Tea polyphenols are the main secondary metabolites of tea plant (Camellia sinensis). They are closely related to the metabolism, growth and quality of tea plant. The synthesis of tea tree phenolic involves a variety of enzymes. At present, the development and application of SSR molecular marker primers for specific enzymes in the tea plant synthesis pathway are still rare. This study was based on the whole genome sequence of tea plant; 158 pairs of SSR molecular marker primers were developed based on 12 key enzyme genes and their flanking sequences of tea plant phenolic synthesis pathway. Among them, the most SSR primers were obtained from flavonoid 3'-hydroxylase gene, with 29 pairs, and the least was cinnamate 4-hydroxylasegene, with only 1 pair. Among the 158 SSR primers, the most were located in the intergenic region, followed by the intron region, upstream region and exon region, and the least were located in the 5' untranslated region. These primers had 122 repeat units, among which dinucleotide was the most, followed by trinucleotide, tetranucleotide and pentanucleotide, and hexanucleotide was the least. 68 pairs of SSR primers were selected from 158 pairs of primers for primer polymorphism screening. The genetic diversity of 72 tea germplasm resources was studied by using 13 pairs of primers with good polymorphism. A total of 105 banding type were obtained, and the duplicate samples of the same sample can be gathered together. The genetic similarity coefficient (GS) of 72 tea germplasm resources ranged from 0.812 0 to 0.984 9. The results showed that the selected primers could distinguish the germplasm resources well and had good accuracy. This study can provide a basis for core germplasm construction, new variety selection and variety identification based on phenolic substances in tea plant, and also provide a reference method for developing SSR molecular markers for specific genes.

Histone methylation plays an important role in regulating gene expression in eukaryotes. The curly leaf protein (CLF), which regulates leaf curling in Arabidopsis, is an important histone methyltransferase and involved in a variety of cell development processes. In this study, the functions of OsCLF were preliminarily studied in rice (Oryza sativa). The overexpression vector pCAMBIA1300-35S-OsCLF was constructed and identified by double restriction endonuclease digestion and sequencing, and then transformed into Arabidopsis thaliana clf mutant. Phenotypic and biochemistry function analysis revealed that OsCLF of rice had the histone H3K27 tri-methyltransferase function, which was similar to CLF of A. thaliana, and the phenotypes of clf mutant could be functional complemented to wild type's by OsCLF. Fluorescence quantitative PCR analysis results showed that the relative expression level of OsCLF was higher under long-day condition than that in short-day condition, and its expression was significantly induced by aluminum stress (P<0.01). After that, the RNA interference vector pYLRNA-OsCLF-RNAi was transferred into wild type rice. Phenotype analysis indicated that the plant height, flag leaf length and width, pollen viability and seed setting rate of OsCLF-RNAi transgenic plants were all significantly decreased, but the tiller number and chlorophyll content were remarkablely increased than those of wild type rice (P<0.01). In addition, the OsCLF RNAi transgenic rice lines were more tolerant to aluminum stress than wild type rice, which indicated that OsCLF negatively regulated aluminum tolerance related genes' expression. This study preliminarily revealed the biological processes of rice growth and development involved by OsCLF gene, and provides a reference basis for further study on the epigenetic mechanism of these processes.

The FAF gene family is one of the unique transcription factors in the plant kingdom and plays a role in regulating the size of meristem. In order to study the effect of FAF gene on branch formation in Brassica napus, genome-wide identification and bioinformatics analysis of FAF family in B. napus were performed in this study. In addition, qPCR was used to investigate the spatiotemporal expression of BnFAFs in the 3 pairs of extreme phenotype materials with different branch number. The results showed that there were 17 BnFAFs distributing on 13 chromosomes in B. napus. Based on the similarity of amino acid sequence, BnFAFs were divided into 4 subfamilies Ⅰ ~ Ⅳ by referring to Arabidopsis FAFs. Gene structure and protein structure identification showed that members of Ⅳ subfamilies were rather differentiated, while other subfamily members were relatively conserved. qPCR analysis showed that the expression pattern of BnFAF4-4 and BnFAF4-7 were consistent, and the relative expression of BnFAF4-4 and BnFAF4-7 in stem, upper axillary bud and middle axillary bud of multi-branched materials were significantly higher than that of less branched materials in bolting stage suggesting that these genes may affect the formation of branches. This study laid a foundation for further improvement of branch traits by using FAF gene in B. napus.

GRAS (gibberellic acid insensitive (GAI), repressor of GAI (RGA), and scarecrow (SCR)) gene family widely exists in plants and plays important role in response to stress, plant growth and development. Vigna radiata is a traditional cash crop in China, with strong resistance to adversity, and it is one of the rominent crops to improved saline-alkali soil. In this study, 58 GRAS genes in Vigna radiata were identified and bioinformatics and expression analysis under abiotic stress were carried out. The results showed that GRAS genes were unevenly distributed on chromosomes 1~11 of Vigna radiata; By phylogenetic analysis, GRAS genes were divided into 8 subfamilies (PAT1, DELLA, SCL3, LISCL, LS, SCR, SHR and HAM), GRAS proteins of the same subfamily had similar protein motifs; Genetic structure analysis showed that almost all GRAS genes in Vigna radiata had no introns, and it was highly conserved in different species; The promoter region contained abscisic acid, ethylene, methyl jasmonate response elements and stress responseelements; qPCR results showed that GRAS genes in leaves could respond to abiotic stresses, such as salt, alkali, drought and low temperature stress in different degree and the expression of GRAS genes under stress was tissue specific. PAT1 subfamily gene played an important role in response to stress. The results would provide reference for analyse the functions of GRAS family genes in stress resistance in Vigna radiata.

Curcuma alismatifolia is a newly emerging tropical bulbous flower in China. However, there is very little known genetic and genetic information about this species, especially the research on bract color genes were blank, which hinders molecular breeding of C. alismatifolia flower color. Sequencing based on the PacBio platform is a third-generation sequencing technology that combines multiple advantages such as rapidness, high throughput, low cost, direct read sequence without PCR amplification, and ultra-long read length advantages. In order to obtain key genes of carotenoid biosynthesis pathway in C. alismatifolia, PacBio platform sequencing was used to sequence full-length transcriptome of C. alismatifolia 'Chiangmai pink', and key genes of the carotenoid biosynthesis pathway were isolated by re-sequencing separation and verified and real-time quantitative PCR (qPCR) method. The experiment obtained a total of 1 189 636 759 bp read bases of insert and a total of 494 242 reads of insert. The mean read length of these inserts were 1 562 bp (1~2 kb range), 2 646 bp (2~3 kb range) and 2 897 bp (3~6 kb range), respectively. Finally, 64 471 high- quality full-length transcript sequences (HQFL polished consensus) were obtained, included 22 107 in the range of 1~2 kb, 40 843 in the range of 2~3 kb, and 1 521 in the range of 3~6 kb. The obtained sequences were functionally annotated and classified using public databases including NR (non-redundant protein database), KOG (eukaryotic orthologous groups), Swiss Prot (Swissprot protein database), COG (cluster of orthologous groups), GO (gene ontology), KEGG, etc. The results showed that a total of 56 215 sequences were annotated. In the KOG classification statistics, a total of 6 608 Unigenes were annotated and divided into 25 categories; in the WEGO functional annotation analysis, 31 789 Unigenes were annotated to the GO database, and all distributed in the 3 major categories of biological process, molecular function, and cellular component with 39 functional groups. In the KEGG functional annotation, 15 785 of the 44 813 Unigenes were annotated, all of which were distributed in 334 metabolic pathway branches. According to the annotation results and re-sequencing, full-length cDNA sequences of 11 key genes in the carotenoid synthesis pathway were obtained, which were CHYB1 (beta-carotene 3-hydroxylase 1), ZDS1 (zeta-carotenedesaturase 1), ZEP1 (zeaxanthin epoxidase 1). PSY1 (phytoene synthase 1), PDS1 (phytoene desaturase 1), CRTLSO1 (prolycopene isomerase 1), LCY-B1 (lycopene beta-cyclase 1), LCYE1 (lycopene epsilon cyclase 1), GPPS1 (geranyl geranyl pyrotenoid synclease1), CCDs1 (carotenoid cleavage dioxygenase1) and carotenoid 9, 10 (9', 10') -cleavage dioxygenase 1. Finally, three genes, PSY1, LCY-B1 and ZEP1, were screened out for tissue expression function verification. PSY1, which was located upstream of the carotenoid synthesis pathway, had the highest expression in sterile bracts and the lowest expression in florets. The gene expressions of LCY-B1 and ZEP1 which were closer to the middle and lower ends of the carotenoid synthesis pathway, were basically the same, and both had the highest expression in florets, followed by sterile bracts. Acquisition of these genes is conducive to improvement of bract color breeding by using molecular regulation methods in the later stage, which will lay the molecular basis for the cultivation of new bract color varieties.

Calmodulin-like (CML) is a calcium-binding protein in plant cells that is involved in signal transduction during growth, development, and stress. In order to explore the expression pattern of CML family genes in Erianthus fulvus under cold stress, 39 EfCML family genes were identified based on transcriptome data, and bioinformatics and expression analysis under cold stress were carried out. The analysis of the physicochemical properties of the proteins showed the number of amino acid residues, molecular weight, and isoelectric point of EfCMLs proteins was 59~312, 6.240 21~34.017 78 kD, and 3.99~11.14, respectively. The predicted results of subcellular localization showed that the 39 EfCMLs were located in the cytoplasm, cell membrane, and nucleus, respectively. Conservative domain analysis showed that EfCML family proteins contained 1~4 typical EF-hand domains. Motif analysis showed that EfCML family proteins contained 1~4 typical Ca2+ binding motifs. Phylogenetic tree analysis showed that EfCMLs proteins could be divided into 11 groups. Expression analysis of cold stress showed that low temperature could induce differential expression of EfCML family genes, with EfCML1 and EfCML2 were consistently up-regulated, and reached peak value at 72 h cold stress with 27 and 35 times higher than that of the control (P<0.01), showing the most active state. This study provides basic data for in-depth analysis of the cold stress regulation mechanism of E. fulvus CML genes.

Insulin like growth factor 1 (IGF1) is a single-chain peptide hormone that affects muscle development by regulating the synthesis of skeletal muscle proteins, and is important for cell proliferation and other physiological functions. Three healthy adult (2~3 years old) Guanling cattle (Bos taurus) were selected and 12 tissue samples were collected from heart, liver, spleen, lung, kidney, foreleg, hindleg, longest dorsal muscle, rhomboid, gluteus, stomach and small intestine for RNA extraction, respectively, and the longest dorsal muscle was successfully cultured in Guanling myogenic cells. The relative mRNA expression of IGF1 gene in different tissues was detected by qPCR; the physical and chemical properties, secondary and tertiary structures, and subcellular localization of IGF1 protein in Guanling bovine were analyzed by online software; meanwhile, three pairs of short hairpin RNA (shRNA) interference sequences and one pair of negative control sequences of IGF1 gene were designed by online software, and sequenced with pGPU6-GFP-Neo. IGF1 interference vector was sequenced and transfected into adult myoblasts. qPCR was used to detect and screen the most efficient interfering vector and analyze the effect of the interfering vector on IGF1 gene expression. The relative mRNA expression of cyclin-dependent kinases 2 (CDK2) and cyclin-dependent kinases 1 (Cyclin D1) was measured by qPCR, and the proliferation of myogenic cells was detected by CCK8. The results showed that IGF1 gene was expressed in 12 tissues including heart, foreleg, hindleg, longest back, rhomboid and gluteus muscles of Guanling cattle, and the relative mRNA expression of liver was the highest, which was significantly higher than that of other tissues (P＜0.01). The IGF1 protein had a molecular formula of C744H1186N214O216S15, a relative molecular mass of 17.06581 kD and a theoretical isoelectric point of 9.36, and was a basic unstable protein; After the IGF1 gene was inhibited, the expression of cyclin D1 and CDK2, was significantly lower than that of shRNA-NC (P＜0.01). The results of cell proliferation detection by CCK8 method showed that the proliferation of myogenic cells at 24, and 72 h was significantly lower than that of the control group after IGF1 gene inhibited (P＜0.05), and was inhibited for extremely significant difference at 48 h (P＜0.01). Silencing IGF1 gene expression in vitro affected the proliferative capacity of myogenic cells and inhibited the expression of genes related to myogenic cell proliferation. This study provides basic data for further research on the effect of IGF1 gene on the muscle growth and development of Guanling cattle and its regulatory mechanism

LncRNA was widely transcribed in the mammalian genome and promote the proliferation, invasion and metastasis of skin malignant melanoma through a variety of regulatory pathways. To further explore the effect of LncRNA TCONS_00153149 gene on the process of melanin deposition in Youzhou Black goat (Capra hircus), the tissue expression profile of LncRNA TCONS_00153149 were detected using qPCR method in different tissues of Youzhou dark goat and Yudong white goat, as well as the LncRNA TCONS_ 00153149 expression characteristics were detected in B16-F10 cells at differentiation stage stages, the LncRNA TCONS_00153149 lentiviral vector was constructed and transfected into B16-F10 melanocytes to explore the effect on the process of melanin deposition. The results demonstrated that LncRNA TCONS_00153149 was widely expressed in different tissues of the goat, especially with significantly higher expression in the skin in Youzhou black goat than that of Yudong white goat (P＜0.01). Comparison analysis illustrated that the expression pattern of the LncRNA TCONS_00153149 was increasing in the early stage of melanocyte differentiation, and reached the highest relative expression on the 3rd day, and then decreasing after that, the expression was the lowest on the 7th day. The expression on the 3rd day was significantly higher than that of the 0, 1, and 7 days (P＜0.01), showed that LncRNA TCONS_00153149 have a positive regulatory relationship to melanin deposition. The transfection experiment found that the relative expression of LncRNA TCONS_ 00153149 gene was extremely significantly increased (P＜0.01), and the melanin content was significantly increased compared with the negative control group (NC), these results verified the significant promotion effect of LncRNA TCONS_00153149 gene on melanin deposition. Besides, The relative expression of TYRosinase related protein 1 (TYRP1), microphthalmia-associated transcription factor (MITF), TYRosinase- related protein 2 (TYRP2) was significantly increased (P＜0.05), and the relative expression of agouti signaling protein (ASIP) was extremely significantly reduced (P＜0.01). Combined with the results of the interaction between LncRNA and target genes by RNA2.0.0 software, this study showed that the LncRNA TCONS_00153149 gene had a positively effect on the deposition of melanin by targeting and enhancing the function of melanin candidate genes (TYRP1, TYRP2, MITF, ASIP). This study provides data for the study of the LncRNA regulation theory related to goat skin coloring.

Cell death inducing DNA fragmentation factor 45 like effector a (CIDEa) is a kind of protein closely related to lipid metabolism. It is involved in the regulation of lipolysis, the growth and fusion of lipid droplets and the maturation of very low density lipoprotein. In order to determine the biological function of CIDEa in dairy goat mammary gland, the mRNA level of CIDEa in mammary gland was detected using qPCR during lactation and dry period. Then, siRNA mediated interference technique was used to study the effect of CIDEa on lipid synthesis and the expression of related genes. The results showed that the expression of CIDEa mRNA in mammary gland was higher in lactation than that in dry period (P＜0.05). The siRNAs targeting CIDEa gene were transfected into dairy goat mammary epithelial cells, and the mRNA level of CIDEa was inhibited by 60%~88% (P＜0.01). The accumulation of lipid droplets and the content of triglyceride in cells were inhibited after interfering CIDEa expression. In addition, interfering CIDEa expression significantly upregulated the expression of hormone sensitive lipase (HSL) and fatty acid synthase (FASN) (P＜0.05), and also promoted the expression of sterol regulatory element binding protein 1 (SREBP1) (P＜0.05). Furthermore, the expression of fatty acid binding protein 3 (FABP3) and diacylglycerol transferase gene 1 (DGAT1) (P＜0.05) were inhibited when interfering CIDEa gene expression. The results showed that CIDEa promoted the synthesis of lipid droplets in dairy goat mammary epithelial cells, which provides a theoretical basis for further study the molecular mechanism of CIDEa gene regulating milk lipid synthesis in dairy goat.

As a muscle growth inhibitor, myostain (MSTN) is a member of TGF- β superfamily. MSTN mutation will cause excessive muscle growth and a "double muscle" phenotype. The gene is of great significance for improving muscle yield and lean meat rate of animals. The current study aimed to analyze the correlation between MSTN gene and meat production traits of Tibetan sheep (Ovis aries), six 6-month-old Tibetan sheep with similar body weight and no disease were randomly selected in this study. After slaughtering, slaughter performance, meat quality and meat nutritional components of Tibetan sheep were determined. The mRNA expression levels of MSTN gene in brachial triceps, quadriceps femoris, longissimus dorsi and semitendinosus of Tibetan sheep were detected by qPCR, and the correlation between the expression level of MSTN gene and meat production traits was analyzed. The results showed that all slaughter performance indexes were no significant difference between ram and ewe. The slaughter rates of ram and ewe were 51.32% and 52.01%, respectively, as well as the carcass weights were 22.08 and 20.03 kg, respectively. The results of meat quality analysis showed that there was no significant difference between male and female sheep except shear force (P<0.05). There were no significant differences in the contents of crude protein, crude fat, crude ash and water between ram and ewe. The protein contents were 23.14% and 22.17% for ram and ewe, respectively, and the crude fat contents of ram and ewe were 2.46% and 2.87%, respectively. MSTN gene was expressed in all muscle tissues of Tibetan sheep, but the differences of the expression levels in all muscle tissues were not significant. By analyzing the correlation between the expression level of MSTN gene and each index, the mRNA expression level of MSTN gene in arm triceps of Tibetan sheep was significantly positively correlated with the live weight before slaughter (P<0.05, 1.000); The expression level in longissimus dorsi muscle was significantly positively correlated with the redness value (P<0.05, 0.831), and it was extremely significantly negatively correlated with cooked meat rate (P<0.01, -0.958); The expression level in quadriceps femoris muscle was significantly negative correlated with cooked meat rate (P<0.01,-1.000); There was no significant correlation between MSTN gene expression in semitendinosus muscle and meat production indexes. Expression of MSTN gene was correlated with meat producing traits of Tibetan sheep. This study provides theoretical basis for the study of the regulation mechanism of MSTN gene on meat quality of Tibetan sheep.

Shanbei white cashmere goat (Capra hircus) and Ziwuling black goat are the main goats in the northern of Shaanxi, but their growth performance needs to be improved. Especially, the population size of the latter decreased sharply, so it is urgent to determine the genetic variation of its population. Genetic variation of major histocompatibility complex (MHC) genes can be used to evaluate population genetic diversity, and MHC genes are also associated with body size traits of domestic animals. The aim of this study was to investigate the polymorphism of major histocompatibility complex (MHC) gene exon3 in Shanbei white cashmere goat and Ziwuling black goat, and to evaluate the population development trend of Ziwuling black goat, and to explore the correlation between MHC gene and growth performance or cashmere traits in Shanbei white cashmere goats. In this study, the genetic variation of exon3 of MHC gene of 600 Shanbei white cashmere goats and 30 Ziwuling black goats was analyzed by using high-throughput sequencing method, and the correlation between MHC gene and growth performance was studied. Exon3 sequences of 2 MHC genes were obtained, including 4 DQA1 alleles and 22 DQB2 alleles. The heterozygosity and polymorphism of Ziwuling black goat were low, and the population development trend was not optimistic, so effective protection measures should be taken. Moreover, Shanbei white cashmere goats had higher polymorphism, the BB genotype of DQA1 gene was significantly higher than the other 2 genotypes in terms of height at hip cross and wool length (P=0.035, P=0.021), and the AA genotype had the smallest cashmere fineness (P=0.003). These alleles could be used as an effective molecular marker for growth performance improvement and ultrafine cashmere goat breeding, respectively. The results of this study are helpful to promote molecular markerassisted selection breeding of Shanbei white cashmere goat, and the protection, development and utilization of Ziwuling black goat germplasm resources.

The speckle-type POZ (pox virus and zinc finger protein) protein (SPOP) is a member of Cullin 3 family of E3 ubiquitin ligase, which promotes the ubiquitination and degradation of substrate by interacting with substrate protein, thus affecting the biological function of substrate protein. In this study, the recombinant eukaryotic expression vector pCMV-HA-SPOP of chicken (Gallus gallus) SPOP gene was constructed, and pCMV-HA-SPOP and empty vector pCMV-HA were transfected into DF-1 respectively, and the total cellular proteins were extracted. Co-immunoprecipitation (Co-IP) combined with mass spectrometry was used to screen and identify the cellular proteins interacting with SPOP protein in chickens, and GO function annotation, KEGG signaling pathway and protein interaction network analysis were carried out. Furthermore, the interaction between chicken SPOP protein and the selected cell protein 26S proteasome non ATPase regulatory subunit 11 (PSMD11) was verified by fluorescence co-localization and Co-IP test. The results showed that the recombinant protein HA-SPOP was correctly expressed in cells, and mainly located in nucleus and cytoplasm. A total of 158 cellular proteins interacting with chicken SPOP protein were screened by Co-IP combined mass spectrometry, which mainly distributed in nucleus, cytoskeleton and cytoplasm, and participated in biological processes such as enzyme activity, nucleic acid binding, protein process, and signal pathways such as metabolism, ribosome composition and biological regulation. There were complex interaction networks among cellular proteins interacting with chicken SPOP protein, among which proteasome activator complex subunit 3 (PSME3), PSMD11 and H2A histone family, member Z (H2AFZ) may have direct interaction with chicken SPOP protein. The results of fluorescence co-localization and Co-IP assay showed that chicken SPOP protein could interact with PSMD11 protein and alter its intracellular localization. This study provides research foundation for further exploring the biological function of chicken SPOP protein.

Duck (Anas platyrhynchos) interferon α (IFNα) has a broad spectrum of antiviral activity, and A. platyrhynchos avian β-defensin 2 (AvBD2) has a broad spectrum of antibacterial activity. In this study, Pichia pastoris was used to secrete and express A. platyrhynchos IFNα -AvBD2 fusion protein, and the biological activity of IFNα -AvBD2 was tested, which would be of great significance for the prevention and control of duck epidemic diseases under existing unresistant breeding conditions. The previous work has successfully constructed the prokaryotic expression plasmid pET32a-AplIFNα -AvBD2, and successfully expressed it in Escherichia coli. In this study, the gene fragment of mature protein AplIFNα -AvBD2 was amplified by PCR using laboratory-preserved pET32a-AplIFNα-AvBD2 as a template to construct recombinant plasmid pPICZαA- AplIFNα -AvBD2; the linearized recombinant plasmid was electroporated into Pichia pastoris KM71H competent cells, and the positive recombinant bacteria were induced by methanol to express, and the biological activity of the expression product in vitro was studied. The results of SDS-PAGE and Western blot showed that the molecular weight of AplIFNα -AvBD2 recombinant protein was about 24 kD; agar diffusion method and microdilution method showed that the recombinant protein had strong antibacterial activity against Staphylococcus aureus and E. coli; cytopathic effect inhibition method detected that the antiviral activity of the recombinant protein was 1.2×105 U/mL, and the specific activity was 5.2×105 U/mg; MTT cytotoxicity test showed that the recombinant protein was non-toxic to African green monkey kidney cells (Vero). The above results indicate that AplIFNα -AvBD2 achieves secretory expression in P. pastoris, with both antibacterial activity and antiviral activity. This study lays the foundation for further application of AplIFNα- AvBD2.

Rice-fish co-culture system with considerable economic and ecological benefits requires improvement because of its low fish yield. Based on the Sanjiang model of rice-fish co-culture system, Jinbian carp (Cyprinus carpio var. Jinbian) was reared with tilapia (Oreochromis niloticus, GIFT strain) and the breeding density was increased in the current research. The influence of increased density and polyculture on the economic and ecological benefits of the rice-fish co-culture system was investigated. It was observed that after 145 days of the experiment, weight gain rate and specific growth rate (415.3% and 1.2, respectively) of carp in high density integrated culture group (IDh) were significantly higher than those in low density integrated culture group (IDl) and monoculture group (SD), and weight gain rate (4918.0%) of tilapia in IDh was significantly higher than those in IDl (P<0.05). The density and polyculture of the rice-fish co-culture system affected the activities of the digestive enzymes amylase, lipase and liver catalase. The activity of carp digestive enzymes amylase in IDl was significantly higher than that in IDh and SD, but the activity of carp liver catalase in IDh was significantly lower than that in IDh and SD (P<0.05). The activities of tilapia digestive enzymes amylase and lipase in IDh were significantly higher than those in IDl (P<0.05). In addition, the density and polyculture of the rice-fish co-culture system influenced the water quality of paddy and pond, such as more ammonia-nitrogen (NH4-N) of SD, nitrate-nitrogen (NO3-N) and nitrite-nitrogen (NO2-N) of IDl, total nitrogen (TN), total phosphorus (TP), chemical oxygen demand (CODCr) and organic matter (OM) of IDh in paddy were decreased. More NH4-N, NO3-N and NO2-N of SD, TN, CODCr and OM of IDh, TP of IDl in the pond were decreased. In summary, the increased density and polyculture of rice-fish co-culture systems in this study did not harm the growth performance of fish and the water quality of paddy and pond. It actually promoted mass circulation and improved the economic benefits to a certain extent. Based on this study, a higher density of fish in the rice-fish co-culture system could be tried to enhance the economic benefits.

The northern region is one of the main crop growing areas in China. However, due to the cold and dry climate and the sandy soil, the agricultural residues are decomposed slowly or even cannot be completely degraded, not only making it unable to be used as a fertilizer source in the current season, but also causing the phenomenon that straw and subsequent crops compete for nitrogen and eventually affect the growth of crops. Stacked straws are burned in large quantities, which cause waste of resources and serious air pollution. In order to solve the problem of straw degradation in cold regions, a strain that could efficiently degrade cellulose at low temperature was isolated through the method of Congo red staining and low-temperature screening. The molecular biological identification of the isolation was carried out based on the 18S rDNA sequence analysis. The fermentation conditions of the strain and the characteristics of the cellulase produced were optimized through single factor experiments and response surface method (RSM). Besides, the straw degradation ratio and the dynamic enzyme activity in the low-temperature fermentation progress were determined to investigate the biodegradability of the target strain on rice straw at 10 ℃. The results showed that the isolated strain was identified as Penicillium (Penicillium sp. JiTF01, GenBank No. MZ285877), which could degrade cellulose at 10 ℃ . The optimal fermentation conditions for JiTF01 to produce cellulase at low temperature were pH of 6.67, inoculation amount of 2.89% and incubation time of 12.13 days, and the maximum cellulase activity obtained under the optimized conditions was 31.05 U/mL. Also, strain JiTF01 had been proved that it could exhibit high cellulase activity under acidic conditions, and the enzymatic activity and stability of the cellulase produced by JiTF01 could retain 41.93% and 94.35% at pH 2, respectively. In addition, it was found that the degradation ratio of rice straw could reach 45.24% after 21 days of fermentation by strain JiTF01 at 10 ℃ . The conclusions found in this study are significance to the production and application of cellulase in the future and have important implications for the disposal of straw resources in cold regions.

Listeria monocytogenes (LM) is a human-animal Gram-positive bacterium with a wide range of adaptations to the external environment. In order to understand the regulatory role of sRNA rli82 on the environmental adaptability and biofilm generation of LM. In this study, the rli82 gene deletion mutant strain LM- Δrli82 and the complementation strain LM- Δrli82/rli82 were constructed using homologous recombination technology and the differences in growth and biofilm generation of these strains under different stress environment were determined, respectively. Moreover, bioinformatics was used to predict the potential target genes regulated by rli82, and the transcript levels of genes related to environmental stress and biofilm formation were measured by qPCR. The results showed that rli82 gene owns 70 bp in length, with a secondary structure of 2 neck ring. The potential target gene was opuCC. LM- Δrli82 significantly enhanced environmental adaptation capacity under different osmotic pressure (4% and 8% NaCl) when compared to LM EGD-e and LM- Δrli82/rli82. The environmental adaptation capacity was significantly lower under stress conditions of 3.8% ethanol, pH4, pH9 and 30 ℃ . However, the biofilm formation of the 3 strains was not significantly different. The qPCR showed a significant decrease in the relative expression of genes related to environmental stress but no significant difference in biofilm related genes. The results demonstrate that the sRNA rli82 plays an important role in regulating the adaptive capacity of LM to environmental stress, and set a foundation for studying the molecular mechanism of sRNA rli82 regulating environmental stress in LM.

Sugarcane (Saccharum officinarum), as an important global sugar crop and industrial raw material crop, has been successfully commercialized in more than 100 countries. The high-quality and sustainable development of the sugarcane industry has important strategic significance for the effective supply of global sugar and the sustainable development of the green energy economy. In recent years, the sugarcane industry, which relies on traditional hybrid breeding technology, has encountered development bottlenecks. With the rapid development of modern biological technology, genetic modification technology has emerged in many fields. Genetic modification technology refers to the specific modification or improvement of target genes through genetic engineering, so as to realize the change of functional traits. Sugarcane is one of the crops with the highest safety level of GMOs, reporter genes and selectable marker genes based on functional genomics have promoted the breeding of high-quality sugarcane varieties in terms of disease resistance, insect resistance, herbicide resistance, cold resistance, and drought resistance. In addition, transgenic technology has also achieved fruitful results in increase sucrose content, improving sucrose quality, and research on energy sugarcane based on bioreactors. Gene editing technologies represented by genome assembly, genomics- assisted breeding (GAB), multi-allelic mutagenesis and creation of loss-of-function phenotypes have performed well in sugarcane functional genome research and new material creation. This paper comprehensively reviews the research progress of genetic modification technology in the above-mentioned sugarcane fields, focusing on the development and commercialization of multivalent fusion transgenic sugarcane with multiple excellent traits. This review provides reference for the breeding and quality improvement of high-quality sugarcane varieties in my country, and promote the high-quality and sustainable development of sugarcane industry of China.

Monitoring the persistence and dynamic changes of transgenes in the soil from genetically modified (GM) crop is an important issue in assessing the potential risks of GM crops. The present study applied a novel droplet digital polymerase chain reaction (ddPCR) method to quantitatively detect the copy number concentrations of exogenous gene CP4-EPSPS (Agrobacterium tumefaciens strain CP4 5-enolpyruvyl shikimate-3-phosphate synthase) and reference gene zSSIIb (Zea mays starch synthase isoform zSTSII-2) fragments in the rhizosphere soil of herbicide-resistant transgenic maize (Zea mays) 'CC-2' and its non-transgenic control line 'Zheng-58', at different growth stages of maize (seedling stage, jointing stage, silking stage, milk-ripe stage and full-ripe stage). The limit of quantitation (LOQ) of ddPCR for the CP4-EPSPS and zSSIIb system was (0.48±0.07) and (0.22±0.04) copies/µL, respectively. The copy number concentrations of CP4-EPSPS were lower than the limit of quantitation in the rhizosphere soil of GM maize 'CC-2' at all growth stages, and the positive detection rate of CP4-EPSPS obviously declined with increasing growth stage. There was no significant difference between CP4-EPSPS and zSSIIb concentrations in rhizosphere soil of 'CC-2'. At different growth stage, there was a similar change tendency in the positive detection rate and concentration of zSSIIb gene fragments in rhizosphere soil of 'CC-2' and 'Zheng-58', but the copy number concentration of zSSIIb in rhizosphere soil of 'Zheng-58' was significantly higher than that of 'CC-2' at the jointing stage (P<0.05). Test results showed that the transgene fragments of GM maize could enter into the soil environment through root exudates, but the concentration was very low and showed a decreasing trend with the growth stage, which indicates that the ddPCR method is suitable for sensitive and precise quantitative analysis of the persistent plant DNA fragments in soil environment. This study provided a novel method and reference for quantitative detection of transgene from GM crops in soil.

The CRISPR/Cas9 system is a simple and efficient gene editing system that has been rapidly developed in recent years. In mammals, research on knocking out multiple genes simultaneously is still scarce. In order to optimize the construction of a knock-out system targeting multiple genes in mammals, the existing CRISPR/Cas9 vector was upgraded in this study, a multi-targets knock-out plasmid based on CRISPR/Cas9 system cascading by 4 small guide RNA (sgRNA) expression cassettes mediated by U6 promoters was constructed. Sirtuin 3 gene (Sirt3) and Perilipin 1 gene (Plin1) of Sus scrofa were chosen to assess the upgraded plasmid. 2 sgRNA targeting Plin1 and Sirt3 were selected respectively, and the knock-out vectors were constructed by inserting the sgRNA based on the upgraded plasmid. In this study, PK15 were divided into 4 groups. The control group CON was the cells transfected by the upgraded plasmid without any targets. The group S2 was the cells cotransfected 2 vectors targeting the Sirt3, while the group P2 was cotransfected 2 vectors targeting the Plin1, and the group S2P2 was cotransfected the vector contains all the targets. Detect the target gene mutation rate of each experimental group cell at the cell genomic DNA level, and use qPCR and Western blot to determine expression of RNA and protein of the target gene expression of each group of cells. The results showed that mutations were detected in the cells of each experimental group. Mutation rates of Sirt3 gene in S2P2 and S2 were 33% and 26%, respectively. Plin1 gene mutation rates in S2P2 and P2 groups were 33% and 24%, respectively. And compared with the CON group, the target mRNA and protein expression levels decreased in all experimental groups, the difference was extremely significant (P＜0.01). Among them, there was no significant difference in Sirt3 gene expression between S2 group and S2P2 group and in Plin1 gene expression between P2 group and S2P2 group. In this study, a vector that can simultaneously knockout porcine Sirt3 and Plin1 genes was constructed through the improved CRISPR/Cas9 vector system. This research is helpful to the subsequent study of multi-gene function.