an open reading frame of 1512 bp encoding a protein containing 503 amino acids.The theoretical molecular weight of the encoded protein is 55.40kDa,which is predicted to be an unstable hydrophilic protein;the protein has no signal peptide and may only exist one transmembrane region.Similar to the PHR1 gene of other plants,the CIPHR1 gene of Chinese fir has a highly conserved MYB-CC domain and is a new member of the MYB-CC family of transcription factors.Phylogenetic tree analysis revealed that the sequence of CIPHR1 was highly homologous to the sesame SiPHRl and the oil-free alfalfaAmPHR1.2.The stable internal reference genes of different tissues of Chinese fir were screened.The most stable internal reference genes analyzed by the geNorm,Normfinder and BestKeeper software were EFl? and Actin in the roots and stems;geNorm and Normfinder analysis showed that the stable internal reference genes in the leaves were UBQ and EFl?;BestKeeper showed the most stable reference genes n the leaves were EFl?and Actin.The expression of CICesAl and ClCesA2 was analyzed by different internal reference genes,and the results showed that the relative expression of CICesA 1 and ClCesA2 in Chinese fir was mainly stem>leaf>root.Among the 7 candidate genes,Actin and EF1? were stably expressed in different tissues of Chinese fir,which is suitable as an internal reference gene for expression analysis.3.CIPHR1 gene was expressed in all tissues of Chinese fir,with the highest expression was in roots,followed by leaves and stems.Under low phosphorus and non-phosphorus stress cultivation,the expression of CIPHRl gene in root for 1-7d was lower than that of control for 0 d.The expression level of ClPHR1 gene in stems and leaves increased at 1 d after low phosphorus cultivation,then decreased at 3 d and increased again at 7 days.In particular,the expression level of ClPHR1 in the stem was relatively obvious.Under the non-phosphorus stress cultivation,the expression level of ClPHR1 in the stem was higher than that in 0 d,and the expression in leaves was also increased first,then decreased and then increased,and the change was obvious.Overall,the expression of CIPHR1 gene showed a dynamic change.4.The target gene CIPHR1 was fused with N-terminal or C-terminal of the carrier PGReenII-62-SK-EGFP protein to construct the transiently expressed fluorescent vector PGReenII-62-SK-PHR1-EGFP.Through transient transfection,the protein is introduced into the plant cells and traced into the organelles.Fluorescence observation showed that the fluorescent signal of the target gene CIPHR1 was located in the nucleus,and no fluorescent signal was found in other organelles.That is,the subcell localization of ClPHR1 in the nucleus and was a MYB-CC transcription factor expressed in the nucleus.5.The specific primers of the enzyme cut site linked to the target gene ClPHR1 and the carrier pCAMBIA-1300-35S-GFP were designed to combine the target gene with the vector to construct the ClPHR1 gene overexpression vector pCAMBIA-1300-35S-PHR1-GFP.The overexpressed vector was introduced into Arabidopsis by agrobacterium tumefaciens infection.Overexpressed of ClPHR1 transgenic Arabidopsis positive plants was obtained through hygromycin resistance screening and PCR detection.The expression of P transporter gene in the young roots of the overexpressed strain was analyzed.Under the total phosphorus culture and the low phosphorus culture,the expression level of AtPht1;1,AtPht1;2,AtPht1;8 and AtPht1;9 in the overexpressed lines was significantly higher than that of the wild type,while the expression level of AtPht1;4 and AtPht1;5 was closer.That means the CIPHR1 gene had a regulatory effect on the expression of AtPhtl;1,AtPht1;2,AtPhtl;8 and AtPht1;9,and no regulate of AtPht1;4 and AtPhtl;5 expression.