To clarify the basic characteristics of the complete chloroplast genome of Syringa villosa subsp. wolfii， the sequence information of chloroplast genome was analyzed by high-throughput sequencing technology， and its systematic evolution position was discussed. The results showed that：（1）The total length of the S. villosa subsp. wolfii chloroplast genome was 156 517 bp， which had a typical tetrad structure and had 131 functional genes， including 36 tRNA genes， 8 rRNA genes and 87 protein-coding genes respectively. （2）The relative synonymous codon usage（RSCU） analysis of the protein-coding region of the chloroplast genome showed that there were 31 codons with RSCU>1， of which 21 ended with A/U base； and there were 34 codons with RSCU<1， including 22 codons ending with G/C base. （3）In the chloroplast genome of S. villosa subsp. wolfii， 334 scattered repeats were detected， including 170 forward repeats and 164 palindromic repeats， and 227 SSR loci were discovered， and PCR primers were designed successfully for 226 of these loci. （4）Phylogenetic tree analysis based on the maximum likelihood method showed that S. villosa subsp. wolfii was the most closely to the S. yunnanensis. In this study， repeated sequences， IR boundaries and phylogeny of chloroplast genome of S. villosa subsp. wolfii were analyzed to provide reference data for molecular markers， phylogenetic classification， species identification and evaluation and DNA barcode development.
In order to clarify the potential functions of bZIP transcription factor family members in Macadamia integrifolia under stress response， MibZIP1 was cloned from ‘Guire 1’ fruit. Combined with the analysis of transcription factor structure and expression patterns under different treatments， it was found that the full length of MibZIP1 was 1 157 bp， and the ORF was 927 bp， and encoded 308 aa，and had a typical bZIP Superfamily domain. Phylogenetic analysis showed that MibZIP1， TsbZIP60 and NnbZIP60 were closely related. The results of tissue expression analysis showed that MibZIP1 expression was the lowest in leaves of ‘Guire 1’ and the highest in branches of ‘695’. The expression of MibZIP1 was significantly up-regulated in ‘Guire 1’ leaves treated by zeatin， salicylic acid， ethephon and abscisic acid respectively， but was unchanged treated by gibberellin and hydrogen peroxide respectively. It was speculated that MibZIP1 was related to the stress tolerance of M. integrifolia， which provided theoretical guidance for the development of new cultivation and hormone regulation techniques.
Sequence variation and gene composition of chloroplast genome could effectively reflect the phylogenetic and evolutionary relationships among plants. In this study， DNA libraries were prepared and sequenced by the Illumina HiSeq， chloroplast genomes of five species of Parnassia and allied genera were assembled respectively， and the structural characteristics， sequence genetic variation and codon preference of protein coding genes of Parnassia chloroplast genome with related species were compared and analyzed. The results showed that the chloroplast genome of Parnassia was conserved tetrad structure， and pseudogenization of several genes were found in P. palustris， but the chloroplast gene composition of other species was the same， encoding 115 genes. Compared with the relative species， the intron of rpl16 gene were lost in all species of Parnassia. The ratio of nonsynonymous/synonymous substitution rate of protein coding genes was low， and chloroplast genes might experience purification selection. Codon preference was consistent with the phylogenetic relationship shown by protein coding sequences. This study reveals that selection pressure may play an important role in the evolution of protein-coding genes in the chloroplast genome of Parnassia， which could enhance the understanding about evolution and adaptation of this genus.
In order to clarify the characteristics and sequences variation of the chloroplast genome of Yucca， a comparative genomics of the chloroplasts was performed， and a phylogenetic tree based on the chloroplast genome was constructed. The chloroplast genome of Y. treculeana was obtained by high-throughput sequencing technology， and combined with the published chloroplast genome of Yucca， the chloroplast genomes of six species of Yucca were studied by comparative genomics， including basic structure， SSR， boundary contraction and expansion， sequence variation and phylogenetic analysis respectively. The results showed that the chloroplast genome size， gene type and number of the six Yucca species were similar， and the interspecific genome structure was conserved. Several repeated sequences were detected in the chloroplast genome of Yucca， among which SSR loci were mostly composed of mononucleotide， dinucleotide and tetranucleotide， and preferred A and T bases. Three regions with high variation（psbK-psbl-trnS-GCU， rpl20-rps12 and ccsA-ndhD） were identified from the chloroplast genomes of six Yucca species according to the nucleic acid polymorphism index（π）≥0.008. The phylogenetic relationships constructed from the chloroplast genome and LSC+SSC region sequences were basically consistent， and the phylogenetic relationships among six Yucca species were confirmed， among which Y. treculeana and Y. queretaroensis were most closely related. The sequencing obtained Y. treculeana chloroplast genome， revealed the genome characteristics and sequence variation of six Yucca species， and clarified the relatedness among various species. The results could provide reference for subsequent molecular marker development and phylogenetic research.
In order to cultivate rice varieties resistant to Xanthomonas oryzae pv. oryzae， Rhizoctonia solani， Magnaporthe oryzae and Fusarium fujikuroi， the method of mining resistance genes is an important way to select resistant varieties. RT-PCR method was used to clone the LcWRKY40 gene （MN187915） from the leaves of Leymus chinensis. The result showed that the CDS was 1 053 bp in length and encoded 350 amino acids with a molecular weight of 38.1 kDa. The results of bioinformatics analysis showed that the primary structure of LcWRKY40 contained WRKY domain， zinc finger protein domain and nuclear localization sequence. Phylogenetic tree construction and motif analysis showed that the phylogenetic relationship between LcWRKY40 and HvWRKY38 was closed. The results of subcellular localization in tobacco showed that the LcWRKY40 protein was located in the nucleus， verified by the software prediction. qRT-PCR tissue specific expression analysis showed that LcWRKY40 gene was expressed in root， stem， leaf， leaf sheath， Lemma and anther of Leymus chinensis respectively， but the expression level was the highest in leaf but the lowest in Lemma. Transgenic LcWRKY40 tobacco plants and wild-type tobacco plants were inoculated with X. oryzae pv. Oryzae， R. solani， M. oryzae and F. fujikuroi， respectively， which showed that transgenic LcWRKY40 tobacco plants could alleviate the four pathogens in different degrees， and showed high resistance to M. oryzae and F. fujikuroi. Therefore， it was speculated that LcWRKY40 protein played a key regulatory role in signal pathways such as resisting disease stress and improved the resistance of plant pathogens， which layed a molecular foundation for the study of LcWRKY40 function and abiotic stress.
APC/C is an E3 ubiquitin-ligase complex that is involved in regulating cell-cycle progression. In order to reveal the function of the APC/C complex in Taraxacum kok-saghyz Rodin（TKS）， TkAPC10 was identified， and its expression pattern was analyzed to preliminarily determine its function in this study. The ORF of TkAPC10 was 579 bp， encoding 192 amino acids. Its genomic DNA sequence was 1 092 bp， contained 6 exons and 5 introns. Genome analysis showed that TkAPC10 was a single copy in TKS genome. In addition to the TATA-box and CAAT-box enhancer elements， TkAPC10 promoter contained cis-acting elements related to ABA， JA， light， and stress response. Phylogenetic analysis suggested that APC10 from different species showed high homology. The similarity between TKAPC10 and lettuce LsAPC10 was up to 99%， and the similarity to APC10 from other Compositae plants was also over 95%. The expression pattern of TKAPC10 was analyzed by qRT-PCR， and the results indicated that TKAPC10 expression in the tissues with vigorous cell division（such as flowers， leaves and roots） was significantly higher than that with relatively slower cell division （such as pedicel）. Under exogenous ABA treatment， the transcript level of TKAPC10 was obviously decreased， while it was significantly increased after MeJA and ET treatments. Under PEG6000 and mannitol treatments， the expression of TKAPC10 was obviously decreased， but its expression was markedly induced by salt stress treatment. The results in this study suggested that TKAPC10 was involved in the regulation of cell division， hormone signaling and abiotic stress response in TKS.
To investigate the role of WRKY transcription factors（TFs） in sensing saline-alkali stress signals and maintaining their tolerance function through physiological and biochemical regulatory pathways， and the WRKY42 gene of Amorpha fruticosa was cloned and the expression pattern in response to salt（NaCl） and alkali（NaHCO3） stress and tissue organs was analyzed， and the salinity tolerance function was also studied its by overexpression in tobacco. In this study， the AfWRKY42 gene was cloned based on transcriptome sequencing data of A. fruticosa L. under stress. Bioinformatics analysis showed that AfWRKY42 contained a WRKY structural domain， two low-complexity regions and a helix region. Phylogenetic tree analysis of amino acids revealed that AfWRKY42 was most closely related to WRKY47 of Cajanus cajan and WRKY42 of Mucuna pruriens. The localization of AfWRKY42 protein in mesophyll protoplasts of Arabidopsis thaliana was confirmed in nucleus by a transient gene expression system. Quantitative analysis of AfWRKY42 gene showed the highest expression in the shoot epidermis of Sophora japonica. Detection of expression patterns in roots and leaves treated with NaCl and NaHCO3 showed an overall increasing trend induced by it， suggesting that overall stress induced an increase in AfWRKY42 gene expression， and AfWRKY42 gene was associated with the regulation of salinity tolerance in plants. Analysis of salinity tolerance in 35S-initiated overexpressing T3 generation of tobacco lines transgenic for AfWRKY42 gene showed that the transgenic tobacco lines showed increased resistance after salinity stress treatment， it had higher chlorophyll and electrical conductivity and significantly lower malondialdehyde content than the wild type， indicating that AfWRKY42 played an important regulatory role in response to salinity stress. It would provide a WRKY transcription factor candidate gene for salinity resistance breeding and lay the foundation for improving the resistance of A. fruticosa and other plants.
To investigate the potential function of ubiquitin carboxyl terminal hydrolases（UCHs） in the ubiquitination process of laticiferous of Hevea brasiliensis， the full-length sequences of two UCHs family members（HbUCH-L3 and HbUCH-L5） were isolated from Hevea brasiliensis， and both had typical UCHs domains. The open reading frame of HbUCH-L3 and HbUCH-L5 were 993 bp and 558 bp， and encoded 330 and 185 amino acids， respectively. The results of qRT-PCR showed that HbUCH-L3 and HbUCH-L5 were constitutively expressed in all tissues， but was low in latex. In vitro ubiquitination substrates cleavage of recombinant HbUCHs showed that both HbUCH-L3 and HbUCH-L5 had the function of hydrolyzing ubiquitin. HbUCHs significantly reduced the overall ubiquitination level of C-serum proteins； while， the deubiquitinating activity of HbUCH-L3 was higher than that of HbUCH-L5. Therefore， it was speculated that UCHs played a role in maintaining the dynamic balance of laticiferous ubiquitination and thus played a specific biological function， but the exact mechanism was still unclear.
To explore the role of SSR1 gene encoding a functionally unknown mitochondrial protein of Arabidopsis thaliana in plant growth and stress response， ssr1-2 and its suppressor mutants EMS143 and EMS145 were used to track the growth of their roots and aboveground parts， as well as their proline sensitivity and iron homeostasis， and the effects of ssr1-2 short root phenotype on aboveground growth of seedlings were analyzed by micrografting. The results showed that the primary root length of ssr1-2 was shorter and the root architecture was similar to the fibrous root system. The growth of their shoots was also retarded， but appeared considerably later than the short-root phenotype. The results of micrografting revealed that the roots of ssr1-2 could restrict the growth of the wild-type shoots and， and conversely， the shoot of ssr1-2 could also affect the growth of the wild-type roots， but the former had a bigger effect. ssr1-2 showed a hypersensitive phenotype to proline in seed germination， root length and leaf chlorophyll content. In addition， ssr1-2 appeared insensitive to Fe nutrition， that was， the stimulating effect of Fe salt on seedling growth was significantly less than that of wild-type WS， indicating a significant decrease in its ability to utilize Fe. The results suggested that SSR1 might be involved in the regulation of A. thaliana root growth by affecting the utilization of iron nutrient， implying that the impairment of the mitochondrial iron utilization machinery might contribute significantly to the enhanced inhibitory effect of proline on plant growth and development.
In order to explore the role of WALLS ARE THIN（WAT1） in wood formation and response to stress in woody plants， bioinformatics tools was used for analysis， and quantitative Real-time PCR（qRT-PCR） was used to investigate the expression patterns of EgrWAT1L and EgrWAT1S in different tissues， internodes and in response to different stresses， and the gene EgrWAT1S and its other transcript EgrWAT1L were cloned from Eucalyptus grandis. The results showed that the EgrWAT1S was highly expressed in phloem， while EgrWAT1L was mainly expressed in roots， and the expression patterns were significantly different under methyl jasmonate（MeJA） and salicylic acid（SA） treatment， salt stress， phosphorus（P） and boron（B） deficiency， and even opposite under MeJA and SA. These results suggested that EgrWAT1L might affect EgrWAT1S expression through transcriptional regulation and further protein translation in response to hormone and stress treatments. The studies provided a basis for further elucidate the function in the growth and development of EgrWAT1 gene and also provided a possibility for future molecular breeding of Eucalyptus.
To investigate the effect of β-amyrin synthase gene（Aeβ-AS） on triterpenoid saponin synthesis of Aralia elata， the Aeβ-AS gene was cloned and genetically transformed into tobacco. The expression difference of Aeβ-AS gene in different parts of transgenic tobacco was analyzed， and the expression levels of Aeβ-AS gene， upstream and downstream key enzyme genes and total triterpene content were detected respectively. The results showed that the plant overexpression vector PROKⅡ-Aeβ-AS was successfully constructed and transferred into wild type tobacco， and seven transgenic lines were obtained and expressed at the mRNA level， and the expression level in leaves was higher than that in roots and stems. In transgenic tobacco， the expression levels of Aeβ-AS gene and its upstream and downstream key enzyme genes were higher than those of wild type， and the relative expression levels of NtFPS and NtSS genes of strain L21 were the highest， and the relative expression levels of NtSE and Aeβ-AS genes of strain L30 were the highest. Compared with wild-type tobacco， the total triterpene content of GM tobacco was significantly increased（1.1-1.6 times）. The results showed that synthesis of Aeβ-AS gene and heterologous transformation in tobacco could significantly increase the total triterpene content in transgenic tobacco.
To clarify the function of FmCCoAOMT gene， the expression pattern of FmCCoAOMT gene was analyzed by qRT-PCR using Fraxinus mandshurica as material in this study. The results showed that FmCCoAOMT gene was tissue specific. The expression level was the highest in the xylem of both female and male perennial plants， and was 9.4 times higher than that in the bark of new shoots in male plants， and was 13.1 times higher than that of leaf in female plants. The leaf disk method was used to obtain transgenic tobacco plants. Compared with the wild type， the lignin content of overexpressing FmCCoAOMT was increased by 41%， while the cellulose and hemicellulose contents were decreased by 32% and 52%， respectively. Tobacco overexpressing FmCCoAOMT after abiotic stress treatment compared to wild type， the activity of POD and SOD increased， the content of MDA decreased， and the content of reactive oxygen decreased after stress treatment. After NaCl treatment， POD activity was 83% higher than that of control group. After NaCl， ABA and mannitol treatments， SOD activity increased by 56.6%， 44.2% and 19.0%， and MDA content decreased by 77.2%， 11.7% and 47.6%， respectively. The results indicated that FmCCoAOMT gene might be involved in lignin synthesis of F. mandshurica， promoted abiotic stress tolerance.
To reveal the role of transcription factor PsnbHLH162 of Populus simonii × P. nigra under abiotic stresses， and to explore the signal transduction process of genes in plants， the signal transduction process of this gene in plants is explored to provide the theoretical basis for the acquisition of excellent stress-resistant tree species in the future. In this experiment， PsnbHLH162 gene was cloned and obtained from P. simonii × P. nigra as material， and the target gene and promoter were analyzed by bioinformatics； and the function of genes in response to abiotic stresses was analyzed by fluorescence quantitative PCR. The results showed that the cDNA of PsnbHLH162 gene was 537 bp， and the N-terminal of the gene contained a highly conserved HLH domain， and the encoded protein might be a stable hydrophilic protein without trans-membrane region， localized in the nucleus and had no transcriptional activation activity. The promoter region contained a variety of ABA-responsive， auxin-responsive， light-responsive and circadian elements， confirming that this gene participated in abiotic stress response. The results of fluorescence quantitative PCR showed that under salt stress， the gene expression in root tissue was the highest compared with stem and leaf tissue； under low temperature stress， gene expression in stem tissue was the highest compared with leaf and root tissue. It was found that the expression of PsnbHLH162 could be induced by salt and low temperature in plants.
To explore the role of H2S signal in regulating stomatal movement in alfalfa and the relationship between H2S and Ca2+ during this process， wild type and the calcium transporter mutants of Medicago truncatula were used as experimental materials， and the transcriptional level， cellular level and physiological level were studied respectively， and the expression levels of related genes were compared by qRT-PCR， the content of Ca2+in vivo was detected by fluorescent probe， the content of H2S was measured by electrode method， and the stomatal aperture was observed by microscope respectively. The results showed that compared with the wild type， the content of H2S in the mutant NF3011 and NF2734 decreased significantly； the H2S signal inhibited the expression of calcium transporter encoding gene MTR_6g027580， the exogenous physiological concentration of H2S fumigation could induce stomatal closure of Medicago truncatula， and the combination of treatment with LaCl3， Ca2+ channel blocker， had no effect on stomatal movement of wild type， but opposite in mutant； the concentration of Ca2+ in guard cells was measured by fluorescence probe， which were consistent with the change law of stomatal aperture. In conclusion， the content of Ca2+ in guard cells of leaves is induced by H2S， whose stomatal aperture become smaller at the same time. In this process， the change of intracellular Ca2+ concentration mainly depend on Ca2+ transporter， and small partly depend on Ca2+ channel. The results of this study not only enrich the mechanism of H2S signaling in theory， but also have the potential to be applied to alfalfa production and other crops.
The root tip of six cultivars of Freesia hybrida was used as the plant material for karyotype analysis to reveal their karyotype characteristics. Then cluster analysis was performed to clarify the similarity among the six cultivars and to explore their relationship. The results showed that the six freesia cultivars were all tetraploid with 44 chromosomes. The karyotype formula of six cultivars of F. hybrida were as follows respectively： 2n=4x=44=33m+11sm for ‘Gold River’， 2n=4x=44=1M+34m+9sm for ‘White River’， 2n=4x=44=35m+9sm for ‘Castor’， 2n=4x=44=32m+12sm for ‘SN Hongtaige’， 2n=4x=44=38m+6sm for ‘Pink Passion’ ， and 2n=4x=44=37m+7sm for ‘Red River’. The karyotype asymmetry coefficient ranged from 57.00% to 60.07%. The karyotype types were all 2B， and the degree of evolution was medium，which was in the transition stage from symmetry to asymmetric. The evolution trend of karyotype was： ‘Pink Passion’→’Red River’→‘Gold River’→‘White River’→‘Castor’→‘SN Hongtaige’. The clustering results showed that when the genetic distance was 15， it could be divided into two categories： the first category included ‘Gold River’， ‘SN Hongtaige’ and ‘Castor’， the second category included ‘Pink Passion’， ‘Red River’ and ‘White River’. The difference of its karyotype parameters could be used as the basis for the classification of different cultivars of freesia plants， provided theoretical reference for its cytological inheritance and breeding practice in future.
To reveal the function of transcription factor PsnbZIP1 from Populus simonii × P. nigra under abiotic stresses， P.simonii × P. nigra was used as material， and the PsnbZIP1 transcription factor gene was cloned with an ORF length of 432 bp， and the molecular mechanism of PsnbZIP1 under salt stress was analyzed. The expression pattern of PsnbZIP1 in P. simonii × P. nigra was analyzed under 150 mmol·L-1 NaCl by q-PCR， it was found that the expression of the gene increased rapidly. The PsnbZIP1 was predicted to be a hydrophilic unstable protein without trans-membrane structure but with signal peptide by bioinformatics analysis. The sub-cellular localization of the gene was observed by Agrobacterium mediated transient expression in tobacco. The results showed that the gene was a nuclear localization protein. It was confirmed by Y2HGold yeast competent cells that the gene had no transcriptional activation activity in yeast. The analysis of the promoter sequence of the PsnbZIP1 gene showed that the promoter contained auxin response elements， ABA response elements， light response elements and seed-specific cis-acting regulatory elements respectively， which might play a role in plant growth and development and response to stress.The promoter also included the MYB binding site and MYBHv1 binding site involved in drought induction， indicating that the gene was likely to interact with some of MYB involved in drought induction.
The bHLH proteins are the second large family of transcription factors， which play important roles in plant growth development， physiological metabolism and stress responses. In this study， Hippophae neurocarpa and H.tibetana were used as materials， the positive selection of the transcription factor bHLH94 gene was screened out using transcriptome sequencing. Based on the analysis of gene sequence and expression of HnbHLH94 and HtbHLH94， the response mechanism of bHLH94 gene in H.neurocarpa and H.tibetana adapted to altitude was studied. The HnbHLH94 and HtbHLH94 gene encoded 338 and 335 amino acids respectively. Sanger sequencing method verified the correctness of sequences， however there were 10 non-synonymous mutation sites outside the DNA binding domain of the two genes， which was speculated to be related to the adaptive evolution of the gene. As the altitude increased， the HnbHLH94 gene expression decreased， while the HtbHLH94 gene expression increased based on qRT-PCR technology. It suggested that gene might provide a molecular basis for adaptation to altitude， drought， freezing and radiation respectively， and the sequence structure and expression of HnbHLH94 and HtbHLH94 genes responded to the habitat conditions of elevation.
To reveal the structure and function of natural rubber biosynthase interacting proteins in natural rubber biosynthesis， the total protein of latex rubber particles of Hevea brasiliensis was used as material， and an unknown functional protein containing DUF1262 domain was captured by immunoprecipitation assay using an antibody targeting CPT， a key enzyme for natural rubber biosynthesis. Bioinformatics analysis showed that there were 50 gene sequences encoding DUF1262 domain proteins in the rubber tree genome， and protein interaction network analysis suggested that DUF1262 domain proteins might be involved in signal transduction and transcriptional regulation. Fluorescence quantitative PCR results showed that the transcription of the gene encoding this protein was widely distributed in roots， leaves， flowers， branches， latex and other tissues respectively， andthe expression was low in latex and but high in bark. Salicylic acid， abscisic acid， hydrogen peroxide and drought treatment could enhance the transcription level of this gene in leaves respectively. This study demonstrated that the DUF1262 protein was involved in stress response and other physiological processes of rubber tree， providing new clues for revealing the regulation mechanism of latex biosynthesis.
A total of 18 new StCRKs（cysteine-rich receptor-like kinases， CRKs） family genes were identified in potato genome using online tools such as Pfam. These StCRKs localize on chromosome 1， 2， 11 and 12， and all contain conserved CRK domains. In the promoter regions of these StCRKs genes， there are five plant hormones responsive elements， the circadian control element， biotic and abiotic responsive elements， and seed-specific regulation element. The tissue-specific expression pattern were measured using qRT-PCR method in root， stem， leave， and flower tissues in potato during flowering time. The results showed different StCRKs expressed in different tissues. After salicylic acid analogue BTH and 4 ℃ treatment， it was found that 10 StCRKs genes could be induced by BTH and 13 StCRKs genes were able to respond to the low temperature signal. The results provide clues for further elucidation functions of StCRKs on biotic and abiotic stresses.
In order to clarify the function of StNPR4 in potato， the CDS and promoter sequence of StNPR4 were cloned and bioinformatics analysis was conducted. Tissue expression pattern was performed by qRT-PCR， and a binary expression vector of StNPR4 driven by its own promoter was constructed and transformed into potato. Transgenic plants were used to examine the responses to salicylic acid， Phytophthora infestans and high salt stress respectively. The results showed that StNPR4 had typical functional domains in NPR1 family with cis-acting elements on the promoter in response to biotic and abiotic stresses. The StNPR4 expression level was highest in leaves StNPR4 was inductively expressed by SA and the induced expression was higher in transgenic plants than in the control. StNPR4 transgenic potato plants showed enhanced resistance to Ph. infestans and higher rooting rate under high salt stress. This indicated that StNPR4 played an important role not only in biotic stress but also in abiotic stress of potato.
