Interleukin-1β gene polymorphism associated with hepatocellular carcinoma in hepatitis B virus infection

AIM： To examine the effect of interleukin-l-beta （IL-113） promoter region C-511T and IL-1 receptor antagonist （IL-1RN） polymorphism among the patients with chronic hepatitis B virus （HBV） infection （HCC and non-HCC）. METHODS： Genomic DNA from 136 Thai patients with chronic HBV infection （HCC =46 and non-HCC= 90） and 152 healthy individuals was genotyped for IL-113 gene polymorphism （-511） using polymerase chain reaction with sequence specific primers （PCR-SSP）. The variable number of tandem repeats （VNTR） of IL-1RN gene was assessed by a PCR-based assay. The association between these genes and status of the disease was evaluated by X^2 test. RESULTS： IL-1B-511 genotype c/c was found to be significantly different in patients with HCC when compared with healthy individuals （P = 0.036, OR = 2.29, 95%CI = 1.05-4.97） and patients without HCC （P=0.036, OR= 2.52, 95%CI=1.05-6.04）. Analysis of allele frequencies of IL-1B-511 showed that IL-1B-511 C allele was also significantly increased in patients with HCC, compared to that in healthy control （P=0.033, OR= 1.72, 95%CI=1.04-2.84）. However, no significant association in IL-1RN gene was found between the two groups. CONCLUSION： IL-1B-511C allele, which may be associated with high IL-1B production in the liver, is a genetic marker for the development of HCC in chronic hepatitis B patients in Thai population.

EFFECT OF INTERLEUKIN-1β ON GROWTH HORMONE GENE EXPRESSION AND ITS POSSIBLE MOLECULAR MECHANISM IN RAT MtT/S SOMATOTROPH CELLS

Objective To elucidate the effect of interleukin-1β （IL- 1β） on human growth hormone （hGH） gene expression in a rat somatotropic pituitary cell line MtT/S. Methods Stably transfected MtT/S cells were firstly established by transfecting 484-Lucl plasmid which contained hGH gene promoter --484 to ＋30 bp and luciferase reporter gene. The effect of IL-1β on hGH gene expression was determined by assaying the luciferase activities. RT-PCR method was also used to determine whether IL-1 recepor mRNA was expressed in MtT/S cells. Results The 10^3 U/mL IL-1β stimulated secretion and synthesis of GH, and promoted the 5＇-promoter activity of GH gene in stably transfected MtT/SGL cells with the action of 1.38 times above the control. Among inhibitors of signaling transduction pathways, mitogen-activated protein kinase kinase （MAPKK/MEK） inhibitor PD98059 （40 μmol/L） and p38 mitogen-activated protein kinase （MAPK） inhibitor SB203580 （5 μmol/L） completely blocked the stimulatory effect of IL-1μ, and phosphatidylinositol-3-kinase （PI3-K） inhibitor LY294002 partly abolished the effect of IL-1μ. Western blot analysis further confirmed the activation of phosphorylated MEK and p38 MAPK in MtT/SGL cells. Neither over-expression of Pit- 1 nor inhibition of Pit- 1 expression affected induction of hGH promoter activity by IL-1μ. A series of deletion constructs of hGH promoter were created to identify the DNA sequence that mediated the effect of IL-1β, and results showed that the stimulatory effect of IL-1β was abolished following deletion of the --196 to -- 132 bp fragment. Conclusions IL-1β promotes GH secretion and synthesis in rat MtT/S somatotroph cells. The stimulatory effect of IL-1β on hGH gene promoter appears to require the activation of MEK, p38 MAPK, PI3-K, and a fragment of promoter sequence that spans the -196 to -132 bp of the gene, but it may be unlinked with Pit-1 protein.

Interleukin-1 and TNF-α polymorphisms and Helicobacter pylori in a Brazilian Amazon population

AIM： To study the association between Interleukin-1 （IL-1） and tumor necrosis factor （TNF）-α polymorphisms, infection by Helicobacter pylori （H pylori） and the development of gastrointestinal diseases.METHODS： Genomic DNA was extracted from the peripheral blood of 177 patients with various gastrointestinal diseases and from 100 healthy volunteers. The polymorphisms in IL-1β and TNF-α genes were analyzed using the polymerase chain reactionrestriction fragment length polymorphism method （PCRRFLP） and those from IL-1RN with PCR. The presence of infection due to H pylori and the presence of the CagA toxin were detected by serology. The histopathological parameters in the gastric biopsies of the patients were according to the Sydney classification.RESULTS： A comparison of the frequencies of the different polymorphisms studied among the patients and the control group demonstrated that the allele IL- 1RN＊2 was more frequent among patients with gastric ulcers and adenocarcinoma. Carriers of the allele IL- RN＊2 and those with reactive serology for anti-CagA IgG had a greater risk of developing peptic ulcer and gastric adenocarcinoma, as well as a higher degree of inflammation and neutrophilic activity in the gastricCONCLUSION： Our results indicate a positive association between IL-1RN gene polymorphism and infection by positive H pylori CagA strains and the development of gastric ulcers and adenocarcinoma.

HBV X Gene Transfection Upregulates IL-1β and IL-6 Gene Expression and Induces Rat Glomerular Mesangial Cell Proliferation

The X gene of HBV encodes a 17-kD protein, termed HBx, which has been shown to function as a transcriptional trans-activator of a variety of viral and cellular promoter/enhancer elements. The aim of this study was to investigate the effect of HBx on gene expression of interleukin （IL）-1β and IL-6, and proliferation of rat mesangial cells in vitro. The X gene of HBV was amplified by PCR assay, and inserted into the eukaryotic expression vector pCI-neo. The structure of recombinant pCI-neo-X plasmid was proved by restrict endonuclease digestion and sequencing analysis. pCI-neo-X was transfected into cultured rat mesangial cell line in vitro via liposome. HBx expression in transfected mesangial cells was detected by Western blot. The IL-1β and IL-6 mRNA expression in those cells was assayed by semiquantitative RT-PCR. Mesangial cell proliferation was tested by MTT. The results showed that HBx was obviously expressed in cultured mesangial cell line at 36th and 48th h after transfection. The expression of IL-1β and IL-6 mRNA was simultaneously increased. The cell proliferation was also obvious at the same time. It was concluded that HBx gene transfection could induce IL-1β and IL-6 gene expression and mesangial cell proliferation. HBx may play a critical role in mesangial cell proliferation through upregulation of the IL-1β and IL-6 gene expression.

MEK AND p38 MAPK-DEPENDANT PATHWAYS ARE INVOLOVED IN THE POSITIVE EFFECT OF INTERLEUKIN-6 ON HUMAN GROWTH HORMONE GENE EXPRESSION IN RAT MtT/S SOMATOTROPH CELLS

Objective To investigate the effect of interleukin-6(IL-6)on the human growth hormone(hGH)gene expression in a rat somatotropic pituitary cell line MtT/S.Methods The plasmids containing various lengths of hGH gene 5'-promoter fragments were constructed.Stably transfected MtT/S cells were created by cotransfecting the above plasmids and pcDNA3.1(+)with DMRIE-C transfection reagent.After the administration of these cells with IL-6 and/or various inhibitors of signaling transduction pathways,the luciferase activities in MtT/S cells lysis were assayed to demonstrate the effects of IL-6 on hGH gene promoter activity and possibly involved mechanism.Results The 103 U/mL IL-6 stimulated GH secretion and synthesis,and promoted the 5'-promoter activity of GH gene in stably transfected MtT/SGL cells with the action of 1.69 times above the control.Among inhibitors of signaling transduction pathways,mitogen-activated protein kinase kinase(MAPKK/MEK)inhibitor PD98059(40 μmol/L)and p38 mitogen-activated protein kinase(MAPK)inhibitor SB203580(5 μmol/L)completely blocked the stimulatory effect of IL-6.Western blot analysis further confirmed the activation of phosphorylated MEK and p38 MAPK in MtT/SGL cells.Neither over-expression of Pit-1 nor inhibition of Pit-1 expression affected IL-6 induction of hGH promoter activity.A series of deletion constructs of hGH promoter were created to identify the DNA sequence that mediated the effect of IL-6.The results showed that the stimulatory effect of IL-6 was abolished following deletion of the-196 to-132 bp fragment.Conclusions IL-6 promotes GH secretion and synthesis by rat MtT/S somatotroph cells.The stimulatory effect of IL-6 on hGH gene promoter appears to require the activation of MEK and p38 MAPK,and a fragment of promoter sequence that spans the-196 to-132 bp of the gene,but may be unlinked with Pit-1 protein.

Expression of GST-IL-1 fusion gene

Two GST-IL-1 fusion genes were constructed by inserting different cDNA fragments of human interleukin1 (IL-1) into the 3'-terminus of GST gene in the fusion protein expression vector pGEX-4T. After IPTG induction ,SDS-PAGE was employed to detect the gene expression. No corresponding protein encoded by GST gene fused with the whole-length 816 bp IL-1 cDNA was observed, nor was free GST protein. However, the fusion protein of GST and IL-1 cDNA without the 189 bp at the 5'- terminus was detected, amounting to 30% of the total bacterial protein expressed. This might suggest that the sequence of 1-189 bp of IL-1 cDNA affected the expression of the fusion gene. That is to say, the downstream sequence distant from the translation start codon AUG in the target gene could significantly affect the expression of the fusion gene.

Inhibition of Osteoarthritis in Rats by Electroporation with Interleukin-1 Receptor Antagonist

Gene therapy constitutes a promising strategy for the treatment of osteoarthritis (OA). We assessed the use of electroporation (EP) of non-viral gene vectors, and compared its efficacy with that of adeno-associated virus (AAV) vectors. EP- and AAV-mediated delivery of human interleukin-1 receptor antagonist (hIL-1Ra) was localized performed in the joints of rats following induction of OA. mRNA levels for hIL-1Ra, IL-1β, TNF-α, MMP-13 and ADAMTS-4 in the cartilage and synovial tissues were analyzed. Structural analyses of the subchondral bone at the medial femoral condyle were performed by Micro-CT after treatment. Knee joint specimens were staining with hematoxylin and eosin and Saffron O. Induction of hIL-1Ra by both EP and AAV inhibited inflammatory-induced sub-chondral bone reconstruction, and effectively suppressed IL-1β activity, as evidenced by decreased expression of MMP-13 and ADAMTS-4. Histological analyses revealed significant protection of cartilage, proteoglycan by EP and AAV. hIL-1Ra expression was similar in both the EP and AAV groups. Notably, this gene is not easier degraded transduced by EP compared with AAV. Taken together, these results show that EP offers transfection efficiency comparable to that of AAV, with the potential for longer gene expression, making EP a promising candidate for efficient non-viral delivery of OA gene therapy.

PI3K/AKT/FoxO信号通路在胰岛素类似生长因子1调节神经细胞PRNP基因表达中的作用

目的探讨磷脂酰肌醇3激酶(PI3K)/蛋白激酶B(AKT)/叉头状转录因子O亚家族蛋白(Fox O)信号通路在胰岛素类似生长因子1(IGF-1)调节神经细胞PC12细胞株PRNP基因表达中发挥的作用。方法将对数生长期的PC12细胞分为IGF-1组、实验1组和实验2组和对照组。IGF-1组加入终浓度80 ng/m L的IGF-1;实验1、2组先加入25、50μmol/L的PI3K/AKT/Fox O信号通路抑制剂LY294002,37℃培养30 min后再滴入80 ng/m L的IGF-1。对照组不加药物。继续培养24 h。采用实时荧光定量PCR法测算各组PC12细胞PRNP mRNA相对表达量,采用Western blotting法测算各组PC12细胞AKT、磷酸化AKT(p AKT)、Fox O3a和磷酸化Fox O3a(p Fox O3a)蛋白相对表达量。结果 IGF-1组PC12细胞PRNP mRNA及p AKT、p Fox O3a蛋白相对表达量高于对照组(P均<0.05)。实验1、2组PC12细胞PRNP mRNA及p AKT、p Fox O3a蛋白相对表达量低于IGF-1组(P均<0.05),且实验2组低于实验1组(P均<0.05)。结论 IGF-1通过磷酸化PI3K/AKT/Fox O信号通路蛋白AKT、Fox O3a调节PC12细胞PRNP基因表达。

奥利亚罗非鱼DMRT1,DMO基因片段的克隆及序列分析

根据其他鱼类DMRT基因中的保守序列设计了一对简并引物,利用RT-PCR扩增了雌雄奥利亚罗非鱼的DMRT基因,并对其扩增产物进行了克隆与测序。结果在雌雄奥利亚罗非鱼个体中获得了两个不同的片段,分别命名为DMO,DMRT1基因。序列同源性分析表明,奥利亚罗非鱼DMRT1,DMO cNA系列的同源性为61.78%,氨基酸同源性为86%,说明了DMRT基因存在性别差异。与尼罗罗非鱼、红鳍东方豚、虹鳟、青鱼将等鱼类DM保守区相比,氨基酸序列同源性为86%-100%,这充分显示了DM-RT基因在进化上的高度保守性。

Study of the mechanisms of acupuncture and moxibustion treatmentfor ulcerative colitis ratsinview of the gene expression of cytokines

AIM:To observe the effect of acupuncture and moxibustion on the expression of IL-1beta and IL-6 mRNA in ulcerative colitis rats.METHODS:The SD rat ulcerative colitis model was created by immunological method associated with local stimulation. Colonic mucosa was prepared from human fresh surgical colonic specimens, homogenized by adding appropriate amount of normal saline and centrifuged at 3000r/min. The supernatant was collected for measurement of protein conentration and then mixed with Freund adjuvant. This antigen fluid was first injected into the plantae of the model group rats, and then into their plantae, dorsa, inguina and abdominal cavities (noFreund adjuvant for the last injection) again on the 10th, 17th, 24th and 31st day. When a certain titer of serum anti colonic antibody was reached, 2% formalin and antigen fluid (no Freund adjuvant) were administered separately by enema. The ulcerative colitis rat model was thus set up. The animals were randomly divided into four groups: model control group (MC, n = 8), electro acupuncture group (EA, n = 8), herbs partition moxibustion group (HPM 8), normal control group (NC,n = 8). HPM: Moxa cones made of refined mugwort floss were placed on the medicinal pad (medicinal pad dispensing: Radix Aconiti praeparata, cortex Cinnamomi, etc) for Qihai (RN 6) and Tianshu (ST 25, bilateral) and ignited. Two moxa cones were used for each acupoint once a day and 14 times in all. EA: Tianshu (bilateral) and Qihai were stimulated by the intermittent pulse with 2Hz frequency, 4mA intensity for 20 minutes once a day and 14 times in all. After treatment, rats of all four groups were killed simultaneously. The spleen was separated and the distal colon was dissected. Total tissue RNA was isolated by the guanidinium thiocyanate phenol chloroform extraction method. RT-PCR technique was used to study the expression of IL-1 beta and IL-6 mRNA.RESULTS:IL-1 beta and IL-6 mRNAs were not detected in the spleen and colonic mucosa of the NC rats, whereas they were significantly expressed in that of the MC rats.IL-1 beta and IL-6 mRNAs were markedly lower in the EA and HPM rats than that in MC rats. There was no significant difference between the levels of IL-1 beta and IL-6 mRNAs in the EA and HPM rats. The expressions of IL-1 beta and IL-6 mRNAs were nearly the same in the spleen and colon of all groups.CONCLUSION:Acupuncture and moxibustion greatly inhibited the expression of IL-1 beta and IL-6 mRNA in the experimental ulcerative colitis rats.

Adhesion molecule and proinflammatory cytokine gene expression in hepatic sinusoidal endothelial cells following cecal ligation and puncture

INTRODUCTIONMultiple organ dysfunction syndrome (MODS) isthought to be a frequent consequence of sepsis[1-3].Despite substantial advances in our knowledge and understanding of the basic pathophysiologic mechanisms[4-7], in critically ill patients infections and sepsis are still associated with a high mortality[8,9].

Molecular detection of monocyte chemotactic protein-1 polymorphism in spontaneous bacterial peritonitis patients

AIM: To investigate the association of the functional monocyte chemotactic protein-1 (MCP-1) promoter polymorphism (A-2518G) with spontaneous bacterial peritonitis (SBP).

Molecular cloning and expression analysis of interleukin - 1β from Japanese sea perch(Lateolabrax japonicus )

The technology of homology cloning and anchored PCR was used to clone the IL-1β gene from the Japanese sea perch （Lateolabrax iaponicus）. The full-length cDNA of sea perch IL-1β was 1 310 bp, including a 5＇ untranslated regiop （UTR） of 136 bp, a 3＇ UTR ot 430 bp, and an ORF of 774 bp encoding a polypeptide of 258 amino acids with an estimated molecular mass of 29.31 kDa. The searches for nucleotides and protein sequence similarities with the BLAST analysis indicated that the deduced amino acid sequence of sea perch IL-1β was homological to the IL-1β in other fish species and even the mammalian. Conserved signature sequences of the IL-1β gene family were found in the sea perch IL-1β deduced amino acid sequence. Temporal expressions of the IL-1β gene in LPS or iridovirus challenged group and in control group were measured by the semi-quantitative RT-PCR. The mRNA transcripts of IL-1β could be detected in head-kidney, spleen, liver, gill and heart of the healthy individuals, and the expression level of IL-1β in head-kidney, spleen and gill was higher than that in liver and heart, but it was hard to be detected in the brain. After being stimulated by the LPS or iridovirus, the IL-1β expression in most of examined tissues was up-regulated, and also could be detected in the brain. These results indicated that the expression of sea perch IL-1β was constitutive and could be up-regulated by immune effector stimulation. Therefore the sea perch IL-1β could play a critical role in the host-pathogen interaction.

Promoter methylation status of hMLH1,MGMT,and CDKN2A/p16 in colorectal adenomas

AIM:To investigate aberrant DNA methylation of CpG islands and subsequent low-or high-level DNA microsatellite instability(MSI)which is assumed to drive colon carcinogenesis. METHODS:DNA of healthy individuals,adenoma(tu-bular or villous/tubulovillous)patients,and colorectal carcinoma patients who underwent colonoscopy was used for assessing the prevalence of aberrant DNA methylation of human DNA mismatch repair gene mutator L homologue 1(hMLH1),Cyclin-dependent kinase inhibitor 2A(CDKN2A/p16),and O-6-methylguanine DNA methyltransferase(MGMT),as well as their rela- tion to MSI. RESULTS:The frequency of promoter methylation for each locus increased in the sequence healthy tissue/adenoma/carcinoma.MGMT showed the highest frequency in each group.MGMT and CDKN2A/p16 presented a statistically significant increase in promoter methylation between the less and more tumorigenic forms of colorectal adenomas(tubular vs tubullovillous and villous adenomas).All patients with tubulovillous/villous adenomas,as well as all colorectal cancer patients,showed promoter methylation in at least one of the examined loci.These findings suggest a potentially crucial role for methylation in the polyp/adenoma to cancer progres- sion in colorectal carcinogenesis.MSI and methylation seem to be interdependent,as simultaneous hMLH1, CDKN2A/p16,and MGMT promoter methylation was present in 8/9 colorectal cancer patients showing the MSI phenotype. CONCLUSION:Methylation analysis of hMLH1,CD- KN2A/p16,and MGMT revealed specific methylation profiles for tubular adenomas,tubulovillous/villous adenomas,and colorectal cancers,supporting the use of these alterations in assessment of colorectal tumorigenesis.

贵州省彝族、瑶族及汉族乙肝病毒易感性与白介素-10基因的相关性

目的:研究贵州省彝族、瑶族以及汉族人群白介素-10(IL-10)基因多态性与乙型肝炎病毒易感性的相关性。方法:对研究人群中肝病毒感染者和非感染者采用聚合酶链式反应-限制性片段长度多态性(PCRRFLP)方法分析IL-10基因启动子区-592位点多态性,序列特异性引物-聚合酶链式反应(PCR-SSP)方法分析IL-10基因启动子区-819位点多态性,短串联重复序列或微卫星DNA-聚合酶链式反应(STRs-PCR)方法分析IL-10基因启动子区IL-10.G和IL-10.R两个STR位点。结果:IL-10基因位点单倍型与HBV感染的相关分析表明,贵州省彝族乙肝病毒感染组(HBsAg、抗-HBs或抗-HBc中任一项阳性者)IL-10.G.461bp(20CA)-592A-819T单倍型频率显著高于乙肝病毒非感染组(P<0.05),非感染组IL-10.G.459bp(19CA)-592 C-819 C单倍型频率显著高于感染组(P<0.05);贵州省汉族乙肝病毒非感染组IL-10.G.459bp(19CA)-592 A-819 T单倍型频率显著高于感染组(P<0.05),贵州省瑶族乙肝病毒感染组与非感染组之间各单倍型频率分布差异均无统计学意义(P>0.05)。结论:IL-10-592、IL-10-819以及IL-10.G、IL-10.R位点多态性在贵州世居彝族、瑶族以及汉族中有不同的分布,并且可能与该人群对乙肝病毒的易感性相关。

花生AhFAD2-1基因与油酸/亚油酸比值的关系

花生AhFAD2-1由位于不同基因组上的两个非等位基因AhFAD2-1A和AhFAD2-1B共同编码,这两个基因的突变引起酶结构、酶活性或表达调控的变化,共同导致高油酸性状的产生。本研究通过对13个不同花生品种（系）的AhFAD2-1基因进行测序和比对分析,查找点突变或插入位点,寻找与高油酸性状关联的基因位点。结果表明：E11、花育30、鲁花12、豫花15、河北高油的基因型是OL1OL1OL2OL2,其相应的O/L值为1.01-1.40;鲁花14、花育17、花育19、花育23、E12S的基因型是ol1ol1OL2OL2,其中E12S较特殊O/L值为9.05,其他品种O/L值为1.54-1.97;E16、E18和花育32号的基因型是ol1ol1ol2ol2,其相应O/L值为12.3-41.85。本研究结果对于高油酸性状的分子鉴定以及高油酸花生新品种的培育具有一定的参考价值。

1-(5-异喹啉磺酰基)-2-甲基哌嗪(H-7)对肺纤维化大鼠肺单核细胞趋化蛋白-1 mRNA表达的影响

研究蛋白激酶C(PKC)抑制剂 1 (5 异喹啉磺酰基 ) 2 甲基哌嗪 (H 7)对肺纤维化大鼠单核细胞趋化蛋白 1(MCP 1)mRNA表达的影响 ,为临床治疗肺纤维化提供理论基础 .博来霉素致大鼠肺纤维化动物模型 ,腹腔注射H 7后 ,定量RT PCR方法检测其对肺组织MCP 1mRNA表达的影响 ;测定实验组和对照组肺组织匀浆内羟脯氨酸的含量 ,观察肺组织病理学变化并计数单核细胞 .结果发现 ,H 7能显著抑制大鼠肺纤维化组织MCP 1mRNA表达和单核细胞的聚集以及羟脯氨酸的含量 .结果表明 ,H 7通过抑制肺组织MCP 1mRNA的表达 ,对博来霉素致肺纤维化有明显的防治作用 .

‘红根甘肃桃1号’抗南方根结线虫活性物质分析

【目的】探讨‘红根甘肃桃1号’抗南方根结线虫的生理机制。【方法】选取对南方根结线虫免疫的‘红根甘肃桃1号’、高抗的‘白根甘肃桃1号’、高感的‘贝蕾’以及‘红根甘肃桃1号’与‘贝蕾’的回交群体BC1为试验材料,采用广泛靶向代谢组技术测定样品间的代谢组成分,并利用转录组分析了黄酮醇合成途径关键基因在‘红根甘肃桃1号’与‘贝蕾’中的表达。【结果】在不同样品间,共检出17类、427种代谢产物,比较这些物质在BC1亲本和抗/感病混合池材料以及‘红根甘肃桃1号’和‘白根甘肃桃1号’种质间的含量变化,鉴定出4种差异的代谢物质,其中甲基槲皮素O-己糖苷的相对含量在‘红根甘肃桃1号’中是‘白根甘肃桃1号’的31.15倍。转录组分析表明,黄酮醇合成酶基因在不同抗性种质间表达有明显差异,其关键基因为Prupe.1G502300。【结论】差异代谢物质甲基槲皮素O-己糖苷可能作为一种重要的抗线虫物质,参与了‘红根甘肃桃1号’对南方根结线虫的免疫反应,该研究为解析‘红根甘肃桃1号’抗根结线虫的生理和分子机制奠定理论基础。

桑椹成熟期间1-脱氧野尻霉素和主要花青素含量及相关基因表达的变化规律研究

桑椹具有较高的营养价值和保健功能,其主要生物活性成分包括生物碱1-脱氧野尻霉素(1-deoxynojirimycin,DNJ)以及矢车菊素-3-葡萄糖苷(cyanidin-3-O-glucoside,C3G)和矢车菊素-3-芸香糖苷(cyanidin-3-O-rutinoside,C3R)等花青素。利用高效液相色谱法检测桑椹成熟期间DNJ、C3G、C3R含量,用实时荧光定量PCR法检测相关基因表达水平的变化情况。结果显示,在桑椹变红前,DNJ含量较高,质量分数最高达到0.246%,但C3G和C3R未能被检测到;随着桑椹的成熟,DNJ含量迅速下降,到完全成熟后质量分数仅有0.006%,而C3G和C3R含量逐渐升高并趋于稳定,最终C3G质量分数稳定在1%左右,C3R质量分数稳定在0.6%左右。DNJ合成路径上相关的赖氨酸脱羧酶(lysine decarboxylase,LDC)和铜胺氧化酶(copper amine oxidase,CAO)编码基因LDC、CAO1、CAO2、CAO4的表达量在桑椹未变红前为逐渐升高,后迅速下降,而CAO3表达量一直逐渐降低;花青素合成关键酶基因4CL和CHI表达量在桑椹变红前逐渐升高,后迅速降低,而CHS表达量在桑椹变红前较低,后迅速升高并持续维持较高水平。研究结果不仅为药用桑椹的采收提供了指导,也为果桑分子育种提供了理论依据。