Investigation On The Function Of PaHK3a And PaHK3b Gene In Populus Alba × P.glandulosa(84k)

Plant responses to drought or salt stresses through a series of physiological and biochemical reactions to survive and maintain normal growth.During this process,signal transducers and transcription factors play important roles in receiving and transacting stress signal to regulate the expression of downstream genes.Histidine kinase belongs to the family of protein kinase and is an important part of the two-component system of plants.It can bind to cytokinins and regulate plant growth and development,such as plant cell differentiation,leaf aging regulation and root elongation.Previous study showed that histidine kinase also played an important role in plant resistance to abiotic stress such as drought and high salinity.Perennial woody plants have a long life and need to deal with abiotic stress during their lifetime,and the anti-stress function of histidine kinase in woody plants has not been reported.In this study,the in vitro leaves of Populus alba×P.glandulosa‘84K’were used as plant materials,and the expression of histidine kinase genes PaHK3a and PaHK3b under different plant hormone treatments and abiotic stresses was detected,and the drought and salt tolerance of PaHK3b was preliminarily identified by prokaryotic expression method.Further,the expression vectors of PaHK3a and PaHK3b gene CRISPR/Cas9 gene editing plants were constructed,and the genetic transformation of‘84K’poplar was carried out and transgenic plants were obtained.The main research results are as follows:1.The CDS and promoter region of PaHK3b gene was cloned,and the conservative structure domain and promoter cis-acting element were analyzed.The PaHK3b gene encodes a typical cytokinin receptor domain,such as CHASE,and the promoter region contains multiple cis-acting elements involved in abiotic stress and hormone signal transduction,such as:defense and stress response elements TC-rich repeats,gibberellin response elements GARE-motif,etc.These results showed that the possible role of PaHK3b gene in poplar response to plant hormone and abiotic stress.2.The expression patterns of PaHK3a and PaHK3b genes in leaf of‘84K’plant were detected under abiotic stress and plant hormone treatment.The results of q RT-PCR showed that PaHK3a and PaHK3b genes were expressed in roots,stems and leaf,with leaf had the highest expression.The expression of PaHK3a and PaHK3b genes were up-regulated under abiotic stress such as high temperature,low temperature,drought and salt stress.Under the treatments of plant exogenous hormones（6-BA,ABA,GA3 and SA）,the expression of PaHK3a and PaHK3b genes were down-regulated,while under the treatment of IBA,the expression of PaHK3a and PaHK3b genes was not significantly different from that of the control.The results showed that both PaHK3b and PaHK3a participated in the signal response of plant hormones and played an important role in poplar response to abiotic stresses.3.The physiological parameters（MDA content,SOD and POD activity etc.）and the expression of PaHK3a and PaHK3b genes were detected in leaf of‘84K’plants under artificial drought and salt stress in greenhouse.At different stress times of drought and salt treatment,the PaHK3a、PaHK3b gene in leaves of greenhouse grown‘84K’plants increased significantly,with the style of increased first then decreased;and the MDA content in leaves also had the similar style.SOD and POD activity were measured in drought and high salt,MDA content increased first and then decreased;SOD activity in leaves increase constantly during drought and salt stresses,and POD activity first increased and then decreased under drought stress,while increased constantly during salt stress.The correlation analysis between physiological indexes and PaHK3a、PaHK3b gene expression found that under drought and salt stress,the expression of PaHK3a、PaHK3b gene was positively correlated with MDA content,SOD and POD activity in leaves.Therefore,PaHK3a and PaHK3b genes were closely related to‘84K’poplar physiological indexes,indicating that PaHK3a and PaHK3b genes play an important regulatory role in poplar drought tolerance and salt tolerance.4.By constructing prokaryotic expression vector and transferring it into Escherichia coli,the antistress function of PaHK3b gene was preliminarily identified.The growth of the E.coli strain with PaHK3b prokaryotic expression construction in LB liquid medium supplemented with 5%PEG6000 were faster than the control.Meanwhile,the growth of the E.coli strain with PaHK3b prokaryotic expression construction on solid LB contained 50～150 mmol·L^-1Na Cl was better than the control.Therefore,the results of prokaryotic expression confirmed that the PaHK3b gene played an important regulatory role in response of poplar to drought and salt stresses.5.YLCRISPR/Cas9-DH-PaHK3a and p YLCRISPR/Cas9-DH-PaHK3b expression vectors were constructed,and transgenic plants were obtained through agrobacterium-mediated transformation genetic transformationof‘84K’leaf disc.Two targeted sites were designed respectively in PaHK3a and PaHK3b genes,and the gene editing expression vectors（YLCRISPR/Cas9-DH-PaHK3a and YLCRISPR/Cas9-DH-PaHK3b）were constructed and then genectic transformation of‘84K’polar were performed.After screening for hygromycin resistance,750 esistant buds and 80 resistant plants of PaHK3a gene editing were obtained,and 900 esistant buds and 107 resistant plants of PaHK3b gene editing were obtained.Finally,38 transgenic plants were of PaHK3b editing were obtained by PCR detection.In summary,this study preliminarily showed that the poplar histidine kinase genes PaHK3a and PaHK3b were involved in the response of poplar to plant hormones and played an important role in poplar response to abiotic stresses.Results of this study laid a foundation for in-depth study of the functions of PaHK3a and PaHK3b genes,and provided candidate genes for stress resistance molecular breeding of poplar.