In order to clarify the mechanism of MADS-box transcription factor gene family member in flowering and floral organ development in Macadamia integrifolia， aMiMADS-box gene was cloned from the leaves of M. integrifolia “GUIRE1” cultivar by PCR technique and analyzed its structure and function by Bioinformatics method. The cloned cDNA sequence of MiMADS-box gene was 1 179 bp in length， 729 bp open reading frame （ORF）， encoded 242 amino acids. MiMADS-box， an unstable hydrophilic protein that had no transmembrane structure， no signal peptide and localized in the nucleus， had MADS superfamily and K-box superfamily conserved domains belonging to the MIKCc-type family. Phylogenetic analysis clustered MiMADS-box and AGL7/AP1， AGL10/CAL1， AGL8/FUL， AGL79 into FUL-AP1（SQUA） subfamily. The transcriptome data were used to analyze the expression patterns of MiMADS-box gene in branch， flower and leave of M. integrifolia “GUIRE1” and “695” cultivar. Results indicated that MiMADS-box had the lowest expression in the flower of “GUIRE1”， but the expression level of “695” was the highest. The green fluorescent protein fusion vector pGREEN-MiMADS-box-ORF-GFP was constructed by homologous recombination. It was speculated that MiMADS-box was closely related to flowering and floral organ development. The study provided a theoretical and technical guide for clarification the role MiMADS-box in flowering regulation mechanism of M. integrifolia.
Hydrogen sulfide（H2S） signaling plays an important role in seed germination and stress response. To explore the influence of soaking seeds with sodium hydrosulfide（NaHS， a donor of H2S） on the seed germination of crops under saline-alkali stress， naked oat（Avena nude） seeds were selected as experimental material which were soaked with different concentration（0， 50， 100， 200 and 400 μmol·L-1） of NaHS for 8 h， and then germinated in different concentrations of mixed saline-alkaline（the mole ratio of NaCl∶Na2SO4∶Na2CO3∶NaHCO3 is 12∶8∶1∶9） solution（0， 15， 30， 45 and 60 mmol·L-1） in petri dishes in incubator. Changes in germination percentage（Gp）， germination index（Gi）， vigor index（Vi）， mean germination time（MGT）， dry weight of seedlings（SDW） and comprehensive evaluation value of membership function（D） were analyzed. The effects of different concentrations of saline-alkali and NaHS and their interaction on Gp， Gi， Vi， MGT， SDW and D were significant（P<0.05） or extremely significant（P<0.01）. The Gp， Gi， Vi， MGT， SDW and D decreased significantly with the increase of saline-alkali concentration. Compared with 0 μmol·L-1 NaHS（CK）， 50 and 100 μmol·L-1 NaHS increased the Gp， Gi， Vi and D under 0 mmol·L-1 saline-alkali stress to different degrees. The 50 μmol·L-1 NaHS significantly increased the Gp under 30 mmol·L-1 saline-alkali stress. The 50 and 100 μmol·L-1 NaHS increased Gi under 15， 30 and 45 mmol·L-1 saline-alkali stress to different degrees. The 50 μmol·L-1 NaHS significantly increased Vi under 30 mmol·L-1 saline-alkali stress. The 50 and 100 μmol·L-1 NaHS significantly increased D under 45 mmol·L-1 saline-alkali stress. However， the germination index values of other treatments were not significantly different or significantly reduced. These results suggest that soaking the seed with NaHS of appropriate concentration could relieve the inhibition of saline-alkali stress on seed germination of naked oats， optimal effects were reached at NaHS concentrations of 50 μmol·L-1 under saline-alkali stress at below 45 mmol·L-1， 200 and 400 mmol·L-1 NaHS aggravated the inhibition of salinity-alkali stress on seed germination， and NaHS had no palliative effect on the inhibition of seed germination of naked oat under high concentration of salinity-alkali stress（60 mmol·L-1）.
By using Nicotiana benthamiana as plant material， the influence of the different Agrobacterium strains（LBA4404， EHA105， GV3101）， bacterial concentration and infection time on GFP（as the reporter gene） the fluorescence transient expression after transformation were analyzed respectively. The results showed that the expression of GFP with different Agrobacterium strains were various in the optimal concentration and time for transient： the highest transient expression efficiency with LBA4404 strain was achieved when the OD600 of bacterial suspension was 0.8； while the highest transient expression efficiency with EHA105 and GV3101 strains was achieved when the OD600 of the bacterial suspension reached 0.6. The level of transient expression by strain LBA4404 was the highest at 2 days later Agrobacterium infiltration while the level of transient expression by strains EHA105 and GV3101 was the highest at 4 days later Agrobacterium infiltration. The comparative analysis among these different strains showed that the transient expression efficiency by using LBA4404 strain was the highest. The above results indicated that Agrobacterium strains and transformation conditions such as bacterial concentration and infection time were important factors that affected the transient expression efficiency.
Tabebuia and Handroanthus species are famous woody plants in the world， which plays an important role in urban landscaping. To comprehensively understand the genetic diversity of cultivated species of Tabebuia and Handroanthus in China， as well as to establish a reliable phenotypic identification method， the phenotypic variation and genetic diversity of 18 leave traits among 6 species in total 812 individual trees cultivated in 11 cities in the southeast and south of China were analyzed respectively， and the 6 species（Tabebuia aurea， T. rosea， Handroanthus heptaphyllus， H.impetiginosus， H. chrysotrichus and H. chrysanthus） were compared as the control respectively and grown in Jianfengling Tropical Forestry Experimental Station， Institute of Tropical Forestry， Chinese Academy of Forestry. The results showed that： ①All the 18 phenotypic traits of species were significantly different among species and individual trees， and the phenotypic differentiation coefficient and interspecific repeatability of the 9 phenotypic traits， such as compound leaf length， leaflet length， leaflet width， leaflet length/width， leaf apex length/width， Leaf margin， leaf texture， leaf coat or not， leaf shape， were all over 60%， indicated that the high genetic correlation in interspecific variation and could be available taxonomy and genetic analysis of these species； ②The individual trees commonly cultivated in southeast China could be divided into 4 categories and 10 types， where the class A included T. aurea， the class B included H. heptaphyllus， T. rosea， H. impetiginosus， the class C included H. chrysotrichus and class D included H. chrysanthus， respectively. Additionally， different types with class B， C， D are divergent in leaf shape， leaf size， leaf length， leaf texture， margin serrate state and coat， suggested a certain diversity variation of species. This study found 9 reliable traits available to species identification and evaluated the genetic diversity of species in urban landscape cultivated in southeast and south China. These results would provide reference for the future study in classification， cultivation， production and application of Tabebuia and Handroanthus species in China.
In order to identify and verify crucial genes that regulate the stress resistance of poplar， based on the RNA-seq data of poplar（Populus davidiana × P.alba var. Pyramidlis， cv ‘Shanxin’） leaves induced by Trichodermaasperellumon or Alternaria alternata， a key responding gene was cloned and named as PdPapWRKY51. In silico analysis showed that the coded protein of PdpapWRKY51 was a IIc class transcription factor of the WRKY family and a non-transmembrane hydrophilic protein localized in the nucleus. The tissue-specific expression profile of PdPapWRKY51 in poplar seedlings was investigated through real-time quantitative polymerase chain reaction（RT-qPCR）. The results showed that PdPapWRKY51 was broadly expressed in plants and the highest expression was found in roots. The expression of PdPapWRKY51 was investigated after 48 hours of induction by salt， alkali， PEG（Polyethylene glycol）， five soil-borne plant fungal pathogens or phytohormones respectively. The results showed that the PdPapWRKY51 expression level was greatly affected by alkali stress. Fusarium oxysporum， Cytospora chrysosperma or A. alternata induction significantly up-regulated PdPapWRKY51 expression in the apex respectively. F. oxysporum induction significantly up-regulated PdPapWRKY51 in the leaf. C. chrysosperma and A. alternata induced significantly higher PdPapWRKY51 expression in the root. PdPapWRKY51 expression could be broadly induced by SA（salicylic acid） in plants. When induced by JA（jasmonic acid） or ABA （abscisic acid）， the PdPapWRKY51 expression was up-regulated in the apex but down-regulated in the root. The results revealed the tissue-specific expression patterns of PdPapWRKY51 gene in respond to multiple induction， and would provide a basis for further elucidating the function of PdPapWRKY51 and insights into breeding novel stress-resistant poplar cultivars through modifying PdPapWRKY51 expression.
In order to understand the function of transcription factor LobHLH34 and explore its expression pattern in different tissues and under different stress conditions， the full-length sequence of LobHLH34 gene was obtained from transcriptome data of root， stem and leaf of Larix olgensis， by using the primers designed. The complete open reading frame（ORF） of LobHLH34 gene was 696 bp and encoded 231 amino acids. Subcellular expression vector was constructed and transformed into protoplasts of Populus trichocarpa. The result showed that the LobHLH34 gene was located in the cell nucleus under the laser confocal microscope. The results of phylogenetic tree analysis showed that larch had the closest genetic relationship with Picea asperata and Selaginella tamariscina. By qRT-PCR， the tissue-specific expression of LobHLH34 gene and its response to abiotic stress were analyzed respectively. The results showed that LobHLH34 gene was expressed in the roots， stems and leave， and the lowest expression level was found in the stem and the highest relative expression in the leaf respectively. The expression level of LobHLH34 was different in different organs under NaCl， PEG and ABA stress. It was speculated that LobHLH34 gene could be involved in the process of plant growth， development and respond to stress， and it had special expressional pattern in different organs.
Being the polar carrier of IAA， PIN family proteins played a key role in plant embryonic development， organ development and tropic growth， especially in the formation of plant phyllodes， veins and vascular tissue differentiation.In order to identify the response characteristics BpPIN5 gene to exogenous hormones， the upstream 1447 bp sequence of BpPIN5 was cloned from the genome DNA of Betula platyphylla. The cis-acting elements in the sequence were predicted by PLACE online software. The results showed that the promoter sequence of BpPIN5 contained different types of auxin responsive elements such as auxin， gibberellin， Salicylic acid， abscisic acid， methyl jasmonate， ethylene respectively. Transgenic vector pro-BpPIN5：：GUS to B. platyphylla was constructed. GUS histochemical staining analysis reflicted that BpPIN5 promoter had transcriptional activity in the tip of leaf crack， venule and root of B. platyphylla respectively. After transgenic Betula platyphylla was treated with IAA， GA， MeJA， SA and ABA respectively， the results showed that BpPIN5 promoter responded to the five hormones above at the crack edge of the first leaf， petiole and root tissue of the second leaf， and the response changes were basically the same. The results would help to clarify the function of BpPIN5 in B. platyphylla.
To explore the classification and identification and interspecific relationship of Calanthe， the floral phenotypes and pollen morphological characteristics of 13 species of Calanthe in Tibet were studied. The results showed that： ①the floral color was divided into yellow， yellow-green， yellow-brown and pink， with pink flowers in the majority； ②the inflorescence was raceme， and length and width of various parts of the flower（sepal， petal， labellum， ovary， stigma） showed the difference. The difference of sepal length， petal width， labellum width， and stigma length was the largest， and the P value showed significant differences among various species； ③the pollen masses of these 13 Calanthe species were composed of thousands of single grains， which has no germination hole found， the obvious sticky substance between pollen grains of some species was found. The different size pollen blocks vary shaped mostly oval， oblong， and pear； ④the surface morphological characteristics of pollen blocks in different species were different， the pollen grains were stacked vertically or horizontally， and the shape of the pollen grains were also different； ⑤the appearance differences of pollen grains of different species were obvious， divided into concave type， smooth type， small hole type and perforated type. The results above showed that the flower phenotype and pollen morphological characteristics refer for identification of 13 species of Prairie Calanthe， and played the supplementary role in determining their phylogeny and systematic classification.
Lilium are perennial herbaceous plants with important ornamental value and some species are edible and medicinal. In the present study， the transcriptome of four species of Lilium（L. pumilum， L. davidii， L. lancifolium and L. brownii var. viridulum） was sequenced respectively， and the genes which involved in flower development were analyzed. Combined with the previous transcriptome data of L. brownii， the orthologous genes were selected by comparative transcriptome analysis. Positive selections were screened and their environmental adaptability were analyzed respectively. 44 565， 51 413， 41 638 and 44 716 unigenes were obtained from L. pumilum， L. davidii， L. lancifolium and L. brownii var. viridulum respectively by assembly. In order to understand the function of unigene， the unigenes were compared and annotated in the public database. 13 genes related to the development of flower were identified. A total of 8 247 orthologous genes were obtained in five species， and Ka， Ks and Ka/Ks values were calculated. 33 pairs of homologous genes were strongly positive selected in five species. Subsequently， GO and KEGG enrichment analysis and gene function annotation were carried out， and it was found that these genes were mainly related to plant defense and biosynthesis.
In order to clarify the characteristics of flower bud differentiation of Hippeastrum hybridum and provide theoretical basis for the research of flower development， flowering regulation， cross breeding and systematic classification， anatomical observation and paraffin sectioning techniques were used to study the growth of flower buds， flower organ differentiation， and sex cell differentiation in the H. hybridum variety of ‘Merry Christmas’. The main results were as follows：Each year H. hybridum ‘Merry Christmas’ produced two inflorescence buds， the flower buds in which completed floral organs differentiation in the second year， then bloomed in the third year after low temperature treatment， and of which the second inflorescence aborted occasionally. The floral organ differentiation process included flower primordium differentiation stage， outer perianth primordium differentiation stage， inner perianth primordium differentiation stage， stamen primordium differentiation stage and carpel primordium differentiation stage， the corresponding length of flower buds were about 0.02， 0.05， 0.1， 0.2， 0.3 cm， respectively. The primordia of all floral organs initiated spirally and centripetally. The anthers of H. hybridum were tetrasporangiate， and the anther wall was composed of epidermis， endothecium， middle layer， tapetum from outside to inside. The tapetum belonged to secretory type. The cytokinesis in the microsporocyte meiosis was successive. The microspore tetrads were cruciform and the mature pollen grains were 2-celled. The gynoecium of H. hybridum was tricarpellary and the ovary was inferior with axile placentation and trilocular with two rows of anatropous ovules in each locule. The ovule was bitegmic and crassinucellate. The development of embryo sac was of an Allium type.
Non-structural carbohydrate was the main intermediate storage between photosynthesis and growth of plants， and played an important role in the growth and metabolism of plants. To explore the distribution and seasonal variation of non-structural carbohydrates and the relationship between soluble sugar and starch in roots. The root sample of Larix gmelinii was collected from May to October in 2018， and the samples were divided into 1， 2， 3， 4， and 5 rank by root order method. The concentration of soluble sugar and starch were measured using the phenol-sulfuric acid method and enzymatic hydrolysis method respectively. The results showed that the concentration of soluble sugar was a significant difference between root order， month， and their interaction（P<0.001）. The concentration of soluble sugar in 1-5 orders ranged from 4.42%~14.90%， 4.35%~16.40%， 5.67%~19.70%， 5.91%~33.10% and 6.95%~37.80%， respectively. There was no significant difference in the concentration of starch among different root orders（P>0.05）. However， the concentration of starch was a significant difference among months（P<0.001）. It was ranged 23.36%~48.65% and decreased from May to July， and then increased， peaked in August， and there was no significant difference between October and May in 2018. The results could provide data for explaining the carbon metabolism and growth adaptation strategies of roots and understanding the internal structure and functional heterogeneity of fine root system.
Petal size was one of the main factors affecting the ornamental value of Camellia nitidissima， but its formation mechanism was unclear. In this study， the petal development process of C. nitidissima was divided into five developmental stages： young bud stage（S1）， early bud stage（S2）， turning yellow stage（S3）， semi-blooming stage（S4） and full bloom stage（S5） respectively. The dynamic changes of transcriptome during flower development were analyzed by RNA-seq technique. Using enrichment analysis and trend analysis of differentially expressed genes， it was found that the number of differentially expressed genes in auxin transduction pathway was the largest， and some auxin response genes such as AUX1/LAX cotransporters， AUX/IAA genes and SAURs were significantly up-regulated during flowering respectively， indicated that auxin was an important regulator of petal growth. Transcription factor genes such as MYB， bHLH and zinc finger protein genes were obviously up-regulated or down-regulated respectively， and some downstream functional genes such as xyloglucan endotransglucosylase/hydrolase（XTH）， pectin esterase（PE） and pectin lyase（PL） were also showed significant changes respectively. The results also suggested that these genes may be candidate genes involved in petal development. In addition， the expressions of some genes related to flowering regulation， such as FT， SOC1， AP3， PI， SEP3， etc.， were analyzed during the petal development of C. nitidissima， but the results showed that their expressions were mainly medium and low expression. Besides， the results of KEGG enrichment analysis of highly expressed genes revealed that the synthesis of secondary metabolites was accompanied by the whole petal development of C. nitidissima. All these results would lay a theoretical foundation for further researches of the regulation mechanism of petal development of C. nitidissima.
In order to optimize the process of polyphenols by macroporous resin from Empetrum nigrum aerial parts， moisture content， adsorption rate and desorption rate of six resins（HPD-100， X-5， AB-8， D101， HPD-600 and NKA-Ⅱ） were investigated， and the resin with the best effect was selected. Four factors（sample concentration， ethanol concentration， elution flow rate and elution volume） that have a great influence on the purification process were selected， and the response surface method was used to analyze the optimal process. HPD-600 macroporous resin had the best purification effect on polyphenols yield. The optimal extraction conditions were as follows： sample concentration was 0.84 mg·mL-1， ethanol concentration was 62.15%， elution flow rate was 0.67 mL·min-1， and elution volume was 2.71 BV. Under these conditions， the yield of polyphenols was 229.18 mg·g-1， the purity of polyphenols was increased from 8.11% to 22.56%， and the recovery rate was 67.78%.
In vitro shoots of Phalaenopsis Aphrodite ‘Mantianhong’ which were infected by Cymbidium mosaic virus（CymMV） were used as initial materials in the study. Droplet vitrification protocol of shoot tips was established by screening sucrose concentrations in preculture medium， preculture time and time of exposure to PVS2 （Plant vitrification solution 2， PVS2）. The regenerative shoot tips were induced to form protocorm-like-body（PLB） and differentiated to shoots. After detection by RT-PCR for CymMV， negative shoots were micropropagated and induced to form roots. The results showed that shoot tips being precultured on BM+0.6 mol·L-1 sucrose for 1-2 days was the optimal， resulting 70%-76.7% survival rate and 53.3%-56.7% regeneration rate of cryopreserved shoot tips. The optimal time duration of PVS2 was 60-90 minutes， resulting 73.3%-76.7% survival rate and 50.0%-56.7% regeneration rate of cryopreserved shoot tips. Being tested by RT-PCR， half of the regenerated shoots were negative. The CymMV eradication frequency was 50% by droplet vitrification protocol， which laid a technical and theoretical foundation for CymMV eradication of orchids.
In this experiment， tobacco BY-2 suspension cells were used as materials to investigate the effects of extracellular ATP on the chitosan-induced changes of ROS（reactive oxygen species）level and PAL（phenylalanine ammonia-lyase）activity. The results showed that the tobacco suspension cells mixed with chitosan 5-20 μg·mL-1 increased the intracellular ROS level at adose-dependent manner， and the PAL activity also increased and reached to the maximum at 15 μg·mL-1 chitosan， and then decreased slightly. There was not significant difference in ROS levels and PAL activity in tobacco suspension cells by adding 10-40 μmol·L-1 exogenous ATP. The extracellular ATP（eATP） level gradually decreased with the concentration of chitosan increased. The effects of eATP on the chitosan-induced change of ROS level and PAL activity were further analyzed. The results showed that the exogenous ATP 20 μmol·L-1 added to chitosan-induced tobacco suspension cells effectively alleviated the increases of ROS level and PAL activity in cells. These results showed that effects of extracellular ATP can affect the chitosan-induced changes in ROS level and PAL activity in cells.
By using the spatial replacement time and the typical sampling as methods， taking Eucalyptus robusta plantation in Weiyuan County， Sichuan Province as materials， we comprehensively analyzed the composition of the understory vegetation species and important value， and the aboveground， underground and whole plant biomass of the shrub and herb layers and species diversity index（Shannon-Wiener diversity index H， Simpson dominance index H′， richness index D and Pielou evenness index JSW）， and explored the dynamic changes of species diversity and biomass of undergrowth vegetation at five different forest ages（4， 5， 6， 7， 8 years） and the correlation between them respectively. The results showed that： A total of 210 species were found， belonging to 79 families and 151 genera， with more species in the herb layer than in the shrub layer. The herb layer of Miscanthus floridulus， Dicranopteris pedata and shrub layer of Melastoma malabathricum and Rubus hastifolius all occupied the dominant position at different ages respectively. With the forest age increasing， the density canopy increased， the D value， H value and H′ value of shrub layer all increased first and then decreased respectively； The D， H and H′ values of the herb layer showed a trend of change of the bimodal curve after increased， then decreased， then increased and then decreased. The biomass of the herb layer increased first and then decreased and then increased， the biomass of shrub layer increased first and then decreased. There was a significant positive correlation between species diversity and biomass， the D and H indices of herb layer were the direct factors affecting biomass. The species distribution， composition， species diversity and biomass of undergrowth vegetation in Eucalyptus robusta plantation were different in response to changes in forest age， showing different dynamic characteristic rules， the results provided data support for Eucalyptus robusta forest management in southwest China.
In order to explore the adaptability of Magnolia denudata Desr. to salt stress， nutrient solution was used to simulate salt stress， and the growth traits， the physiological and biochemical response characteristics were determined and analyzed under different stress levels respectively. During the whole stress process， the symptom level of salt damage was reached up to grade 2， the leaves appeared yellow and scorched. With the stress time longer， the relative electric conductivity and contents of MDA and proline in leaves increased respectively. The contents of chlorophyll b， soluble sugar， soluble protein， SOD and POD activity increased first and then decreased. The contents of chlorophyll and chlorophyll a increased first and then decreased under 100 and 200 mmol·L-1 of NaCl stress respectively， but decreased under 300 mmol·L-1 of NaCl stress. M. denudata had certain ability of salt tolerance， and it could be used in mild saline soil.
In order to optimize somatic embryo induction system of Syringa reticulata var. mandshurica， callus induction and somatic embryogenesis from mature zygotic embryo were conducted by adjusting the type and concentration of plant growth regulators. The morphological observation and physiological analysis of the explants in the culture process were carried out. The results showed that： ①Somatic embryo regenerate from mature zygotic embryo explant 30 d cultured later and cotyledonous somatic embryo can be obtained cultured 60 d later； ②BA played a leading role in callus induction， the callus induction rate was reached 100% in the combinations of 0.5 mg·mL-1 BA with 5-6 mg·mL-1 NAA， and the somatic embryo induction rate reached 8% in the combination of 0.5 mg·mL-1 BA and 5 mg·mL-1 NAA； ③Polyphenol content increased sharply at the primary stage of callus formation and kept a relatively high level in the process of culture. The PAL and POD activity increased and the MDA and SOD activity decreased in cotyledonous stage of somatic embryo.
MicroRNAs（miRNAs） are a class of non-coding small RNAs that negatively regulate gene expression. Through high-throughput sequencing miRNAs of Salvia miltiorrhiza， a mature sequence miR858 named Sm-miR858 was obtained. The sequence alignment showed that the Sm-miR858 was highly conserved with the identified miR858 sequences in other plants， and the result of Small RNA northern blotting showed that the Sm-miR858 was expressed in the root， stem and leaf tissues of S. miltiorrhiza， especially relatively high in leaves. To explore the function of Sm-miR858 in S. miltiorrhiza， the Sm-miR858 was predicted first online using biological software. The result of psRNATarget analysis showed that there were 13 potential target genes of Sm-miR858. One was SmPAP1， an important transcription factor， participated in the metabolic regulation on phenolic acid active substances of S. miltiorrhiza. To further verify the targeting effect of Sm-miR858 on SmPAP1， the correlation of co-expression between Sm-miR858 and SmPAP1 was analyzed and verified by Real-time qPCR in the tobacco transient expression system and S. miltiorrhiza tissues and organs. The Real-time qPCR results showed that there was a significant negative correlation co-expression between SmPAP1 and Sm-miR858 in S. miltiorrhiza. Then the Sm-miR858 and SmPAP1 over-expressing plant vectors were constructed， and the transient co-expression of the Sm-miR858 and SmPAP1 was subsequently assayed in tobacco. The results showed that the sm-mir858 overexpression resulted in a significant decrease of the SmPAP1 mRNA expression level， compared with the control， and this indicated that sm-mir858 did actually target and negatively regulate the expression of SmPAP1 in S. miltiorrhiza. Above results laid a solid foundation for elucidating the role of sm-mir858 in the regulation phenolic acids biosynthesis in S. miltiorrhiza.
