| Pinus massoniana Lamb.is distributed in 17 provinces,autonomous regions and municipalities in China.P.massoniana is an important timber tree species in southern China.It has certain stress resistance,especially drought and barren resistance,and has excellent characteristics of strong adaptability and high comprehensive utilization,and is a pioneer species for afforestation.Guangxi is an important production area of P.massoniana in China,with abundant annual precipitation.However,its uneven annual distribution of makes some regions are prone to seasonal drought stress.To make matter worse,the drought is likely to be more severe in the context of global warming,which will threaten P.massoniana habitat,but also reduce the growth of P.massoniana.And thus restricts the development of P.massoniana industry.Effort has been made in studying the physiological and molecular mechanisms of P.massoniana in response to drought stress,However,from the molecular perspective,the mechanism of transcription factors involved in drought resistance P.massoniana is rarely reported.Therefore,in this study,the drought resistant family（19-220）and drought sensitive family（19-214）of P.massoniana were used to investigate the expression patterns of three transcription factors,including WRKY,MYB and AP2/ERF.Combined with the previous drought transcriptome results,four PmWRKYs genes,five Pm MYBs genes and nine Pm AP2/ERFs genes were selected to analyze the expression patterns of genes under tissue specificity,exogenous hormone treatment and drought stress.The function of PmWRKY31 in response to drought was verified.This study will lay the foundation for further research on the molecular functions of WRKY,MYB and AP2/ERF family genes in P.massoniana under drought stress.The main results are as follows:（1）The seedlings of drought-resistant family 19-220 and drought-sensitive family 19-214 were treated with different concentrations of exogenous hormones SA,Me JA and ABA,in which 10 m L were sprayed on the above-ground part（stem and leaf）and 10 m L on the underground part（root）for 4 days,followed by water supplement for drought stress.Subsequently,drought stress was conducted to screen the appropriate concentrations of exogenous SA,Me JA and ABA for improving the drought resistance of P.massoniana.Based on the phenotypic changes,seedling growth rate and other indicators,it was determined that 50 mg/L SA,0.5 mmol/L Me JA and 25 mg/L ABA were the best hormones and concentrations to improve the drought resistance of half-yearly P.massoniana.（2）A total of 43 PmWRKYs genes,86 Pm MYBs genes and 124 Pm AP2/ERFs genes were identified from transcriptome data by bioinformatics software.The nucleic acid sequences and encoding proteins of each gene family were analyzed.These three gene families were classified by phylogenetic tree,PmWRKYs were divided into 7 subfamilies,Pm R2R3-MYBs were divided into 21 subfamilies,and Pm AP2/ERFs were divided into 12 subfamilies.It was found that each gene had its characteristic conserved domain.These results indicate that each gene family had high similarity and conservation in gene structure and conserved domain.（3）Four PmWRKYs genes,five Pm MYBs genes and nine Pm AP2/ERFs genes were selected to analyze the expression among different tissues.The results showed that 18 genes were expressed in all tissues,among which PmWRKY10,PmWRKY22,Pm MYB55,Pm MYB57,Pm AP2/ERF14,Pm AP2/ERF44,Pm AP2/ERF49,Pm AP2/ERF109 and Pm MYB34,Pm AP2/ERF11,Pm AP2/ERF46,Pm AP2/ERF80,Pm AP2/ERF96 were the highly expressed in roots and leaves of the two families,respectively.These genes may be involved in regulating roots or leaves to respond to drought stress.（4）The half-year-old seedlings of different drought resistant families were used to conduct exogenous hormone treatment with 50 mg/L SA,0.5 mmol/L Me JA,25 mg/L ABA,and drought stress experiments.The expression patterns of genes under hormone treatment and drought stress were analyzed by q RT-PCR.The results showed that the expression of 18 genes varies under drought treatment.Most genes were up-regulated after stress,which was positively regulated by drought stress.However,PmWRKY22 and Pm AP2/ERF96 genes were down-regulated after drought,which were negative regulatory genes.The expression patterns of PmWRKYs,Pm MYBs and Pm AP2/ERFs were different under drought stress by using exogenous hormones SA,Me JA and ABA,and each gene was up-regulated in response to at least one exogenous hormone under drought stress.Comprehensive analysis of PmWRKY6 and PmWRKY30 genes of PmWRKYs,five Pm MYBs genes,Pm AP2/ERF11,Pm AP2/ERF80,Pm AP2/ERF44 and Pm AP2/ERF77 genes of Pm AP2/ERFs genes were significantly induced by three kinds of hormones.It is speculated that the gene may be related to the pathway of hormone signal transduction in response to drought stress,and the gene expression after hormone treatment is lower than that of CK2.Therefore,it is speculated that PmWRKYs,Pm MYBs and Pm AP2/ERFs genes may positively or negatively regulate corresponding signal transduction pathways,thereby improving drought resistance of P.massoniana.（5）The results showed that the drought tolerance of transgenic tobacco was significantly higher than that of wild tobacco under drought stress,and the MDA content was significantly lower than that of wild tobacco,and the Pro accumulation was significantly higher than that of wild tobacco.We proposed that PmWRKY31 gene increased the expression of drought resistance related genes,and preliminarily verified the function of PmWRKY31 gene in P.massoniana. |
