Genome-Wide Analysis of the F3′5′H Gene Family in Blueberry(Vaccinium corymbosum L.)Provides Insights into the Regulation of Anthocyanin Biosynthesis

The F3′5′H gene family plays an important role in regulating anthocyanin biosynthesis,abiotic stress,and hormone signaling.In this study,14 F3′5′H genes were identified from the blueberry genome.The chromosomal distribution,physicochemical properties,F3′5′H domain,conserved motifs,cis-acting elements,and intron/exon compositions were analyzed.The functional prediction analysis of these VcF3′5′Hs indicated that their biological functions included light response and other secondary metabolites.The results of qRT-PCR showed that VcF3′5′Hs(especially VcF3′5′H4)were highly expressed at the ripening stage.Subcellular localization revealed that VcF3′5′H4 may be located in the endoplasmic reticulum.Co-expression analysis showed that the VcF3′5′H gene family was related to anthocyanin.This research provides an overview of the blueberry F3′5′H family and helps verify the role of these genes in regulating anthocyanin biosynthesis.

人F3-Restin基因的克隆与表达

目的:克隆人F3-Restin基因,表达有生物学活性的新型融合蛋白F3-Restin。方法:采用亚克隆技术构建融合表达载体PET32a(+)-F3-Restin,原核诱导表达,经Ni2+-NTA琼脂糖树脂亲和层析纯化,透析去盐。结果:在20℃、10h的条件下IPTG原核诱导表达、纯化,得到分子量约为40kD的高纯度的可溶性蛋白F3-Restin。结论:成功地克隆了人F3-Restin基因,构建融合表达载体PET32a(+)-F3-Restin,并表达出新型融合蛋白F3-Restin,为进一步研究F3-Restin蛋白抗肿瘤血管生长的活性及作用机理奠定了良好的基础。

重组蛋白F3-Restin生物学活性的初步研究

目的:表达有生物学活性的新型融合蛋白F3-Restin,并纯化、鉴定,探讨F3-Restin融合肽的生物学活性。方法:在20℃、10h的条件下,IPTG诱导融合表达载体PET32a(+)-F3-Restin原核表达、纯化,得到分子量约为40kD的高纯度的可溶性蛋白F3-Restin,检测与HUVEC的亲和活性,CAM试验检测抗血管活性。结果:重组蛋白F3-Restin具有特异结合血管内皮细胞的能力,并能抑制新血管的生成。结论:初步表明新型融合蛋白F3-Restin仍具特异结合血管内皮细胞,抑制新血管生长的能力。

粉尘螨主要变应原基因Der f1和Der f3改组的研究

目的:探索粉尘螨变应原基因Der f1和Der f3改组条件,以期获得粉尘螨变应原融合突变基因。方法:RT-PCR方法扩增粉尘螨变应原Der f 1和Der f3基因,用DnaseⅠ分别酶解Der f 1和Der f3基因10、15、20、25、30 min;酶解相同时间的Der f1和Der f3基因片段两两混合,采用DNAshuffling技术对粉尘螨变应原Der f1和Der f3基因进行重组,琼脂糖凝胶电泳检测重组子。结果:以Der f 2 F和Der f2 R为引物在酶切10、15、20、25、30min均可扩增出清晰条带;以Der f1 F和Der f1 R、Der f1 F和Der f2 R、Der f2 F和Der f1 R为引物在酶切10、15、20、25、30min的模板中均无条带出现;而其他引物组合则在不同酶切时间的模板中均出现条带。结论:通过多种条件的组合,可获得多个粉尘螨变应原基因Der f1和Der f3间的融合基因,为大规模制备高效、低价的哮喘疫苗奠定了基础。

Cloning, sequence analysis and expression in E. coli of the group 3 allergen of Dermatophagoides farinae

Background The dust mites, which are mostly represented by Dermatophagoides spp. （Acari： Pyroglyphidae）, are the major sources of indoor allergens. Identification and characterization of these mite allergen molecules are an important step in the development of new effective diagnostic procedures and possible therapeutic strategies for allergic disorders associated with dust mites. Methods Total RNA was extracted from Dermatophagoides farinae. The gene coding for Der f 3 was amplified by RT-PCR with the primers designed based on previous sequence published in GenBank. The target gene was cloned intermediately into pMD19-T plasmid and finally into plasmid pET28a （＋）, expressed in E. coil BL21 at the aid of the inducer isopropyI-D-thiogalactopyranoside （IPTG）. The physicochemical properties, spatial structure of the allergen were analyzed with bioinformatics software. Results The cDNA coding for group 3 allergen of Dermatophagoides farinae from China was cloned and expressed successfully. Sequencing analysis showed that there were nineteen mismatched nucleotides in five Der f3 cDNA clones in comparison with the reference （GenBank Accession No. AY283291）, which resulted in deduced amino acid sequence incompatibility in eleven residues. Bioinformatics analysis revealed that the Der f 3 pro-protein was an extracellular hydrophobic protein, consisting of 259 amino acids with a 16 amino acid signal peptide. The protein was deduced to have three chymotrypsin active sites （53-68 AA, 108-122 AA and 205-217 AA）, one N-glycosylation site, one cAMP- and cGMP-dependent protein kinase phosphorylation site, four protein kinase C phosphorylation sites, two casein kinase II phosphorylation sites, and five N-myristoylation sites. Conclusions Der f3 is an extracellular hydrophobic protein which possesses multiple activation and phosphorylation sites. Polymorphism may exist in the Der f3 gene but this needs to be further confirmed in the future.

The Expression of <i>TIMP</i>1, <i>TIMP</i>2, <i>VCAN</i>, <i>SPARC</i>, <i>CLEC</i>3<i>B</i>and <i>E</i>2<i>F</i>1 in Subcutaneous Adipose Tissue of Obese Males and Glucose Intolerance

We investigated the expression of TIMP1, TIMP2, SPARC, VCAN, and CLEC3B genes, encoded matricellular proteins with pleiotropic functions, and glucose intolerance in obese male subjects with normal and impaired glucose tolerance. The purpose of this study was to examine the association between the gene expressions and glucose intolerance in obesity. The results indicate that obesity leads to significant increase of TIMP1, TIMP2, E2F1 and CLEC3B gene expressions in subcutaneous adipose tissue, especially TIMP2 gene. However, more significant increase of the expression of TIMP1 and TIMP2 was found in adipose tissue of obese patients with glucose intolerance. No significant changes were found in the expression of VCAN and SPARC genes in adipose tissue of obese subjects with normal glucose tolerance but increased in the group of obese subjects with glucose intolerance. At the same time, the E2F1 and CLEC3B gene expressions were decreased in adipose tissue of obese patients with glucose intolerance. Results of this study provide evidence that changes in the expression of genes encoded TIMP1, TIMP2, VCAN, SPARC, E2F1 and CLEC3B in subcutaneous adipose tissue of obese individuals associate with glucose intolerance.

人3型副流感病毒F蛋白在Bac-to-Bac杆状病毒表达系统中表达的研究

目的用Bac-to-Bac杆状病毒表达系统(Bac-to-Bac■baculovirus expression systems, Bac-to-Bac BES)表达人3型副流感病毒(human parainfluenza virus type 3, HPIV-3)的重组融合蛋白(fusion protein, F),并研究密码子优化、疏水性区域、SUMOStar融合标签及gp67分泌信号肽对目的蛋白表达的影响,同时研究F蛋白的抗原性与免疫原性。方法全基因合成HPIV-3 F蛋白昆虫细胞密码子优化基因序列,设计特异性引物,经PCR扩增获得HPIV-3全长优化型F基因(op-F)、去除N端和C端疏水区域的截短优化型F基因(opt-F)和截短野生型F基因(wtt-F);将以上基因分别构建至pFastBac1、pI-SUMOStar、pI-secSUMOStar 3种载体,采用Bac-to-Bac杆状病毒表达系统对各目的基因对应的蛋白进行表达。利用SDS-PAGE、Western blot等方法对各目的蛋白的表达及特异性进行检测。用纯化的重组opt-F蛋白免疫青紫蓝兔,制备F蛋白特异性抗体。结果 ①对HPIV-3的opt-F基因及wtt-F基因的表达对比显示,opt-F基因在3种载体中都得到了良好表达,而wtt-F基因在3种载体中均未表达,说明密码子优化对目的基因的表达具有重要作用;②对HPIV-3的op-F基因及opt-F基因在pFastBac1载体中的表达对比显示,opt-F基因得到了良好表达,而op-F基因未表达,说明疏水性区域可显著影响目的基因的表达;③对HPIV-3的opt-F基因在pFastBac1及pI-SUMOStar载体中的表达对比显示,SUMOStar融合标签可使opt-F基因的表达量明显提高;④对HPIV-3的opt-F基因在pI-SUMOStar及pI-secSUMOStar载体中的表达研究显示,添加gp67分泌信号肽后未检测到opt-F基因的可溶性表达,说明gp67信号肽对目的蛋白无促可溶性表达效果。⑤纯化的opt-F/pI-SUMOStar蛋白免疫青紫蓝兔,得到了滴度为1∶8 192的抗血清。Western blot显示,此血清可以特异性结合HPIV-3病毒的F蛋白。结论密码子优化、N/C端疏水区域的去除以及SUMOStar融合标签的嵌合,可以显著提高F蛋白在Sf9昆虫细胞中的表达。

三七3-羟基-3-甲基戊二酸单酰辅酶A还原酶基因的克隆和生物信息学分析

目的克隆三七总皂苷甲羟戊酸合成途径中的1个关键酶3-羟基-3-甲基戊二酸单酰辅酶A还原酶(3-hydroxy-3-methylglutaryl coenzyme-A reductase,HMGR)基因,为进一步研究HMGR蛋白的功能及开展三七皂苷的合成生物学研究奠定基础。方法根据NCBI上已公布的同属人参的HMGR基因全长序列(登录号GU565097.1)设计特异性引物。利用RT-PCR技术,以三七愈伤组织总RNA反转录的cDNA为模板扩增基因片段;并对其编码的蛋白进行生物信息学预测和基因表达分析。结果序列分析表明,获得的三七HMGR(命名为PnHMGR)基因的cDNA序列大小为1 893 bp,该序列与人参、刺五加和杜仲中HMGR基因的一致性分别达到98%、93%、80%,且含有大小为1 725 bp的开放阅读框,经Genbank查询为一新的cDNA。生物信息学预测PnHMGR基因编码蛋白包含2个跨膜区,不含信号肽,具有HMGR催化作用的活性中心结构域。实时荧光定量PCR检测到PnHMGR基因在三七细胞生长30 d表达量最高。结论首次从药用植物三七中克隆得到HMGR基因的编码区序列,为进一步鉴定PnHMGR的功能和开展三七皂苷的合成生物学研究奠定了基础。

粉尘螨变应原第3组分基因克隆及序列多态性分析

目的获得粉尘螨变应原第3组分(Der f 3)的编码基因并了解其序列多态性。方法根据GenBank已公布的Der f 3核酸序列设计引物,用RT-PCR扩增获得其编码基因,插入pMD19-T载体,进行序列测定和多态性分析。结果获得粉尘螨变应原Der f 3编码基因为780bp。5株Der f 3基因cDNA克隆的核苷酸序列与参考序列(GenBankNo.AY 283291,GenBank No.D63858,GenBank No.EU312162,GenBank No.EU312163)相比,有19处cDNA序列发生突变,但有7处突变至少在3个cDNA克隆中序列一致;推导出氨基酸序列后进行比对,有11处氨基酸序列发生变化,有4处与上述7个位点相一致。结论获得了粉尘螨变应原Der f 3编码基因且证明其序列具有多态性,为进一步开展相关研究奠定了基础。