Coronatine (COR) is a kind of novel plant growth regulator, which is environmentally friendly and can be used to effectively control plant growth and enhance plant resistance. At present, coronatine is mainly produced by microbial fermentation, however low fermentation yield is the biggest problem that limits the application of coronatine. To improve the yield of coronatine, the method of semicontinuous fermentation using Pseudomonas syringae Z2-6 was established, and related fermentation conditions were optimized. Optimization includes the initial removal time, and removal volume. The results showed that the beginning of removal at the 10 d post inoculate (dpi) could extend the production cycle of coronatine, which extended for more than 4 d than the beginning at 8 or 9 dpi, and kept the synthesis of coronatine maintaining at a high level. Coronatine production efficiency with time interval of 1 d was higher than 2 d. When the amount of removal was not more than 30%, the growth of bacteria was not be affected. The highest coronatine yield was obtained at the amount of removal of 30%. In conclusion, when 30% of the fermentation broth was removed from 10 dpi every day and equal volume fresh medium was supplemented, the production of coronatine reached the highest level. The yield of coronatine was increased from 11.2 mg/(L·d) to 22.1 mg/(L·d), and the production efficiency was increased by 98%. This method of semicontinuous fermentation could be used to improve the production efficiency of coronatine, reduce the production cost, and provide support for the further popularization of coronatine.

Natural transformation can be used to construct deficient mutant of Haemophilus parasuis (HPS), and it has significance on the study of pathogenesis and virulent factors. The experimental conditions of natural transformation are optimized by control variable methods. This research was conducted to obtain the optimal conditions for natural transformation of HPS. The transformation frequency was compared under the differential conditions, which including the bacterial phases (OD600=0.15, 0.40, 0.70, 1.00, 1.30, 1.50, 1.60, 1.90, 2.00), bacterial incubation time (T1=7, 9, 11, 13, 15, 17, 19 h), bacterial concentration (1/100 OD600=0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7), the concentration of exogenous cyclic adenosine monophosphate (cAMP) (terminal concentration C1=0.0, 0.1, 0.2, 0.4, 0.8, 1.0, 2.0, 4.0, 8.0 mmol/L), plasmid (terminal concentration C2=0.000, 0.005, 0.010, 0.020, 0.040, 0.050, 0.100, 0.250, 0.350 μg/μL), and the incubation time after transformed (T2=0, 1, 2, 3, 4, 5, 6, 7 h). The results indicated that the transformation on agar plates of HPS occurred in a high frequency when the spotting bacteria was in the late-log phage(OD600≈1.5~1.7), the incubation time was 13 h, the concentration of exogenous plasmid was more than 0.1 μg/μL, and the incubation time after transformed was 5 h. The optimal conditions of natural transformation are determined, which lay a foundation for the research of virulence factor and pathogenic mechanism associated with Haemophilus parasuis.

Banna Miniature Inbred Pig (Sus scrofa) is the first large inbred mammal experimental animal. Its gene is highly homozygous, and genetic background is clear. And its gene has great potential in genetic breeding, functional gene research, human disease model and xenotransplantation. However, due to the high degree of inbreeding depression, the survival rate of offspring is low and genetic resources are very precious, and the preservation of the genetic information has scientific and practical significance. In this paper, the lean subline pig and the fat subline pig were taken as the research objects. The two subline pigs' DNA was extracted from agarose pre-embedding lumps, and sheared to the appropriate size (about 36~48 kb) by physical method. And then, the DNA fragments were used to construct the two sublines' Fosmid libraries by CopyControl? Fosmid Library Production kit. There were about 300 000 and 400 000 clones of the lean subline pig's and the fat subline pig's Fosmid library respectively, which covers the genome up to 4.4 and 5.9 fold. Basing on the upstream sequence of lean subline pig' heart-type fatty acid-binding protein gene, we designed the specific primers for grading PCR to screen the target clone in the library. Firstly, the first round PCR reaction using the original library bacteria solutions as template were conducted, which could screen the tube containing the target clones. When the tube which contained the positive target clones was confirmed, and then the positive tube bacterium were cultured. Then, the colonies of every plate were collected to one EP tube, and used as template to do PCR, to continue to screen the target tube. Like this, several rounds grading PCR were done until the target tube which contained 100~1 000 CFU per 10 μL LB medium was screened. Then this positive tube bacterium were cultured on LB medium plates. After 12-16 hours, the single colonies on LB medium plate were transferred to 96-well plate continued to culture as the same conditions as before. Then, the single colony bacteria solutions were used to do PCR to screen positive colony, and sequenced to confirm the single colony of bacteria containing target gene. Five single colonies that were identified by sequence analysis were abtained. Using the above method, we can get the fat subline pig's target single gene clone. We have successfully constructed the two subline pigs' Fosmid libraries, with a high rate of genome coverage, and the probability of screening to the target gene is 99% in theory. These Fosmid libraries can be used to preserve Banna Miniature Inbred Pig's genetic resources and carry out more deeply research on function and regulation of gene.

The copy number of exogenous gene affects the genetic stability and gene expression level in transgenic plants, and the identification and screening of single copy homozygous plants are necessary for the functional analysis of progeny. To fast and accurately identify gene copy of transgenic rice materials harboring hygromycin resistant gene (hygromycin phosphotransferase II (hptII), Taqman based Multiplex qRT-PCR was developed using constructive gene Oryza sativa splicing factor u2af (OsSFu2a) as reference gene. By comparison of amplification efficiency of different primer sets, the primer set of hptII-1 and OsFU2a-2 was the best one. The copy number of 10 transgenic clones was determinated using this system, and the results shown that 7 of 10 were single copy and 3 were double copy. Furthermore, Southern blot and segregation ratio analysis were performed to validate this system, and when compared with traditional singlex qRT-PCR, this system improved the stability and accuracy of detection result. Therefore, this system could be great help for plant biotechnologists to fast, accurately and high-throughput identify endogenous gene copy number and homozygous plant.

The replacement of histone (H2A) with its variants in nucleosome regulates gene expression in plant. Although it is revealed that AtH2A involves in regulation of growth and development in Arabidopsis thaliana, study on function of H2A family members in plant is rare. In this study, A StH2A-1 cDNA was isolated from potato (Solanum tuberosum). A Nicotiana benthamiana transgenic line h2a-1, expressing antisense StH2A-1 cDNA displayed 31% lower leaf H2A contents than wild type (WT), leading to an apical dominance-deficient and late-flowering phenotype. BLAST outcome in Nicotiana benthamiana genome database showed that 4 NbH2As, sharing higher than 97% similarity with StH2A-1 uniquely contain a poly (Aln) motif with various length in the N-terminal of the amino acid sequences. Outcome of the reverse transcription polymerase chain reaction (RT-PCR) showed that accumulation of NbH2A mRNA, encoding NbH2A with N-terminal poly(Aln) was lower in the h2a-1 line than it in WT. The other NbH2As displayed a clearly higher mRNA accumulation in the h2a-1 line than it in WT. This suggested that NbH2As with and without N-terminal poly (Aln) may be complemented one another to function in gene expression regulation. RT-PCR also exhibited that mRNA accumulations of floral genes, including ELF4(EARLY FLOWERING 4), CO(CONSTANS) and FT(FLOWERING LOCUS T) in the h2a-1 line were down-regulated, suggesting that NbH2As with N-terminal poly(Aln) may involve in transcription of those genes. As a conclusion, StH2A-1 is associated with formation of apical dominance and regular flowering and positively regulates plant growth and development. The result may contribute to further understanding on function of H2A in plant growth and development.

In order to investigate the differential gene expression on intramuscular and subcutaneous preadipocytes during differentiation in Congjiang Xiang pig (Sus scrofa), the fat precursors culture model in vitro of Congjiang Xiang pig was established. In this study, Congjiang Xiang pig in five days old was provided, then subcutaneous adipose tissue and the longissimus dorsi muscle was excised. We used collagenase to harvest preadipocytes from longissimus dorsi muscle and subcutaneous adipose tissue respectively. The two preadipocytes were induced differentiation by a series of hormone-induced complexes. The mRNA expression levels of gene involved in adipogenesis, such as peroxisome proliferator-activated receptorsγ (PPARγ), lipoprotein lipase (LPL), fatty acid synthase (FAS), acetyl-CoA carboxylase (ACC), adipose triglyceride lipase (ATGL) and Hormone sensitive lipase (HSL), were compared by real-time PCR in the two preadipocytes. The result showed that the model of preadipocytes in vitro was successfully established from Congjiang pig. The mRNA expression levels of PPARγ, FAS and ACC in intramuscular preadipocytes were significantly a higher at 72 h after the addition of inducer, which a higher than the level in 0 h (P＜0.05). The mRNA expression level of LPL was no significant difference in the various stages, but there was a significant up-regulation of LPL in 144 h after the addition of inducer, and the value was significantly higher than the other stages (P＜0.01). The mRNA expression levels of ATGL and HSL showed the pattern of “up-down-up”in the differentiation phase. In the subcutaneous preadipocytes, the mRNA expression level of PPARγ was the highest in 72 h after induction, and the value was significantly higher than in other stages (P＜0.01). The mRNA expression levels of LPL, FAS, ACC, ATGL and HSL were all higher in 48 and 72 h, and the value was significantly higher than that in 0h (P＜0.01). In summary, this study showed that in intramuscular preadipocytes, the genes related to adipocyte differentiation were more active at the late stage of differentiation, while in subcutaneous preadipocytes, the related genes were more active at the earlier stage of differentiation in vitro. The result prompt that the formation of subcutaneous adipose is much earlier than intramuscular adipose, which may be of great significance in fat deposition mechanism of Congjiang Xiang pig for further study.

Auricularia heimuer is an important and widely cultivated edible fungus in China, and the morphological development and genetic breeding were concerned by researchers. It has brought a lot of inconvenience in breeding, because of introduction and serious synonymy phenomenon. A molecular ID system based on SSR markers for identifying and differentiating of germplasm resource, and it is important to identify and protect the germplasm resources of edible fungi. In this study, SSR markers were employed to clarify the genetic diversity and to establish the SSR ID of 72 accessions of A. heimuer collected from different areas in China, which contain cultivated and wild species. Eight SSR primer pairs were selected from a pool of 65 primer pairs, the cluster results were analysed by the method of UPGMA (unweighted pair-group method with arithmetic means). The results showed that the genetic similarity coefficient were 0.37~1.00, at the 0.56, the tested strains could be divided into 3 groups. The following groups of strains are the synonyms. Au1 to Au11 is series of Hei Shan, Au44 and Au90, Au40 and Au73, Au22 and Au60. They have a close relationship. Based on the results of the UPGMA cluster analysis, amplification patterns SSR molecular ID system, the molecular identities for the resource protection and new varieties of A. heimuer can be construct. The results provide a basis for identification and protection of edible fungi germplasm resources.

In plants, epicuticular wax plays important roles in plant growth and development, and it is indispensable for plants to adaptation to the external environment. Based on the known protein sequences of wax genes in Arabidopsis thaliana, 44 gene copies for 10 wax inducer 2 (WIN2) gene families were identified and analyzed in the genome reference of rapeseed (Brassica napus) ZS11 by the bioinformatics methods. In this study, the cDNA were isolated from 3 oilseed (L1050, P144 and W481) with the differences on epicuticular waxy of leaves before bolting (BB) and after bolting (AB), respectively. Then they were thoroughly cloned by the specific primers from them using the homology-based cloning strategy. However, homology comparison revealed that the nucleotide and protein had high sequence homology among them, but the SNPs and amino acid mutations were stably detected in the sequences of BnWIN2C01. Meanwhile, the expression levels of BnWIN2C01 were detected using the qRT-PCR among the leaves before bolting (BB) and after bolting (AB) in different B. napus. The results showed that the highly differences were found among them, indicating that the specific expression of BnWIN2C01 might be involved in the biosynthesis of epicuticular waxy. Results will be helpful for deciphering the molecular mechanism of epicuticular waxy in B. napus, and provide a reference to improve the rapeseed stress resistance.

WD-repeat protein, containing WD-40 motif, also known as WD40 repeat protein, which plays an essential regulating role during the abiotic stress response of plants. In this study, it was found that a kenaf (Hibiscus cannabinus) transcriptome unigene which was highly similar to the WD40 gene, and then designed the primers to carry out reverse transcription PCR amplification and obtained HcWD40-1(GenBank No.: KX711617) cDNA sequence by sanger sequencing. Bioinformatics analysis showed that the gene open reading frame was 1 356 bp, encoding 451 amino acids, and containing 7 typical WD40 domains. qRT-PCR analysis showed that HcWD40-1 gene was induced by salt and drought stress, and was induced by abscisic acid (ABA) and methyl jasmonate (MeJA). The results suggested that the HcWD40-1 gene was a key hub gene for ABA and MeJA signaling pathways, salt and drought stress responses. The results of the study provide basic data for the study of salt-tolerant, drought-resistant on ABA signal regulation network and MeJA signal regulation network of kenaf.

The fecundity of sheep (Ovis aries) is controlled by the dominant gene, and the bonemorphogenetic protein type IB gene (BMPR-IB) has been identified as one of the dominant genes in multiparous ewes. To provide a theoretical basis for breeding for sheep fecundity and markers, the polymerase chain reaction single strand conformation polymorphism (PCR) was used to analysis the single nucleotide polymorphism (SNP) detection at 1113 loci in the bonemorphogenetic protein type IB (BMPR-IB) gene coding region of 3 breeds of sheep, including 188 Mongolian sheep, 112 Gansu Alpine Merino sheep and 48 Small Tailed Han sheep, then we also analyze the correlation among the polymorphism of 3 breeds of sheep BMPR-IB gene and litter size, in order to provide the material for studying the mechanism of prolificacy in sheep. The result showed that 3 different genotypes were found in Mongolian sheep and Gansu Alpine Merino sheep, including wild type called AA, homozygous mutant called BB and heterozygous mutant called AB, AB was the dominant genotype. 2 different genotypes were found in Small Tailed Han sheep, BB and AB, BB was the dominant genotype. With the analysis of polymorphism information content, it was shown that Small Tail Han sheep belonged to low polymorphism (PIC＜0.25), the other two breeds of sheep belonged to moderate polymorphism (0.25＜PIC＜0.5). There was a significant difference in the 1113 locus of BMPR-IB gene coding region among these 3 different breeds of sheep, this site may be related to the litter size of sheep so it can be used as a marker loci for reproductive traits of sheep and further theoretical basis for the production practice.

WRKY is a class of transcription factor superfamilies found in higher plants. Many studies have shown that WRKY transcription factors play a critical role in biotic and abiotic stress of plants. However, these studies are mainly concentrated in arabidopsis and other model plant species and also have been reported in melon(Cucumis melo) and pepper(Capsicum annuum) and other vegetable crops, while the research of WRKY transcription factor about browning from luffa (Luffa cylindrical) has not been reported in present. The transcriptome database of luffa Fushi-3 fresh-cut browning flesh were obtained by using the transcriptome sequencing (RNA-seq)technology in early , and a total of four Unigene sequences that shared high homology with WRKY protein were screened from the transcriptome databases in this study. Sequence analysis showed that full length of these four luffa WRKY genes (Unigene0018509, Unigene0021412, Unigene0025291 and Unigene0034271) were 2,189、1,990、2,365 and 2,274 bp, respectively, and they all contained an open reading frame(ORF), and the GenBank accession numbers were from KY621843 to KY621846. Wolf Psort prediction indicated that WRKY proteins encoded by the four Unigene were located in the nuclear. Gene phylogenetic analysis and conservative domains analysis showed that four luffa WRKY transcription factors all had a WRKY conserved domain and C2H2 type zinc finger, which suggested that these four WRKY protein were members of the second category of WRKY protein family. The expression levels of these four WRKY genes in different tissues (root, stem, leaf,flower and fruit) and different storage periods of postharvest flesh were analyzed by quantitative Real-time PCR, and the expression patterns and RNA-seq results of four WRKY genes during different time points of fresh-cut browning flesh have also been analyzed. The results showed that all of the four WRKY genes had the tissue-specific expression, and their expression patterns were different. The variation trends of these four WRKY genes on the expression of quantitative Real-time PCR and RNA-seq databases (i.e.，FPKM value) in luffa fresh-cut browning fruit were almost same, and the expression of Unigene0018509, Unigene0021412 and Unigene0025291 were significantly up-regulated from 0 h to 6 h after fresh-cut browning, and the expression of Unigene0034271 was up-regulated during 0 ~ 3 h and then decreased in 6 h. Overall, the expression trends of four WRKY genes were up-regulated in different postharvest and storage browning periods and their expression patterns were not the same. This study may provide a theoretical foundation for further expounding the luffa browning mechanism and breeding of varieties.

Lycopene beta-cyclase (LCYB), which catalyzes the formation of β-carotene and its oxides, is an enzyme act as branching point of the plant carotenoid synthesis pathway. This study investigated the effect of elicitors on the expression of lcyb gene and content of fucoidin in Phaeodactylum tricornutum. Firstly, the full-length cDNA of lcyb had been obtained from P. tricornutum, and bioinformatics analysis was carried out. Then, aethyl jasmonate (MeJA), aacetylsalicylic acid (ASA)，arachidonic acid (AA) and ammonium cerous sulfate (ACS) were used to induce the expression of Ptlcyb gene and the content of fucoxanthin in P. tricornutum. The results showed that the lcyb gene of Phaeodactylum tricornutum obtained by de novo sequencing was same as the sequence on NCBI(GenBank No. XM002176576). The full length of lcyb gene was comprised by 2 060 bp, contanting a 1 980 bp open reading fragment(ORF), which encoded a polypeptide of 659 amio acids. Sequence analysis showed that P. tricornutum LCYB had a dinucleotides-biding site at the N-terminus and a Predicted TM helix at the C-terminus. In addition, signal peptide and the presence of chloroplast peptide han been found, which further proved that LCYB was located in the thylakoid membrane. Phylogenetic tree demostrated that diatom such as P. tricornutum coexisted in an evolutionary branch, which was closely related. Upon the observation of Ptlcyb regulation expression, the expression level of Ptlcyb had significant rise when the concentration was 50 μmol/L MeJA, 10 mg/L ASA, 0.1 mg /L AA and 0. 4 mg/L ACS. At the same time, the content of fucoxanthin in P. tricornutum was significantly higher than that in the control group (P ＜0.01). The variation trend of fucoxanthin content and Ptlcyb expression were basically the same, which showed that Ptlcyb played an important role in the synthesis of fucoxanthin. This study provide theoretical guidance for the regulation of fucoxanthin synthesis and also provide a reference for further improving the content of fucoxanthin by means of metabolic engineering in P. tricornutum.

F-box protein, as one component of SCF ubiquitin-ligase (skp1-cullin1-F-box) complex, is involved in growth, cell division and hormone responses of plants. To better understand the molecular mechanisms of plantlet formation of Kalanchoe daigremontiana, an F-box protein, KdFBX(GenBank No. KU740356), was cloned using rapid amplification of cDNA ends (RACE) PCR. KdFBX gene consists of an ORF of 987 bp which was predicted to encode a 382 amino acid residues long protein of 37.72 kD with an isoelectric point of 4.4. KdFBX showed homology to orthologs from Ricinus communis, Gossypium raimondii and other plants. Phylogenetic analysis showed that KdFBX protein was most related to Arabidopsis thaliana. No obvious transmembrane region, signal peptide or cleavage were observed. KdFBX protein was a water-soluble and non-secreted protein that located in the cytoplasm. 247 amino acids of KdFBX protein were completely matched to the B chain of the ubiquitin ligase substrate complex, and the backbone structure, as well as the side chain structure was reasonable through Ramachandran analysis. Real-time PCR analysis revealed that KdFBX transcript was expressed highly in root (2.5 times compared to petiole) and down-regulated under osmotic stress. This study characterized the novel KdFBX gene from K. daigremontiana for the first time, and the results may be useful for further functional determination of this gene.

GATA transcription factor family, which regulates a variety of biological processes, was widely studied in some plant pathogenic fungi. However, up to date, little is reported about the family in Setosphaeria turcica. In the present research, bioinformatics methods were used to search transcription factor family in the whole genome database of S. turcica. Five members of GATA transcription factor family designated as StGATA1~StGATA5, which scattered in the 5 different scaffolds in the genome, were identified. The analysis based on the physical and chemical properties of the GATA transcription factor family members showed that StGATA3 was acidic, while the other four GATA transcription factors were alkaline. All of the five proteins were unstable proteins. The amino acid number in predicted proteins were between 278 and 1 080. Conservative domain analysis indicated that the transcription factors contained ZnF-GATA domains. Both StGATA3 and StGATA4 contained PAS domain, while StGATA4 contained two PAC domains. Phylogenetic analysis showed that StGATA2 was in a group with Passalora fulva, Penicillium urticae and Aspergillus nidulans, while the other four GATA transcription factors (StGATA1, StGATA3, StGATA4, StGATA5) in S. turcica were assembled into a cluster. Real-time PCR technique was employed to analyze the expression patterns of GATA transcription factor family at different developmental stages in S. turcica. The results showed that StGATA1 and StGATA3 expressed with significant higher level at mycelial growth stage than that of other stages. StGATA2 showed significantly higher level in invading wire stage than that of the other period, and its expression level at mycelial and conidium stage were relative high. StGATA4 and StGATA5 expressed with relative high level at mycelial and conidium stage(P＜0.05).The result will not only lay a foundation for further revealing the functions of GATA transcription factor family in S. turcica, but also provide a identifiable ground for fungus disease prevention and control.

The reference gene expression stability levels are the important factors to determine the reliability of quantitative Real-time polymerase chain reaction (qRT-PCR) results. This study was conducted to explore the selection of stable reference genes in different treatment of Glucosamine (GluN) to ensure the reliability and accuracy of gene expression analysis in caged layers (Gallus gallus domesticus) during the late laying period. A total of 500-day HY-Line layers were selected to the research objects which were randomly divided into four groups, the layers in control group were fed with the diet of the corn-soybean meal basal diet, while the others in experiment groups were fed the basal diet supplemented with 0.4%, 0.6%, 0.8% GluN, respectively. Expression of ten housekeeping genes, ribosomal protein S2 (RPS2), β-Actin, glyceraldehyde-3-phosphate dehydrogenase(GAPDH), hydroxymethyl biliary synthetase(HMBS), TATA-box binding protein(TBP), hypoxanthine phosphoribosyltransferase 1 (HPRT1), tubulin beta class (TUBB), succinate dehydrogenase complex flavoprotein subunit A (SDHA), ribosomal protein L4 (RPL4) and beta-2 microglobulin (B2M) were assessed by qRT-PCR in 8 tissues (heart, liver, lung, kidney, duodenum, pancreas, uterus and tibia), and 3 online reference genes stability assessment tools(geNorm, NormFinder, RefFinder) and the methods of Cycle threshold (Cq) and Delta cycle threshold (Delta CT) were used to the expression stability assessment of 10 reference gene by the qRT-PCR data. The results were shown as follows. The expression stability of same candidate reference gene varied with different treatment and tissues from the analysis of Cq mean value, which explained that the 10 reference genes had a certain expression difference in these 8 tissues. Under the different GluN treatment, the method of Delta CT found that the expression stability of reference genes were not the same, in general, the expression stability of TUBB、β-Actin and RPS2 were better than other genes. From the geNorm software, TBP/RPS2(1.08), RPS2/β-Actin(0.88), TBP/TUBB(1.16) and RPS2/HMBS(0.77) showed the stable expression under different GluN treatment, which showed that the expression stability of TBP, β-Actin and RPS2 were better. The NormFinder program software showed that RPS2 and HMBS were the stable genes. The refFinder online software showed that the RPS2 had most stable expression under 0.0% and 0.8% GluN group. From the comprehensive analysis of above results, RPS2 and TBP genes can apply to the quantitative expression analysis for the caged layers during the late laying period, while the expression stability of RPL4 gene was the worst, and it can not be applied to quantitative expression analysis. This experiment results also provide a theoretical basis for correcting the expression of target genes under the treatment of GluN.

Plant transgenic studies have found that multiple copies insertion often lead to decreased expression of exogenous genes. However, when constructing the vector, the same exogenous gene is constructed on the same vector with series connection. Whether it will improve the expression of exogenous genes after transformation of plants has not been reported. In order to improve the expression of exogenous gene in transgenic plants and explore the effect of superposition of the same target gene on gene expression and transgenic plant growth, in this study, the plant transformation vectors carrying single and double Bacillus thuringiensis(Bt) gene Cry1Ac respectively were transformed into tobacco (Nicotiana tabacum) tissue culture seedlings by Agrobacterium (Agrobacterium tumefaciens)-mediated leaf disc method. Then, the transgenic lines were detected by polymerase chain reaction (PCR), fluorescence quantitative PCR (FQ-PCR) (Absolute quantitative) and Bt toxin and insect resistance tests to identify whether or not the exogenous gene was integrated into the tobacco genome and the expression were carried out. At the same time, morphological indexes and physiological and biochemical indexes of the transgenic plants transformed with the two vectors and non-transgenic plants were observed to research the growth of the transgenic plants. The results showed that nine transgenic single Cry1Ac gene lines and 8 transgenic dual Cry1Ac gene lines were obtained by PCR detection, and the target genes were integrated into the tobacco genome. FQ- PCR and toxin detections showed that the Cry1Ac gene transcriptional abundance of the transgenic dual Cry1Ac gene lines was about 2.6 times higher than that of transgenic single Cry1Ac gene lines, the Cry1Ac toxin content of the transgenic dual Cry1Ac gene lines was about 10 times higher than that of transgenic single Cry1Ac gene lines, and the two types of values of the transgenic dual Cry1Ac gene lines were significantly higher than that of transgenic single Cry1Ac gene lines. The result of indoor insect feeding test showed that with the increase of feeding days, the mortality of cotton bollworm (Helicoverpa armigera) fed with transgenic lines leaves increased gradually. There were some differences among different transgenic lines. The lethal rates of the two kinds of transgenic lines against the first instar larvae of Helicoverpa armigera were 100%. When feeding for three days, the larva mortality rate of transgenic dual Cry1Ac gene lines B1, B4, B6, B7 and transgenic single Cry1Ac gene lines A2, A7 reached 100%. Feeding for four days, the larva mortality rate of B3, B5, B8, A1, A3, A4, A5, and A9 reached 100%. At five days, the larva mortality rate of all transgenic lines reached 100%, while the rate of non-transgenic tobacco was less than 10%. Moreover, the lethal rates of the two kinds of transgenic lines against the second instar larvae of Helicoverpa armigera were also 100%. The larva mortality rate of B7 and B6 reached 100% at feeding for five days and six days respectively, while A2 and A5 reached 100% at feeding for eight days. Overall, the lethal time of the transgenic dual Cry1Ac gene lines was shorter than transgenic single Cry1Ac gene lines, and transgenic dual Cry1Ac gene lines had high insect resistance. The morphological and physiological and biochemical indexes detections showed that there were no significant differences in the parameters of ground diameter, net photosynthetic rate (Pn), stomatal conductance (Cond), intercellular CO2 concentration (Ci), transpiration rate (Tr), maximum fluorescence (Fm), chlorophyll a (Chla), and total chlorophyll content (CT) among transgenic dual Cry1Ac gene lines and CK and transgenic single Cry1Ac gene lines. While there were some differences in other parameters. On the whole, the growth of most transgenic lines was not significantly different from that of the control group, and some of the transgenic dual Cry1Ac gene lines showed dwarfing phenomenon. In general, in this study, compared with transgenic single Cry1Ac gene lines, the exogenous gene expression level of transgenic dual Cry1Ac gene lines was improved, and the plant growth and other aspects were not significantly affected. This study will lay the foundation for exploring ways to improve the expression of exogenous genes and to transform other plants.

Rice (Oryza sativa) is one of the most important grain crops in the world, and the grain quality is a very complex quantitative trait in rice, thus it has great scientific significance and practical application value to improve the grain quality of rice using modern technologies. This paper mainly summarized the new advances in grain quality improvement by applying genomic editing, high throughput sequencing, near infrared spectroscopy, scanning electron microscopy, and other modern technologies in recent years. At the same time, we also highlighted the application prospects of these modern detection and analysis technologies in rice quality researches. Therefore, our results will provide informative references for genetic improvement of grain quality and the breeding of new varieties in rice.

The Chinese-flavor liquor has developed quickly and have achieved higher market share in varied drink styles.With the further study of Chinese liquor, people have a deep understanding of microorganism and their enzyme system used as important factors in producing of Chinese liquor gradually. The importance of the microorganism and enzyme system has been highlighted. In the traditional Chinese liquor-brewing process, the microorganism and their enzyme play vital roles in flavor, quality of product and liquor rate. The flavor materials in Chinese liquor are formed resulting from the mutual overlapping reaction among Mailard reaction and the enzyme reaction of the microorganism species cultured in Daqu (starter), fermented grains and pit mud.The enzyme system and activity of microorganism from the unique brewing technique for Chinese liquor with different flavor were analysed and the latest researched achievement were reviewed in this paper. The relationship between the enzyme system and the biological indexes of liquor, the flavoring substance and the quality was elaborated in this paper. It will provide a scientific reference for improving the quality of liquor and the flavor of liquor, also for making full use of white wine brewing microorganism and enzyme resource.