Growth Strategies Of Quercus Mongolica Fisch.ex Ledeb. Seedlings In Shade Environment And Functions Of QmbHLH81 Gene

Quercus mongolica Fisch.ex Ledeb.is a famous ornamental and timber species with important economic and ecological value.However,in natural forests or natural secondary forests,it is difficult to achieve the regeneration process of Q.mongolica seedlings that grow naturally due to light.In this study,the 1-year old and 2-year old Q.mongolica seedlings was taken as the research object,and the influence of shade on the growth and and photosynthetic characteristics of Q.mongolica seedlings was clarified by artificial setting of different shade intensity.The survival strategy of Q.mongolica seedlings under carbon starvation caused by shade were determined by complete shade and light treatment.By identifying the core transcription factor bHLH family of light response in Q.mongolica and analyzing its expression characteristics under shade,QmbHLH81 gene which is closely related to shading response and photosynthesis regulation of Q.mongolica was obtained.The molecular structure,expression characteristics and gene function of Q.mongolica were studied by gene cloning,bioinformatics analysis,q RT-PCR and genetic transformation of Arabidopsis thaliana.The function and mechanism of QmbHLH81 in light environment adaptation were revealed.This paper aims to clarify the growth adaptation and survival strategies and physiological and molecular regulation mechanisms of Q.mongolica seedlings in shade environment,and provide theoretical basis and practical guidance for artificial intervention in understory seedling regeneration.The main research results are as follows:（1）Shading had a significant effect on the growth and photosynthetic characteristics of Q.mongolica seedlings.Q.mongolica seedlings had the highest biomass,maximum net photosynthetic rate(P_nmax),maximum carboxylation rate(V_cmax),maximum electron transfer rate(J_max)and tribose phosphate utilization rate（TPU）under full light.The maximum optical quantum yield（F_v/F_m）of photosystem II（PSII）was about 0.8,and the heat dissipation ratio（D）of the antenna was significantly higher than that of the shading treatment,indicating that the seedlings of Q.mongolica were not affected by the light suppression,and the energy absorbed by the leaves mainly dissipated the excess energy through the heat dissipation of the antenna.There was no significant difference in net photosynthetic rate（P_n）and apparent quantum efficiency（AQY）of Q.mongolica seedlings under 40%shade compared with that under full light.P_n and AQY significantly decreased when the shade degree was greater than 40%,and the photosynthetic pigment content was significantly higher than that under 40%shade,and the antenna heat dissipation ratio was significantly lower than that under full light.The results showed that Q.mongolica seedlings had a certain tolerance to 40%shade.Under 60%shade,the height-diameter ratio of Q.mongolica seedlings was significantly higher than other treatments,showing the characteristics of"thin height",the root-shoot ratio was significantly lower than other treatments,and more biomass was allocated to the aboveground part.In addition,P_n was significantly lower than that of full light.Besides increasing photosynthetic pigment content and decreasing antenna heat dissipation level,dark respiration rate（R_d）of leaves was significantly decreased to promote the accumulation of carbon assimilation products,so as to ensure the growth of seedlings.The lowest V_cmax,J_max and TPU were found under 80%shade,which indicated that the activities of photosynthetic carbon assimilation enzyme and photosynthetic electron transfer were severely limited,and the P_n could not be maintained,which was not conducive to the growth of seedlings.The ratio of root shoot and root biomass were significantly higher than those of other treatments,and more biomass was allocated to the underground part.（2）The threshold range of non-structural carbohydrate（NSC）concentration for survival of Q.mongolica seedlings under full shade was determined.Q.mongolica seedlings could survive in total darkness for 9 weeks,during which the NSC concentration decreased rapidly,the whole plant starch concentration decreased to 0.8%DM,the whole seedling soluble sugar concentration decreased to 2.0%DM,all seedlings died after 12 weeks of darkness,and the starch concentration of the whole seedling was reduced to 0.4%DM,the concentration of soluble sugar in the whole seedling was reduced to 0.9%DM,and the starch and sugar concentrations in most tissues were lower than 1%DM.The threshold of lethal NSC concentration in different tissues of Q.mongolica seedlings was between 9 wk and 12 wk in dark treatment.It was found that the lowest threshold of starch in xylem of stems of Q.mongolica was between 0.3%DM and 0.7%DM.The lowest threshold of soluble sugar was between 0.16%DM and 0.75%DM.When the NSC concentration of Q.mongolica seedlings was lower than 29%DM,the biomass growth almost stopped.（3）The tissue specific QmbHLH gene involved in shade response of Q.mongolica was excavated.A total of 89 QmbHLH genes were identified,which were unevenly distributed on12 chromosomes and divided into 21 subfamilies.The transcription factors of QmbHLH within the same subfamily had similar physical and chemical properties,gene structure and conserved protein motif pattern,but there were significant differences among different subfamilies.76QmbHLH genes contained photoresponsive elements.The stable reference genes ACT97,HIS4,RPS13 and CYP18 were screened for gene expression analysis in different organs and shade conditions of Q.mongolia.ACT97 was the best reference gene in different organs,while CYP18,HIS4 and RPS13 had the highest stability in leaf,stem and root tissues under shade treatment,respectively.The expression patterns of QmbHLH genes in various tissues of Q.mongolica under shade treatment were analyzed using the screened optimal reference genes and q RT-PCR,and it was found that the expression of QmbHLH genes were induced by shade treatment to different degrees,and the expression patterns of QmbHLH genes varied with shade treatment time or were expressed differently in different organs.（4）The QmbHLH81 gene was cloned successfully.The total length of QmbHLH81 gene was 2139 bp,encoding 712 amino acids.The molecular formula of QmbHLH81 protein is C₃₄₆₅H₅₄₇₀N₉₉₄O₁₁₀₀S₁₈,the relative molecular weight is 79.23 k Da,the theoretical isoelectric point is 5.60,the positive and negative charge residues are 96 and 75,the instability coefficient is 65.96,which is the unstable protein,the fat coefficient is 73.55.The total mean hydrophilicity of the protein was-0.649,which was a hydrophilic protein,and subcellular localization predicted that it was located in the nucleus.The photosynthetic physiological indexes of bhlh81,bhlh81-OE-QmbHLH81,WT and WT-OE-QmbHLH81 plants were compared.It was found that overexpression of QmbHLH81 gene increased chlorophyll content,photosynthetic rate and stomatal conductance in Arabidopsis.The contents of soluble sugar and starch were significantly increased compared with those of WT wild-type plants,and the physiological indexes of bhlh81 mutant could be restored,indicating that QmbHLH81 gene plays an important positive regulatory role in photosynthesis,chlorophyll and photosynthetic product accumulation.