The Molecular Mechanism Of Masson Pine Drought-resistant Germplasm In Response To Drought Stress

Drought is the most extensive abiotic stress factor affecting plant growth and development.Drought usually causes water shortage in the plants'organism,separation of cytoplasm and cell wall,cell membrane and enzyme system will be further destructed,then cell functions disable and metabolism disorder,which will eventually affect the growth and development of forest trees and significantly reduce forest yield.The growth slowdown and biomass reduction which are caused by drought are far higher than other abiotic stress factors such as salt and high temperature.Pinus massoniana is the most important afforestation pioneer species in southern China.Its growth and development often suffers from seasonal drought in the south,which causes forest yield reduction.At present,plenty of studies on drought tolerance of Pinus massoniana have already been done,most of which focus on morphology,physiology and growth.A mount of genes participate in the drought response of plants under drought stress,but there are few reports on the drought resistance genes of Pinus massoniana.The molecular mechanisms that respond to drought are not yet clear.Exploring the drought resistance genes of Pinus massoniana at the molecular level and revealing its drought-tolerant regulation mechanism has become a difficult problem to be solved in the current research work of Pinus massoniana.This is of great significance to accelerate the breeding process of Pinus massoniana drought-resistant germplasm and further improve the Pinus massoniana under drought conditions.For this purpose,this study used three superior families of Pinus massoniana as test materials,and used ISSR molecular markers to screen out seedlings with the same genetic background;observed the differences in growth and physiological changes of three superior families under drought stress,and fuzzy membership function and grey correlation analysis were used to evaluate the drought resistance ability,and the high drought resistance family was screened.The high-throughput sequencing technology was used to construct the transcriptional library of the Pinus massoniana drought-resistant family,and the massive transcript information was obtained.The differential expression analysis of the drought-treated and normal control genes was carried out.The key genes of drought were explored and combined with the dynamic changes of morphology,photosynthetic characteristics,water use efficiency,physiological indexes and endogenous hormones under drought stress,and the molecular mechanism of response to drought was expounded.The main research contents and results are as follows:1.Genetic background consistency test:Pinus massoniana is a genetically rich species.The genetic background can be used to determine the degree of genetic similarity at the molecular level,and effectively eliminate the interference of different genetic genotypes and improve the selection efficiency of materials.In this study,12 polymorphic,reproducible,clear and stable primers were screened from 100 ISSR primers for genetic diversity analysis of three Pinus massoniana families,and based on which we screened out families with the consistent genetic background as analytical material.2.Screening of Pinus massoniana drought resistant genetic resources:Three kinds of Pinus massoniana seedlings with the same genetic background were used as the tested materials and then were planted in the greenhouse by pots to simulate the natural drought environment,and the drought stress was carried out to study the function and adaptive mechanism of growth,antioxidant system and osmotic adjustment of the three superior families during the continuous drought,and evaluate their drought resistance.The results showed that:?1?Continuous drought stress significantly restricted the growth of Pinus massoniana seedlings,and the growth and dry matter accumulation of three families were inhibited.The difference of growth of seedling height,ground diameter growth and dry matter accumulation among different families were significant?P<0.05?.The root-shoot ratio of GX01 and GX02 was significantly higher than that of the control.GZ01 was also higher than the control,but the difference was not significant.It can be seen that under drought stress,the Pinus massoniana seedlings can adapt to drought stress by increasing the growth of the underground part and reducing the growth of aboveground parts.?2?With the deepening of drought stress,the SOD activities of three families of Pinus massoniana increased first and then decreased,and there were significant differences in the stress period from0 to10^th days?P<0.05?among three families.POD activity also increased first and then decreased,without significant differences between families?P>0.05?;GX01 increased first and then decreased in MDA content,GX02 and GZ01 decreased first and then increased,there was a significant difference between different families in the period from 5^th20^th days of drought stress and on the 30^th day?P<0.05?;proline content of GZ01 kept increasing while that of GX02 and GX01 increased first,then decreased and then increased again.The difference was significant between each family in the period from 20^th30^th day of stress?P<0.05?.It showed that the seedlings of Pinus massoniana enhanced the antioxidant capacity by increasing the protective enzyme activity under mild drought stress to alleviate the drought stress injury;and increasing the osmotic adjustment substance reducing the water potential and strengthening the water retention capacity under the moderate and severe drought stress to protect against drought stress;and the protective enzyme activity decreased significantly,the MDA content increased significantly,and the membrane peroxidation was seriously injured under the severe drought stress.?3?By grey correlation analysis,it showed that the correlation between root-shoot ratio,seedling height and drought resistance was the largest,followed by MDA and PRO content,which could be used as the main index to evaluate drought resistance,and the correlation between SOD and POD activities and drought resistance was the smallest;by membership function,the drought resistance of three species of Pinus massoniana seedlings was analyzed as GX01>GX02>GZ01.3.Transcriptome analysis of drought-resistant genetic resources of Pinus massoniana under drought stress:Pinus massoniana seedlings of three different managements under continuous drought?10 days,15 days,25 days of stress?and the water supply,were took as the test materials,and the cDNA library was constructed separately.Through high-throughput sequencing,low-quality reads to remove redundancy,Trinity assembly to obtain Unigenes;by the Blast alignment,Unigenes information were annotated with seven database,Nine,Nt,Swiss-Prot,PFAM,GO,KOG,KEGG,further division of GO gene function,KOG category and KEGG metabolic pathway for successful annotated Unigenes was done;The Misa software was used for batch searching of SSR sites,and the primer 3.0 software for large-scale SSR primer design.The results showed that:?1?A total of 194,821 Unigenes were obtained from the transcriptome of Pinus massoniana under drought stress,of which 101,806 Unigenes were annotated,containing 64,943GO functional annotations,35,880 KOG functional annotations,and 30,882 KEGG annotations.In addition,among the 66,825 NR annotations,24.42%of unigene?16,323?Unigenes are homologous to the phylogenetically related Picea and Pinus sequences,and it was the most in the number of matches in all annotated species,which indicated that the sequence annotation results are better,and the annotation success rate is higher.?2?A total of 6728 SSR loci were found in the entire transcriptome data,distributed in6367 Unigenes,and the SSR frequency was 3.45%.The repeat types were mainly mono-,tri-and double-ucleotides,accounting for 35.82%,33.03%and 25.22%of the total SSR,respectively;the repeat motif were mainly A/T,AT/AT,AG/CT,AGC/CTG,AAG/CTT;the motif length were mainly short sequence SSR around 10²0 bp;The number of repeats was dominant in 5-10 replicates.At the same time,13338 pairs of SSR primers were designed in batches,the effective amplification rate of random primers was 45.5%,which indicated that the SSR primers that we developed in this study have certain validity and versatility,which laied the foundation for subsequent molecularly assisted breeding and genetic diversity research of Pinus massoniana.4.Gene differential expression analysis of Pinus massoniana drought-resistant genetic resources under drought stress:The differential expression analysis of 101806 unigenes in the transcriptome of Pinus massoniana under drought stress was carried out.The GO and KEGG enrichment analysis were performed for differential expression of unigene.At the same time,9drought-related unigenes were randomly selected from the transcriptome for real-time PCR test to verify the reliability of transcriptome data.The results showed that:?1?4300 differentially expressed Unigenes were detected in the transcriptome of Pinus massoniana under drought stress.The differential expression of Unigene at 10,15,and25 days was 3397,1695,and 1550,respectively;the up-regulated expression was 1656,611 and 651,respectively;the down-regulated expressions was 1741,1084,and 899,respectively;the specific differential expression Unigene was 1906,317,and 422,respectively;the total differential expression of Unigene was 687.Among them,156unigenes were involved in biological processes such as signal transduction,defense response,transcriptional regulation,photosynthesis,transmembrane transport,biosynthetic processes,metabolic processes,xidation-reduction processes,and protein phosphorylation.?2?GO enrichment analysis showed that the differentially expressed genes induced by different drought stress levels were involved in three biological processes such as metabolism,redox and photosynthesis,which indicated that drought stress may be reason which caused the above biological function changes.KEGG enrichment analysis showed that under the drought stress,four metabolic pathways such as photosynthesis,plant hormone signaling,phenylalanine metabolism and phenylpropanoid biosynthesis were significantly enriched,which indicated that these four metabolic pathways were related to drought stress responses of Pinus massoniana.?3?Among the 9 randomly selected unigenes,the qRT-PCR changes of 6 genes including DREB,GH3,P450,GPX,MYB,and NIP were consistent with the changes of transcript level DEG;The qRT-PCR amplification times of 3 Unigenes such as P5CS,WRKY,and MCM was higher than that of the transcriptional level DEG,but the variation trend of the two were consistent,which indicated that the data of the transcriptome of this study was reliable.5.Drought resistance genes and molecular mechanisms of drought resistance of Pinus massoniana:Based on the results of gene differential expression analysis,and combined with the dynamic changes of morphology,photosynthetic characteristics,water use efficiency,physiological indicators and endogenous hormones under drought stress,the molecular mechanism of drought resistance of Pinus massoniana was comprehensively analyzed.?1?Through the analysis of gene function,some regulatory genes and functional genes related to drought stress response were discovered.Regulatory genes include:plant physiologic rhythm pathway-related genes HY5 and LHY;ABA-related genes NCED?9-cis-epoxycarotenoid dioxygenase?,PYL,PP2C,SnRK2;growth-related gene ERF,ARF and IAA in phytohormone signal transduction pathway;and plant pathogen interaction defense genes:WRKY.Functional genes include:osmotic adjustment related genes:P5CS?pyrroline-5-carboxylate synthase?,AQP?Aquaporin?,inositol transporter?Inositol transporter?;antioxidant enzyme genes:GPX,GST and GSR.?2?Pinus massoniana initiated the defense system rapidly in the early stage of drought,and resisted drought stress through the interaction of growth,physiology and molecular response to rebuild the system balance,which indicated it had strong drought tolerance.Firstly,the root system of the underground part grew obviously,and the ratio of root to shoot was significantly increased,which promoted the absorption of water by the root system.Secondly,the above-ground part adopted a drought-tolerant strategy of“one increase and one decrease”.On the one hand,in the early stage of drought,the ABA-related gene NCED significantly up-regulated expression,rapid accumulation of ABA and signal transduction,induced transcription factor expression and regulated the expression of downstream functional proteins in response to drought stress.In the middle and late stages of drought,GPX,GST and GSR were significantly up-regulated,with superior oxygen scavenging capacity to mitigate the peroxidation damage caused by drought.During the whole drought stress period,WRKY was significantly up-regulated in early stage,which showed rapid defense ability.At the same time,P5CS was significantly up-regulated,PRO content continued to accumulate,which showed strong osmotic adjustment ability to slow the membrane penetration pressure.Pinus massoniana could delay and reduce body damage caused by drought by implementing a series of strong protection measures.On the other hand,the growth factors such as ARF and IAA are down-regulated;the expression changes of rhythmic factors HY5 and LHY caused changes in circadian rhythm,which had a negative effect on light absorption,photosynthesis and glucose metabolism were significantly weakened,and aquaporin AQP was down-regulated expression,etc.;it indicated that Pinus massoniana resist the drought stress by initiatively reducing the growth and energy consumption of aboveground parts and water consumption.This complex network regulation from morphology,growth,physiology to molecular level is an important survival strategy for Pinus massoniana under drought stress.