Study On The Function Of ClTMO5/ClLHW Gene Promoting Phosphate Uptake In Cunninghamia Lanceolata (Lamb.) Hook

Phosphorus is not only the important environmental factor but also a basic nutrient element that regulates plant growth and development.Cunninghamia lanceolata(Lamb.)Hook(C.lanceolata)is a fast-growing timber species which widely cultivated in areas with severe soil phosphate deficiency zone of 17 provinces and cities in south China,with its cultivation area and wood yield ranking first in the country.It has played an important role in China’s timber production,ecological civilization construction,and local social development.Due to the long-term low phosphate stress of Cunninghamia lanceolata,which has adversely affected its growth and development as well as people’s timber needs,TMO5 and LHW belong to the bHLH transcription factor family.Studies have found that TMO5 and LHW regulate the root architecture of plants to respond to low phosphate stress.However,research on them is mainly focused on the model plant Arabidopsis,and there are few reports on other woody plants and gymnosperms.This study used C.lanceolata as the research material,and two genes,ClTM05 and ClLHW,were cloned from C.lanceolata.Through experiments,the following research results were obtained:(1)Based on mining of the transcriptome data of C.lanceolata roots under different phosphate concentrations,we found two transcription factors,CITM05 and CILHW,that actively responded to the low phosphate stress.Through fluorescence quantitative PCR,we determined that the expression of the two factors was on the high side under the low phosphate conditions.With the increase in phosphate concentration,the overall trend of the expression level decreased.Through gene sequence alignment,protein sequence alignment,phylogenetic tree analysis,and protein three-dimensional structure prediction,the two proteins were finally identified as members of the bHLH transcription factor family.Bioinformatics analysis shows that ClLHW is an unstable protein,but ClTMO5 is a stable protein.The three-dimensional structure prediction shows that two proteins have the conservative d of the bHLH transcription factor family.(2)The expression pattern analysis of ClTM05 and ClLHW showed that both were expressed in the root,stem,leaf,and cone of C.lanceolata,but their expression amount was significantly different.ClLHW is mainly expressed in the root,while ClTMO5 is mainly expressed in the stem,but its expression level in the root is the same as that of ClLHW in the root,indicating that both plays a major role in the plant root.The expression level of ClTMO5 in the plant stem is 1.45 times that of the root,and it is speculated that it also plays an important role in the plant stem.The subcellular localization study showed that the two proteins were in the nucleus,and the yeast one hybridization experiment proved that the two proteins had the transcription activity,it suggested that they are belonging to transcription factors.(3)Through Yeast two-hybrid experiment and a bimolecular fluorescence(BIFC)experiment,the result indicated that the ClTM05 interacts with ClLHW to form a protein complex to play special function in C.lanceolata.The promoter sequences of ClTMO5 and CILHW were cloned from the genome sequence of C.lanceolata by the genome walking method.The promoter sequence of ClTMO5 was 1803 bp in total,which was completed twice.A total of 33 cis-regulating elements were detected in the promoter sequence,and the promoter of CILHW was 1884 bp in total,which was completed twice.A total of 35 cis-regulating elements were detected in the promoter sequence,and it was found that the two genes of C.lanceolata,CITMO5 and CILHW were subject to multiple hormones and stresses,and the transcription factor family,and the binding site P1BS-like of phosphate starvations response factor 1(PHR1),the key gene in response to phosphate stress,was identified.It is speculated that the upstream of CITMO5 and ClLHW in C.lanceolata may be regulated by PHR1.(4)We obtained ClTMO5 and CILHW transgenic Arabidopsis by the Agrobacterium mediated floral-dip method.Compared with the wild type.the related genes in the cytokinin biosynthesis pathway and lignin biosynthesis pathway in the transgenic plants with ClTM05 and ClLHW were upregulated.Under low phosphate stress,the number of lateral roots of the transgenic plants with CITMO5 and ClLHW was greater than that of the wild-type plants.In the phosphate recovery experiment,the phosphorus content of the transgenic plants with CITMO5 and CILHW was higher than that of the wild-type plants.In summary,the research results show that ClTMO5 and CILHW of C.lanceolata regulate the root architecture of plants by regulating the expression level of the cytokinin biosynthesis pathway and lignin biosynthesis pathway genes,which increase the contact area between roots and soil environment,and actively regulate the phosphate absorption of plants from soil.