Cloning of Cotton Delta-12 Oleate Desaturase Gene FAD2-1 and Construction of Its ihpRNA and amiRNA Interference Vectors

Delta-12 oleate desaturase gene （FAD2-1） which converts oleic acid into linoleic acid, is the key enzyme determining the fatty acid composition of cottonseed oil. By employing RT-PCR method, full length cDNA of cotton delta-12 oleate desat- urase gene GhFAD2-1 containing an open reading frame of 1 158 bp was cloned for constructing RNAi vector. A 515 bp long specific fragment of this gene was se- lected for constructing ihpRNA vector under the control of a seed-specific promoter NAPIN, named pFGC1008-NAPIN-FAD2-1; meanwhile miRNA gene-silencing vector pCAMBIA1302-amiRNA-FAD2-1 targeting GhFAD2-1 was also constructed.

Exercise-induced modulation of miR-149-5p and MMP9 in LPS-triggered diabetic myoblast ER stress: licorice glycoside E as a potential therapeutic target

Background:This study explores the relationship between endoplasmic reticulum(ER)stress and diabetes,particularly focusing on the impact of physical exercise on ER stress mechanisms and identifying potential therapeutic drugs and targets for diabetes-related sepsis.The research also incorporates traditional physical therapy perspectives,emphasizing the genomic insights gained from exercise therapy in disease management and prevention.Methods:Gene analysis was conducted on the GSE168796 and GSE94717 datasets to identify ER stress-related genes.Gene interactions and immune cell correlations were mapped using GeneCard and STRING databases.A screening of 2,456 compounds from the TCMSP database was performed to identify potential therapeutic agents,with a focus on their docking potential.Techniques such as luciferase reporter gene assay and RNA interference were used to examine the interactions between microRNA-149-5p and MMP9.Results:The study identified 2,006 differentially expressed genes and 616 miRNAs.Key genes like MMP9,TNF-α,and IL1B were linked to an immunosuppressive state.Licorice glycoside E demonstrated high affinity for MMP9,suggesting its potential effectiveness in treating diabetes.The constructed miRNA network highlighted the regulatory roles of MMP9,IL1B,IFNG,and TNF-α.Experimental evidence confirmed the binding of microRNA-149-5p to MMP9,impacting apoptosis in diabetic cells.Conclusion:The findings highlight the regulatory role of microRNA-149-5p in managing MMP9,a crucial gene in diabetes pathophysiology.Licorice glycoside E emerges as a promising treatment option for diabetes,especially targeting MMP9 affected by ER stress.The study also underscores the significance of physical exercise in modulating ER stress pathways in diabetes management,bridging traditional physical therapy and modern scientific understanding.Our study has limitations.It focuses on the microRNA-149-5p-MMP9 network in sepsis,using cell-based methods without animal or clinical trials.Despite strong in vitro findings,in vivo studies are needed to confirm licorice glycoside E’s therapeutic potential and understand the microRNA-149-5p-MMP9 dynamics in real conditions.

固始-安卡鸡F_2资源群△-9脂肪酸脱氢酶基因第4、5、6外显子SNP及其与脂肪酸相关性研究

利用PCR-SSCP(single strand conformation polymorphism)方法,对固始-安卡鸡F2代资源群△-9脂肪酸脱氢酶基因第4、5、6对外显子多态性进行分析,发现存在一个单核苷酸多态(SNP,single nucleotide polymorphisms)位点。在△-9脂肪酸脱氢酶基因第4外显子编码区(89nt)发生了碱基的转换突变(G→A)。在整个F2代资源群中进行了该多态位点与脂肪酸性状的相关分析,结果表明:在8个脂肪酸性状中,AA型个体十六碳二烯酸(C16∶2)含量显著高于GG型和GA型个体(P<0.05);家系1中AA型的C16∶0、C16∶2、C18∶0含量均显著高于GG型和GA型(P<0.05);家系3中AA型的C14∶0含量显著高于GG型和GA型(P<0.05)。

Expressions of MMP-2,-9,TIMP-1,-2,-3 mRNA in Rat Uterus during Estrous Cycle

Zymography and in situ hybridization were used to investigate matrixmetalloproteinase -2, -9 (MMP -2, MMP-9) activities and expressions of MMP -2, -9 and TIMP1, -2, -3 (tissue inhibitors of matrix metallo-proteinases) mRNA in the rat uterus during estrouscycle. The relative activity was semiquanted by using densitometric analysis. The MMP-2(67 kDa) activity in every stage during estrpus cycle was detected by zymography. MMP-2activity was highest at proestrus; higher at estrus and metaestrus; lowest at diestrus. Throughin situ hybridization, MMP -2, -9, TIMP -1~ -3 mRNA mainly in hasal stroma cells of uterineendometrium were detected. The positive signals of MMP -2 and -9 mRNAs in hasal stromacells were shown stronger at proestrus, estrus and metaestrus while they showed the weakest atdiestrus. The expression of MMP -2 mRNA coincided with MMP -2 activity change. MMP-2and -9 mRNAs were also highly expressed in uterine circular muscle at estrus. Weak signals ofMMP -9 mRNA were detected in uterine luminal and glandular epithelial cells at estrus.TIMP -1 mRNA in hasal stroma cells was shown as the strongest expression at estrus andmetaestrus; stronger at proestrus and the weakest at diestrus. TIMP-2 mRNA in basal stromacells was stronger at estrus and diestrus; weaker at proestrus and metaestrus. TIMP -1 and -2mRNAs were also highly expressed in uterine luminal and glandular epithelial cells at estrus.TIMP -3 mRNA in hasal stroma cells revealed the strongest expression at estrus; stronger atdiestrus and metaestrus and showed the weakest at proestrus. The mRNA was also highlyexpressed in uterine circular muscle at estrus. In short, our present results provide evidencethat MMP -2, -9 and TIMP -1~ -3 were involved in rat uterine endometrium reconstructionduring estrous cycle.

Detailed Deletion Mapping of Chromosome 9p21-22 in Nasopharyngeal Carcinoma

Objective: To further refine the extent of deletion on chromosome 9p21-22 in nasopharyngeal carcinoma (NPC) and provide evidence for discovering new tumor suppressor gene. Methods: Loss of heterozygosity (LOH) on chromosome 9p21-22 was analyzed in 25 paired blood and tumor samples by using 11 high-density microsatellite polymorphic markers. Results: 17 of 25 cases (68.0%) showed LOH at one or more loci. Higher frequencies of LOH were found at four loci: D9S161 (35.0%), D9S1678 (31.5%), D9S263 (33.3%) and D9S1853 (33.3%), where 6 cases had a contiguous stretch of allelic loss. Conclusion: The minimal common region of deletion might be defined between D9S161 and D9S1853 (estimated about 2.7 cM in extent) at 9p21.1, suggesting that inactivation of one or more tumor suppressor genes located in this region may be an important step in NPC.

大头金蝇酰基辅酶AΔ9去饱和酶cDNA克隆与原核表达

为深入研究大头金蝇Chrysomya megacephala (Fabricius)脂肪酸代谢关键功能基因酰基辅酶AΔ9去饱和酶(ACD9des),运用RT-PCR和RACE技术,获得其cDNA全长序列,并对其进行生物信息学分析。大头金蝇ACD9des基因cDNA (GenBank登录号为KF835695)全长1 429 bp,其中开放阅读框(ORF)为1 146 bp,编码381个氨基酸,5'UTR长度为138 bp,3'UTR约为114 bp。ORF编码的蛋白质分子量为43. 47 kD,等电点9. 06,氨基酸序列与其他昆虫酰基辅酶A去饱和酶一致性高达66%-93%,且含有由7个酰基辅酶A去饱和酶蛋白家族特有的保守模式(motif)所构成的指纹(IPR015876)。大头金蝇ACD9des在进化上与葱蝇Delia antiqua最趋于一致。将ACD9des的ORF克隆到原核表达载体p ET-44a(+),并利用Rosetta (DE3)感受态细胞进行ACD9des原核表达。Western Blot分析表明,IPTG诱导表达的特异性蛋白可以与anti-His抗体特异性结合,大小与预期理论值(43. 47 kDa)相符,为ACD9des。该蛋白主要存在于上清溶液中,为可溶性表达。最后利用含250 mM咪唑洗脱液和镍离子亲和层析柱对扩大培养获得的重组蛋白进行了纯化收集。本文的研究结果为大头金蝇功能基因的深入研究提供了坚实的基础。

Antisense-Mediated Depletion of Tomato Chloroplast Omega-3 Fatty Acid Desaturase Enhances Thermal Tolerance

A chloroplast-localized tomato （Lycopersicon esculentum Mill.） ω-3 fatty acid desaturase gene （LeFADT） was isolated and characterized with regard to its sequence, response to various temperatures, and function in antisense transgenic tomato plants. The deduced amino acid sequence had four histidine-rich regions, of which three regions were highly conserved throughout the whole ω-3 fatty acid desaturasegene family. Southern blotting analysis showed that LeFAD7was encoded by a single copy gene and had two homologous genes in the tomato genome. Northern blot showed that LeFAD7 was expressed in all organs and was especially abundant in leaf tissue. Meanwhile, expression of LeFAD7 was induced by chilling stress （4 ℃）, but was inhibited by high temperature （45 ℃）, in leaves. Transgenic tomato plants were produced by integration of the antisense LeFAD7DNA under the control of a CaMV35S promoter into the genome. Antisense transgenic plants with lower 18 ： 3 content could maintain a higher maximal photochemical efficiency （Fv/Fm） and O2 evolution rate than wild-type plants. These results suggested that silence of the LeFAD7 gene alleviated high-temperature stress. There was also a correlation between the low content of 18 ： 3 resulting from silence of the LeFAD7 gene and tolerance to high-temperature stress.

山桐子IpSAD基因家族分析及功能鉴定

【目的】硬脂酰-ACPΔ~9脱氢酶(stearoyl-ACPΔ~9 desaturase,SAD)是决定脂肪酸组成的关键酶,分离和鉴定木本油料植物山桐子(Idesia polycarpa)的SAD基因,可为遗传改良山桐子油的脂肪酸组成提供基因资源。【方法】使用HMM和Blastp鉴定山桐子全基因组中的SAD家族成员;利用MEGA X、MEME和PlantCare等在线软件对其蛋白质理化性质、系统发育关系、基因结构、保守基序和启动子顺式调控元件等进行分析;使用MCSCANX分析山桐子和毛果杨SAD基因之间的共线性关系;基于RNA-seq数据,分析IpSAD各成员的表达模式,通过病毒诱导的基因沉默技术(virus induced gene silencing,VIGS)快速验证它们的生物学功能。【结果】(1)在山桐子基因组中共鉴定到7个IpSAD基因(IpSAD1~7),多重序列比对结果显示各成员间序列相似度较高且结构域保守;(2)系统发育分析表明,IpSAD成员可以分为3个亚组,与拟南芥SAD家族的分类结果一致,拟南芥单不饱和油酸合成关键基因AtSSI2与山桐子IpSAD2、IpSAD3、IpSAD4的亲缘关系较近;(3)启动子区域顺势调控元件预测结果显示IpSAD基因的启动子含有光反应、脱落酸响应等顺式调控元件;(4)IpSAD基因按其组织表达模式可分为3类,第1类和第2类成员在大部分组织中表达量较低,而第3类成员在所有组织中表达量均较高。(5)沉默IpSAD基因使叶片的油酸含量显著降低,其中IpSAD3的沉默可使油酸含量下降76%。【结论】明确了IpSAD家族各成员的基本特征,确定了它们对于油酸合成的生物学功能,并为山桐子油脂生物合成调控机制研究奠定了基础。