Cloning And Functional Identification Of Pl PlWRKY18 And PlWRKY41 Gene In Herbaceous Peony

Herbaceous peony(Paeonia lactiflora Pall.)is a perennial herbaceous plant,it is reputed as “the premier in floral kingdom” in China due to its gorgeous flowers and favor by Chinese people.It is widely planted in gardens in north of China.In the long evolutionary process,peony has formed the characteristics of winter dormancy in order to adapt to the changes of external adverse conditions.Up to now,the research on the function and mechanism of peony dormancy release is not clear enough.Previous studies have shown that WRKY transcription factor plays an important role in stresses,growth and development,and may regulate plant dormancy.Up to now,the relationship between WRKY transcription factor and peony dormancy has not clear enough.Transcriptome data of peony before and after endodormancy release of peony buds were obtained in our laboratory.In this study,two WRKY transcription factors(PlWRKY18 and PlWRKY41)with the changes in expression more than 10-fold were screened out and their functions were identified.Firstly,the expression levels of PlWRKY18 and PlWRKY41 genes in the dormancy release process were detected by quantitative real-time PCR.Then,the full-length ORF sequences of PlWRKY18 and PlWRKY41 genes were successfully isolated from the buds of peony by RT-PCR,and the two genes were comprehensively identified and analyzed by bioinformatics analysis.On this basis,the two genes were transformed into Arabidopsis thaliana by transgenic technology,and the genetic transformation of peony was carried out by virus induced gene silencing(VIGS)technology,so as to explore the functions of the two genes in the aspects of dormancy release,germination,growth and development.In addition,quantitative real-time PCR was used to detect the expression patterns of these two genes under abiotic stress.The main research results are as follows:(1)During the process of 35 d dormancy release of peony buds via chilling,the expression of PlWRKY18 was significantly up-regulated,whereas PlWRKY41 was significantly down-regulated.(2)PlWRKY18(Gen Bank accession number: MG736922),with a total length of 765 bp,encodes 254 amino acids,belonging to Group IIa of WRKY family;PlWRKY41(Gen Bank accession number: MG736921),with a total length of 1056 bp,encodes 351 amino acids,belonging to group III of WRKY family.(3)Overexpression of PlWRKY18 in A.thaliana led to earlier seed germination by 12 hours and advanced bolting by 4-5 d compared with the wild type,whereas overexpression of PlWRKY41 in A.thaliana delayed seed germination by 7 hours and delayed bolting by 1-2 d.(4)In transgenic A.thaliana plants,the expression of eight genes related to gibberellin signal transduction pathway were identified by q RT-PCR.The expressions of GID1 B and GID1 C in PlWRKY18 transgenic lines were significantly up-regulated,whereas the expressions of GAI,RGL1,RGL2 and RGL3 were significantly down-regulated,and there was no significant difference in the transcript levels of SLY1 and RGA.In PlWRKY18 transgenic lines,GID1 B and GID1 C were down-regulated,RGA,GAI and RGL3 were up-regulated.Unexpectedly,SLY1 was significantly up-regulated,while RGL1 and RGL2 were significantly down-regulated.(5)The gene silencing vector mediated by tobacco rattle virus was constructed.After sliencing the two genes in the buds of peony ‘Da Fugui’,respectively.It was found that silencing PlWRKY18 delayed budburst by 10-16 days compared to control plants,whereas silencing PlWRKY41 accelerated budburst by 4-10 days.Moreover,compared with the control group,silencing PlWRKY18 significantly inhibited plant growth within 72 d,while silencing PlWRKY41 significantly promoted plant growth within 54 d,although no significant difference was observed in final plant height between the silenced plants and control plants.Collectively,these results indicated PlWRKY18 was a positive responser and PlWRKY41 was a negative responser for bud dormancy release and vegetative growth of peony.(6)The yeast bait vectors p GBKT7-PlWRKY18 and p GBKT7-PlWRKY41 were constructed,and showed that p GBKT7-PlWRKY18 and p GBKT7-PlWRKY41 were all non-toxic,p GBKT7-PlWRKY18 had self-activation activities that could not be used directly for the next step of point-to-point verification,while p GBKT7-PlWRKY41 could be used for the next step of point-to-point verification.(7)The yeast prey vectors p GADT7-PlWRKY13,p GADT7-PlWRKY18,p GADT7-PlWRKY40,p GADT7-PlWRKY50 were constructed,it can be used for point-to-point verification of the next yeast two hybrid test with p GBKT7-PlWRKY41,in order to find the interaction protein of PlWRKY41 in the process of peony dormancy release.(8)The expression of PlWRKY18 and PlWRKY41 gene in abiotic stress was analyzed by q RT-PCR.It was found that PlWRKY18 gene was up-regulated under low temperature,high temperature and waterlogging stress,and down-regulated under salt stress.PlWRKY41 was up-regulated under low temperature,salt stress and waterlogging stress,and down-regulated under high temperature stress.